Justin Cooper-White

Saliva proteome research: current status and future outlook

Publisher: Informa UK Limited

Date: 21-05-2013

DOI: 10.3109/07388551.2012.687361

Abstract: Human saliva harbours proteins of clinical relevance and about 30% of blood proteins are also present in saliva. This highlights that saliva can be used for clinical applications just as urine or blood. However, the translation of salivary biomarker discoveries into clinical settings is h ered by the dynamics and complexity of the salivary proteome. This review focuses on the current status of technological developments and achievements relating to approaches for unravelling the human salivary proteome. We discuss the dynamics of the salivary proteome, as well as the importance of s le preparation and processing techniques and their influence on downstream protein applications post-translational modifications of salivary proteome and protein: protein interactions. In addition, we describe possible enrichment strategies for discerning post-translational modifications of salivary proteins, the potential utility of selected-reaction-monitoring techniques for biomarker discovery and validation, limitations to proteomics and the biomarker challenge and future perspectives. In summary, we provide recommendations for practical saliva s ling, processing and storage conditions to increase the quality of future studies in an emerging field of saliva clinical proteomics. We propose that the advent of technologies allowing sensitive and high throughput proteome-wide analyses, coupled to well-controlled study design, will allow saliva to enter clinical practice as an alternative to blood-based methods due to its simplistic nature of s ling, non-invasiveness, easy of collection and multiple collections by untrained professionals and cost-effective advantages.

Adipose Tissue Engineering Based on the Controlled Release of Fibroblast Growth Factor-2 in a Collagen Matrix

Publisher: Mary Ann Liebert Inc

Date: 11-2006

DOI: 10.1089/TEN.2006.12.3035

Abstract: Adipose tissue forms when basement membrane extract (Matrigel) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 microL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Poly (glycerol‐sebacate) bioelastomers: 2. Synthesis using Brabender Plasticoder^® as a batch reactor

Publisher: Wiley

Date: 10-09-2015

DOI: 10.1002/APP.42852

Contact angle effects on microdroplet deformation using CFD

Publisher: Elsevier BV

Date: 10-2006

DOI: 10.1016/J.APM.2005.06.011

Quantification of D-dimer levels in human saliva

Publisher: Future Science Ltd

Date: 09-2013

DOI: 10.4155/BIO.13.190

Abstract: Background: Plasma D-dimer tests are currently used to exclude deep vein thrombosis and pulmonary embolism. Human saliva has numerous advantages over blood as a diagnostic s le. The aims of our study were to develop a reliable immunoassay to detect D-dimer levels in saliva, and to determine the correlation between salivary and blood D-dimer levels. Results/methodology: Saliva and blood s les were collected from 40 healthy volunteers. We developed a AlphaLISA ® immunoassay with acceptable analytical performances to quantify D-dimer levels in the s les. The median salivary D-dimer levels were 138.1 ng/ml (morning) and 140.7 ng/ml (afternoon), and the plasma levels were 75.0 ng/ml. Salivary D-dimer levels did not correlate with plasma levels (p = 0.61). Conclusion: For the first time, we have quantified D-dimer levels and found twofold increase in saliva (p 0.05) than in plasma. Further studies are required to demonstrate the clinical relevance/utility of salivary D-dimer in patients with confirmed deep vein thrombosis and/or pulmonary embolism.

A simple method for fabricating 3-D multilayered composite scaffolds

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.ACTBIO.2012.08.015

Abstract: One limitation of electrospinning stems from the charge build-up that occurs during processing, preventing further fibre deposition and limiting the scaffold overall thickness and hence their end-use in tissue engineering applications targeting large tissue defect repair. To overcome this, we have developed a technique in which thermally induced phase separation (TIPS) and electrospinning are combined. Thick three-dimensional, multilayered composite scaffolds were produced by simply stacking in idual polycaprolactone (PCL) microfibrous electrospun discs into a cylindrical holder that was filled with a 3% poly(lactic-co-glycolic acid) (PLGA) solution in dimethylsulfoxide (a good solvent for PLGA but a poor one for PCL). The construct was quenched in liquid nitrogen and the solvent removed by leaching out in cold water. This technique enables the fabrication of scaffolds composed principally of electrospun membranes that have no limit to their thickness. The mechanical properties of these scaffolds were assessed under both quasi-static and dynamic conditions. The multilayered composite scaffolds had similar compressive properties to 5% PCL scaffolds fabricated solely by the TIPS methodology. However, tensile tests demonstrated that the multilayered construct outperformed a scaffold made purely by TIPS, highlighting the contribution of the electrospun component of the composite scaffold to enhancing the overall mechanical property slate. Cell studies revealed cell infiltration principally from the scaffold edges under static seeding conditions. This fabrication methodology permits the rapid construction of thick, strong scaffolds from a range of biodegradable polymers often used in tissue engineering, and will be particularly useful when large dimension electrospun scaffolds are required.

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure

Publisher: Springer Science and Business Media LLC

Date: 08-10-2014

DOI: 10.1007/S10529-013-1341-0

Abstract: The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

A parametric study of droplet deformation through a microfluidic contraction: Low viscosity Newtonian droplets

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1016/J.CES.2006.03.011

Hierarchical “As-Electrospun” Self-Assembled Fibrous Scaffolds Deconvolute Impacts of Chemically Defined Extracellular Matrix- and Cell Adhesion-Type Interactions on Stem Cell Haptokinesis

Publisher: American Chemical Society (ACS)

Date: 05-12-2017

DOI: 10.1021/ACSMACROLETT.7B00834

High-throughput, deterministic single cell trapping and long-term clonal cell culture in microfluidic devices

Publisher: Royal Society of Chemistry (RSC)

Date: 2015

DOI: 10.1039/C4LC01176G

Abstract: In this paper, the design, development and validation of a novel high throughput microfluidic device enabling both the robust and rapid trapping of 100's to 1000's of single cells and their in situ clonal growth is described.

A Comparison of Two Ovine Lumbar Intervertebral Disc Injury Models for the Evaluation and Development of Novel Regenerative Therapies

Publisher: SAGE Publications

Date: 10-06-2018

DOI: 10.1177/2192568218779988

Ascorbate Promotes Epigenetic Activation of CD30 in Human Embryonic Stem Cells    

Publisher: Oxford University Press (OUP)

Date: 10-2010

DOI: 10.1002/STEM.500

Abstract: Human embryonic stem cells (hESCs) and induced pluripotent stem cells have the ability to adapt to various culture conditions. Phenotypic and epigenetic changes brought about by the culture conditions can, however, have significant impacts on their use in research and in clinical applications. Here, we show that diploid hESCs start to express CD30, a biomarker for malignant cells in Hodgkin's disease and embryonal carcinoma cells, when cultured in knockout serum replacement (KOSR)-based medium, but not in fetal calf serum containing medium. We identify the commonly used medium additive, ascorbate, as the sole medium component in KOSR responsible for CD30 induction. Our data show that this epigenetic activation of CD30 expression in hESCs by ascorbate occurs through a dramatic loss of DNA methylation of a CpG island in the CD30 promoter. Analysis of the phenotype and transcriptome of hESCs that overexpress the CD30 signaling domain reveals that CD30 signaling leads to inhibition of apoptosis, enhanced single-cell growth, and transcriptome changes that are associated with cell signaling, lipid metabolism, and tissue development. Collectively, our data show that hESC culture media that contain ascorbate trigger CD30 expression through an epigenetic mechanism and that this provides a survival advantage and transcriptome changes that may help adapt hESCs to in vitro culture conditions.

A novel multishear microdevice for studying cell mechanics

Publisher: Royal Society of Chemistry (RSC)

Date: 2009

DOI: 10.1039/B823180J

Abstract: Shear stresses are known to influence the morphology, and even the fate, of many cell types, including endothelial, smooth muscle, and osteoblast cells. This paper describes a novel shear device for the study of cell mechanics. Unlike all other published shear devices, such as parallel-plate flow chambers, where a single shear stress is evaluated for a single input flow rate, the described device enables the simultaneous evaluation of 10 different shear stresses ranging over two orders of magnitude (0.7-130 dynes cm(-2), 0.07-13 Pa). Human umblical vein endothelial cells (HUVECs) were exposed to the shear stress profiles provided by the device over a 20 h perfusion period, and the secretion level of von Willebrand factor (vWF) was investigated. Confirming previous studies, increasing shear resulted in increased vWF secretion. Furthermore, changes in cell morphology, including cell and nuclear size (area) and perimeter with shear, were analysed. HUVECs under shear stresses ranging from 1-3 dynes cm(-2) (0.1-0.3 Pa) showed similar vWF content, cell and nuclear size and perimeter to static cultures, while cells under shear stresses above 5 dynes cm(-2) (0.5 Pa) showed significantly higher vWF secretion and were at least 30% smaller in cell size. We also note that cells exposed to perfusion rates resulting in a shear stress of 0.7 dynes cm(-2) (0.07 Pa) showed significantly lower levels of vWF and were 35% smaller in size than those under static conditions. Overall, the results confirm the significant utility of this device to rapidly screen cellular responses to simultaneously imposed physiologically relevant ranges of shear stress.

A parametric study of droplet deformation through a microfluidic contraction: Shear thinning liquids

Publisher: Elsevier BV

Date: 05-2007

DOI: 10.1016/J.IJMULTIPHASEFLOW.2006.12.002

Compact tunable microfluidic interferometer

Publisher: Optica Publishing Group

Date: 2004

DOI: 10.1364/OPEX.12.005440

Abstract: We demonstrate a compact tunable filter based on a novel microfluidic single beam Mach-Zehnder interferometer. The optical path difference occurs during propagation across a fluid-air interface (meniscus), the inherent mobility of which provides tunability. Optical losses are minimized by optimizing the meniscus shape through surface treatment. Optical spectra are compared to a 3D beam propagation method simulations and good agreement is found. Tunability, low insertion loss and strength of the resonance are well reproduced. The device performance displays a resonance depth of -28 dB and insertion loss maintained at -4 dB.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1016/J.BIOMATERIALS.2007.10.017

Abstract: Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Fluorescent and Magnetic Mesoporous Hybrid Material: A Chemical and Biological Nanosensor for Hg²⁺ Ions

Publisher: Springer Science and Business Media LLC

Date: 25-02-2016

DOI: 10.1038/SREP21820

Abstract: We introduce “sense, track and separate” approach for the removal of Hg 2+ ion from aqueous media using highly ordered and magnetic mesoporous ferrosilicate nanocages functionalised with rhodamine fluorophore derivative. These functionalised materials offer both fluorescent and magnetic properties in a single system which help not only to selectively sense the Hg 2+ ions with a high precision but also adsorb and separate a significant amount of Hg 2+ ion in aqueous media. We demonstrate that the magnetic affinity of these materials, generated from the ultrafine γ-Fe 2 O 3 nanoparticles present inside the nanochannels of the support, can efficiently be used as a fluorescent tag to sense the Hg 2+ ions present in NIH3T3 fibroblasts live cells and to track the movement of the cells by external magnetic field monitored using confocal fluorescence microscopy. This simple approach of introducing multiple functions in the magnetic mesoporous materials raise the prospect of creating new advanced functional materials by fusing organic, inorganic and biomolecules to create advanced hybrid nanoporous materials which have a potential use not only for sensing and the separation of toxic metal ions but also for cell tracking in bio-separation and the drug delivery.

Differential mesengenic potential and expression of stem cell‐fate modulators in mesenchymal stromal cells from human‐term placenta and bone marrow

Publisher: Wiley

Date: 17-05-2012

DOI: 10.1002/JCP.24014

Abstract: Placenta has attracted increasing attention over the past decade as a stem cell source for regenerative medicine. In particular, the amniochorionic membrane has been shown to harbor populations of mesenchymal stromal cells (MSCs). In this study, we have characterized ex vivo expanded MSCs from the human amniotic (hAMSCs) and chorionic (hCMSCs) membranes of human full-term placentas and adult bone marrow (hBMSCs). Our results show that hAMSCs, hCMSCs, and hBMSCs express typical mesenchymal (CD73, CD90, CD105, CD44, CD146, CD166) and pluripotent (Oct-4, Sox2, Nanog, Lin28, and Klf4) markers but not hematopoietic markers (CD45, CD34). Ex vivo expanded hAMSCs were found to be of fetal origin, while hCMSCs cultures contained only maternal cells. Cell proliferation was significantly higher in hCMSCs, compared to hAMSCs and hBMSCs. Integrin profiling revealed marked differences in the expression of α subunits between the three cell sources. Cadherin receptors were consistently expressed on a subset of progenitors (ranging from 1% to 60%), while N-CAM (CD56) was only expressed in hAMSCs and hCMSCs but not in hBMSCs. When induced to differentiate, hAMSCs and hCMSCs displayed strong chondrogenic and osteogenic differentiation potential but very limited capacity for adipogenic conversion. In contrast, hBMSCs showed strong differentiation potential along the three lineages. These results illustrate how MSCs from different ontological sources display differential expression of cell-fate mediators and mesodermal differentiation capacity.

Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules

Publisher: Springer Science and Business Media LLC

Date: 30-11-2017

DOI: 10.1038/NPROT.2017.116

Abstract: Our understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulse-chase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in in idual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippoc al presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes (iii) the dissection, culturing and transfection of hippoc al neurons in microfluidic devices and (iv) guidance on how to perform the pulse-chase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take ∼1 week.

A defined medium and substrate for expansion of human mesenchymal stromal cell progenitors that enriches for osteo- and chondrogenic precursors.

Publisher: Mary Ann Liebert Inc

Date: 2010

DOI: 10.1089/SCD.2009.0497

Abstract: Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo, but after isolation can be expanded considerably, generating clinically useful numbers of cells. In this study, we demonstrate the use of a defined embryonic stem cell expansion medium, mTeSR (Stem Cell Technologies), for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates, demonstrate tri-lineage differentiation potential, and show similar surface marker profiles (CD29(+), CD44(+), CD49a(+), CD73(+), CD90(+), CD105(+), CD146(+), CD166(+), CD34(-), and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However, expression of early differentiation transcription factors runt-related transcription factor 2, sex-determining region Y box 9, and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated, whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS, adipogenic differentiation was significantly decreased in mTeSR-expanded cells, both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further, this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs, such as bone repair and cartilage generation.

Evaluation of saliva collection devices for the analysis of proteins

Publisher: Elsevier BV

Date: 07-2012

DOI: 10.1016/J.CCA.2012.02.020

Abstract: Human saliva mirrors the body's health and can be collected non-invasively, does not require specialized skills and is suitable for large population based screening programs. The aims were twofold: to evaluate the suitability of commercially available saliva collection devices for quantifying proteins present in saliva and to provide levels for C-reactive protein (CRP), myoglobin, and immunoglobin E (IgE) in saliva of healthy in iduals as a baseline for future studies. Saliva was collected from healthy volunteers (n=17, ages 18-33years). The following collection methods were evaluated: drool Salimetrics® Oral Swab (SOS) Salivette® Cotton and Synthetic (Sarstedt) and Greiner Bio-One Saliva Collection System (GBO SCS®). We used AlphaLISA® assays to measure CRP, IgE and myoglobin levels in human saliva. Significant (p<0.05) differences in the salivary flow rates were observed based on the method of collection, i.e. salivary flow rates were significantly lower (p<0.05) in unstimulated saliva (i.e. drool and SOS), when compared with mechanically stimulated methods (p<0.05) (Salivette® Cotton and Synthetic) and acid stimulated method (p<0.05) (SCS®). Saliva collected using SOS yielded significantly (p<0.05) lower concentrations of myoglobin and CRP, whilst, saliva collected using the Salivette® Cotton and Synthetic swab yielded significantly (p<0.05) lower myoglobin and IgE concentrations respectively. The results demonstrated significantly relevant differences in analyte levels based on the collection method. Significant differences in the salivary flow rates were also observed depending on the saliva collection method. The data provide preliminary baseline values for salivary CRP, myoglobin, and IgE levels in healthy participants and based on the collection method.

miRNAs in head and neck cancer revisited

Publisher: Springer Science and Business Media LLC

Date: 22-01-2013

DOI: 10.1007/S13402-012-0122-4

Abstract: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cause of cancer mortality in the world and the 5th most commonly occurring cancer. Tobacco smoking, alcohol consumption and human papilloma virus (HPV) infections have been associated with the occurrence of HNSCC. Despite advances that have been made in HNSCC treatment, smoking-associated HNSCC patients still exhibit a poor 5 year survival rate (30-50 %) and a concomitant poor quality of life. The major clinical challenge to date lies in the early detection of dysplastic lesions,which can progress to malignancy. In addition, there are currently no tools available to monitor HNSCC patients for early stages of local recurrences or distant metastases. In the recent past, micro-RNAs (miRNA) have been assessed for their role in cancer initiation and progression, including HNSCC. It is now well-established that deregulation of these single stranded, small non-coding, 19-25 nt RNAs can e.g. enhance the expression of oncogenes or subdue the expression of tumor suppressor genes. The aims of this review are three-fold: first to retrieve from the literature miRNAs that have specifically been associated with HNSCC, second to group these miRNAs into those regulating tumor initiation, progression and metastasis, and third to discern miRNAs related to smoking-associated HNSCC versus HPV-associated HNSCC development. This review gives an overview on the miRNAs regulating the development of head and neck cancers. The ultimate establishment of miRNA expression profiles that are HNSCC specific, and miRNAs that orchestrate altered gene and protein expression levels in HNSCC, could pave the way for a better understanding of the mechanism underlying its pathogenesis and the development of novel, targeted therapies.

A microdevice for the creation of patent, three-dimensional endothelial cell-based microcirculatory networks

Publisher: AIP Publishing

Date: 16-08-2011

DOI: 10.1063/1.3609264

Abstract: Microvascular network formation is a significant and challenging goal in the engineering of large three-dimensional artificial tissue structures. We show here the development of a fully patent, 3D endothelial cell (microvascular) microfluidic network that has a single inlet and outlet, created in only 28 h in a microdevice involving fluid flow equivalent to natural vasculature. Our microdevice features a tailored “multi-rung ladder” network, a stylized mimic of an arterial-to-venous pedicle, designed to also allow for systematic and reproducible cell seeding. Immunofluorescence staining revealed a highly contiguous endothelial monolayer (human umbilical vein endothelial cells) throughout the whole network after 24 h of continuous perfusion. This network persisted for up to 72 h of culture, providing a useful template from which the effects of surface chemistry, fluid flow, and environmental conditions on the development of artificial vascular networks ex vivo may be rapidly and robustly evaluated.

Effect of Poly(vinyl alcohol) Macromer Chemistry and Chain Interactions on Hydrogel Mechanical Properties

Publisher: American Chemical Society (ACS)

Date: 21-04-2007

DOI: 10.1021/CM0626381

Electrospinning and crosslinking of low-molecular-weight poly(trimethylene carbonate-co-l-lactide) as an elastomeric scaffold for vascular engineering

Publisher: Elsevier BV

Date: 06-2013

DOI: 10.1016/J.ACTBIO.2013.02.009

Abstract: The growth of suitable tissue to replace natural blood vessels requires a degradable scaffold material that is processable into porous structures with appropriate mechanical and cell growth properties. This study investigates the fabrication of degradable, crosslinkable prepolymers of l-lactide-co-trimethylene carbonate into porous scaffolds by electrospinning. After crosslinking by γ-radiation, dimensionally stable scaffolds were obtained with up to 56% trimethylene carbonate incorporation. The fibrous mats showed Young's moduli closely matching human arteries (0.4-0.8MPa). Repeated cyclic extension yielded negligible change in mechanical properties, demonstrating the potential for use under dynamic physiological conditions. The scaffolds remained elastic and resilient at 30% strain after 84days of degradation in phosphate buffer, while the modulus and ultimate stress and strain progressively decreased. The electrospun mats are mechanically superior to solid films of the same materials. In vitro, human mesenchymal stem cells adhered to and readily proliferated on the three-dimensional fiber network, demonstrating that these polymers may find use in growing artificial blood vessels in vivo.

Targeted, Stimuli-Responsive Delivery of Plasmid DNA and miRNAs Using a Facile Self-Assembled Supramolecular Nanoparticle System

Publisher: American Chemical Society (ACS)

Date: 18-01-2018

DOI: 10.1021/ACS.BIOMAC.7B01462

Circulating Fragments of N-Terminal Pro–B-Type Natriuretic Peptides in Plasma of Heart Failure Patients

Publisher: Oxford University Press (OUP)

Date: 10-2013

DOI: 10.1373/CLINCHEM.2012.200204

Abstract: The use of nonstandardized N-terminal pro–B-type natriuretic peptide (NT-proBNP) assays can contribute to the misdiagnosis of heart failure (HF). Moreover, there is yet to be established a common consensus regarding the circulating forms of NT-proBNP being used in current assays. We aimed to characterize and quantify the various forms of NT-proBNP in the circulation of HF patients. Plasma s les were collected from HF patients (n = 20) at rest and stored at −80 °C. NT-proBNP was enriched from HF patient plasma by use of immunoprecipitation followed by mass spectrometric analysis. Customized homogeneous sandwich AlphaLISA® immunoassays were developed and validated to quantify 6 fragments of NT-proBNP. Mass spectrometry identified the presence of several N- and C-terminally processed forms of circulating NT-proBNP, with physiological proteolysis between Pro2-Leu3, Leu3-Gly4, Pro6-Gly7, and Pro75-Arg76. Consistent with this result, AlphaLISA immunoassays demonstrated that antibodies targeting the extreme N or C termini measured a low apparent concentration of circulating NT-proBNP. The apparent circulating NT-proBNP concentration was increased with antibodies targeting nonglycosylated and nonterminal epitopes (P & 0.05). In plasma collected from HF patients, immunoreactive NT-proBNP was present as multiple N- and C-terminally truncated fragments of the full length NT-proBNP molecule. Immunodetection of NT-proBNP was significantly improved with the use of antibodies that did not target these terminal regions. These findings support the development of a next generation NT-proBNP assay targeting nonterminal epitopes as well as avoiding the central glycosylated region of this molecule.

Generation of a human embryonic stem cell line stably expressing high levels of the fluorescent protein mCherry

Publisher: Baishideng Publishing Group Inc.

Date: 2012

DOI: 10.4252/WJSC.V4.I7.71

Electrochemical quartz crystal microbalance to monitor biofilm growth and properties during bioelectrochemical system inoculation and load conditions

Publisher: The Electrochemical Society

Date: 08-10-2010

DOI: 10.1149/1.3492222

Abstract: BioElectrochemical Systems are an emerging technology of interest. Central to the successful operation of these systems is the development of a biofilm on an electrode surface that facilitates the interaction of microorganisms capable of extra-cellular electron transfer. Porous graphite is typically chosen as the electrode material. However, other materials could contribute to a significant improvement in reactor performance. In order to evaluate these novel materials, a platform methodology is required. In this study, E-QCM-D was evaluated using a gold electrode, a mixed biofilm population and a challenging real influent (fermented solubilised sludge). The results gave real-time feedback on both the acoustic and electrochemical impedance of the working electrode, hence providing information about biofilm thickness, viscoelasticity, kinetics and mass transport. The use of E-QCM-D is novel for this field and has the potential to provide a wealth of new data to help optimise the performance of biofilms in these systems.

Functionalised polycaprolactone films and 3D scaffolds via gamma irradiation-induced grafting

Publisher: Royal Society of Chemistry (RSC)

Date: 2013

DOI: 10.1039/C3TB20267D

Diagnostic potential of saliva: current state and future applications.

Publisher: Oxford University Press (OUP)

Date: 05-2011

DOI: 10.1373/CLINCHEM.2010.153767

Abstract: Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow. As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by in iduals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining s les from children or handicapped or anxious patients, in whom blood s ling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings. Saliva has the potential to become a first-line diagnostic s le of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance.

Determinants of Zika virus host tropism uncovered by deep mutational scanning

Publisher: Springer Science and Business Media LLC

Date: 18-03-2019

DOI: 10.1038/S41564-019-0399-4

Primitive Cardiac Cells from Human Embryonic Stem Cells

Publisher: Mary Ann Liebert Inc

Date: 10-06-2012

DOI: 10.1089/SCD.2011.0254

Abstract: Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.

Focal adhesion dynamics are altered in schizophrenia.

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.BIOPSYCH.2013.01.020

Abstract: Evidence from genetic association studies implicate genes involved in neural migration associated with schizophrenia risk. Neural stem rogenitor cell cultures (neurosphere-derived cells) from olfactory mucosa of schizophrenia patients have significantly dysregulated expression of genes in focal adhesion kinase (FAK) signaling, a key pathway regulating cell adhesion and migration. The aim of this study was to investigate whether olfactory neurosphere-derived cells from schizophrenia patients have altered cell adhesion, cell motility, and focal adhesion dynamics. Olfactory neurosphere-derived cells from nine male schizophrenia patients and nine male healthy control subjects were used. Cells were assayed for cell adhesion and cell motility and analyzed for integrins and FAK proteins. Focal adhesions were counted and measured in fixed cells, and time-lapse imaging was used to assess cell motility and focal adhesion dynamics. Patient-derived cells were less adhesive and more motile than cells derived from healthy control subjects, and their motility was reduced to control cell levels by integrin-blocking antibodies and by inhibition of FAK. Vinculin-stained focal adhesion complexes were significantly smaller and fewer in patient cells. Time-lapse imaging of cells expressing FAK tagged with green fluorescent protein revealed that the disassembly of focal adhesions was significantly faster in patient cells. The evidence for altered motility and focal adhesion dynamics in patient-derived cells is consistent with dysregulated gene expression in the FAK signaling pathway in these cells. Alterations in cell adhesion dynamics and cell motility could bias the trajectory of brain development in schizophrenia.

The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.BIOMATERIALS.2005.12.015

Abstract: The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies-thermally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. Over 6 weeks, in vitro degradation produced changes in SCPL scaffold dimension, mass, internal architecture and mechanical properties. TIPS scaffolds produced far less changes in these parameters providing significant advantages over SCPL. In vivo results were based on a microsurgically created arteriovenous (AV) loop sandwiched between two TIPS scaffolds placed in a polycarbonate chamber under rat groin skin. Histologically, a predominant foreign body giant cell response and reduced vascularity was evident in tissue ingrowth between 2 and 8 weeks in TIPS scaffolds. Tissue death occurred at 8 weeks in the smallest pores. Morphometric comparison of TIPS and SCPL scaffolds indicated slightly better tissue ingrowth but greater loss of scaffold structure in SCPL scaffolds. Although advantageous in vitro, large surface area:volume ratios and varying pore sizes in PLGA TIPS scaffolds mean that effective in vivo (AV loop) utilization will only be achieved if the foreign body response can be significantly reduced so as to allow successful vascularisation, and hence sustained tissue growth, in pores less than 300 microm.

High-Yield RNA-Extraction Method for Saliva

Publisher: Oxford University Press (OUP)

Date: 07-2013

DOI: 10.1373/CLINCHEM.2012.197863

Abstract: The use of salivary diagnostics is increasing because of its noninvasiveness, ease of s ling, and the relatively low risk of contracting infectious organisms. Saliva has been used as a biological fluid to identify and validate RNA targets in head and neck cancer patients. The goal of this study was to develop a robust, easy, and cost-effective method for isolating high yields of total RNA from saliva for downstream expression studies. Oral whole saliva (200 μL) was collected from healthy controls (n = 6) and from patients with head and neck cancer (n = 8). The method developed in-house used QIAzol lysis reagent (Qiagen) to extract RNA from saliva (both cell-free supernatants and cell pellets), followed by isopropyl alcohol precipitation, cDNA synthesis, and real-time PCR analyses for the genes encoding β-actin (“housekeeping” gene) and histatin (a salivary gland–specific gene). The in-house QIAzol lysis reagent produced a high yield of total RNA (0.89–7.1 μg) from saliva (cell-free saliva and cell pellet) after DNase treatment. The ratio of the absorbance measured at 260 nm to that at 280 nm ranged from 1.6 to 1.9. The commercial kit produced a 10-fold lower RNA yield. Using our method with the QIAzol lysis reagent, we were also able to isolate RNA from archived saliva s les that had been stored without RNase inhibitors at −80 °C for & years. Our in-house QIAzol method is robust, is simple, provides RNA at high yields, and can be implemented to allow saliva transcriptomic studies to be translated into a clinical setting.

An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.BIOMATERIALS.2013.08.072

Abstract: Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.

Induction of Human iPSC-Derived Cardiomyocyte Proliferation Revealed by Combinatorial Screening in High Density Microbioreactor Arrays

Publisher: Springer Science and Business Media LLC

Date: 21-04-2016

DOI: 10.1038/SREP24637

Abstract: Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways, however, it remains unclear as to what extent these pathways can be exploited, either in idually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro , but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, we developed a high-density microbioreactor array (HDMA) – a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt, Hedgehog, IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67, and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration.

Tailoring biomaterial scaffolds for osteochondral repair

Publisher: Elsevier BV

Date: 05-2017

DOI: 10.1016/J.IJPHARM.2016.10.035

Transplantation of 3D scaffolds seeded with human embryonic stem cells: Biological features of surrogate tissue and teratoma-forming potential

Publisher: Future Medicine Ltd

Date: 05-2007

DOI: 10.2217/17460751.2.3.289

Abstract: Aim: To generate complex surrogate tissue by transplanting 3D scaffolds seeded with human embryonic stem cells (hESCs) between the liver lobules of severe combined immunodeficient (SCID) mice and to assess the teratoma-forming potential. Materials & methods: 3D poly-(lactic-co-glycolic acid) (PLGA) scaffolds coated with laminin were seeded with hESCs and then transplanted between the liver lobules of SCID mice. After a period of in vivo differentiation, the scaffolds were retrieved and analyzed using reverse transcription polymerase chain reaction, immunofluorescent staining and scanning electron microscopy. Results: A proportion of the hESCs within the scaffolds differentiated into cells that produced proteins characteristic of specific tissues, including endoderm and pancreatic markers glucogon-like peptide-1 receptor, islet amyloid polypeptide and Insulin. Markers of hepatic and neuronal lineages were also investigated. Major matrix proteins abundant in multiple tissue types, including collagen I, laminin and collagen IV, were found to be profuse within the scaffold pores. Transplantation of the seeded scaffolds between liver lobules also resulted in extensive vascularization both from host blood vessel incursion and the differentiation of hESCs into endothelial progenitor cells. An investigation of teratoma-forming potential demonstrated that transplantation of 3D scaffolds seeded with hESCs will, under certain conditions, lead to the growth of teratomas. Discussion: Transplantation of 3D scaffolds seeded with hESCs between liver lobules resulted in the development of surrogate tissue containing cells that produced proteins representing the pancreatic, hepatic and neuronal lineages, the assembly of an extracellular matrix structure and the formation of a vasculature. hESCs seeded within 3D scaffolds and transplanted into SCID mice were capable of forming teratomas. However, the formation and progression of teratoma growth is shown to be dependant on both the site of transplantation and the treatment of cells prior to transplantation.

Single-cell transcriptome profiling reveals multicellular ecosystem of nucleus pulposus during degeneration progression

Publisher: Cold Spring Harbor Laboratory

Date: 26-05-2021

DOI: 10.1101/2021.05.25.445620

Abstract: Degeneration of the nucleus pulposus (NP) is a major contributor to intervertebral disc degeneration (IVDD) and low back pain. However, the underlying molecular complexity and cellular heterogeneity remain poorly understood. Here, we first reported a comprehensive single-cell resolution transcriptional landscape of human NP. Six novel human nucleus pulposus cell (NPCs) populations were identified by distinct molecular signatures. The potential functional differences among NPC subpopulations were analyzed at the single-cell level. Predictive genes, transcriptional factors, and signal pathways with respect to degeneration grades were analyzed. We reported that fibroNPCs, one of our identified subpopulations, might be a population for NP regeneration. CD90+NPCs were observed to be progenitor cells in degenerative NP tissues. NP-infiltrating immune cells comprise a previously unrecognized ersity of cell types, including granulocytic myeloid-derived suppressor cells (G-MDSCs). We uncovered CD11b, OLR1, and CD24 as surface markers of NP-derived G-MDSCs. The G-MDSCs were also found to be enriched in mildly degenerated (grade I and II) NP tissues compared to severely degenerated (grade III and IV) NP tissues. Their immunosuppressive function and protective effects for NPCs were revealed. Collectively, this study revealed the NPC type complexity and phenotypic characteristics in NP, providing new insights and clues for IVDD treatment.

Multivariate patterning of human pluripotent cells under perfusion reveals critical roles of induced paracrine factors in kidney organoid development

Publisher: American Association for the Advancement of Science (AAAS)

Date: 10-01-2020

DOI: 10.1126/SCIADV.AAW2746

Abstract: Multivariate patterning of perfused pluripotent cells reveals critical roles of induced paracrine factors in kidney organoids.

Tissue-Engineered Breast Reconstruction

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 12-2011

DOI: 10.1097/PRS.0B013E318230C5B2

Low biofouling chitosan-hyaluronic acid multilayers with ultra-low friction coefficients

Publisher: American Chemical Society (ACS)

Date: 07-04-2009

DOI: 10.1021/BM801079A

Abstract: Resistance to biofouling is an advantageous material property in a variety of biomedical and biofluid processing applications. Protein-resisting surface coatings must also be resistant to wear and degradation and in certain applications good aqueous lubricating properties are required. We show that cross-linked polyelectrolyte multilayers, consisting of chitosan and hyaluronan on polydimethylsiloxane (PDMS) surfaces, form a highly lubricating film that is resistant to wear and protein adsorption. The multilayer film shows much stronger resistance to protein adsorption from human whole saliva than both hydrophobic and hydrophilic PDMS surfaces the latter two showed identical adsorbed salivary film thicknesses. The boundary friction coefficient under aqueous conditions was extremely low (mu approximately 0.01) between multilayer-coated PDMS substrates and the film is robust against dry rubbing and many hours of tribological experiments in a range of aqueous lubricants. The origins of the assembly's low friction coefficients and robustness are discussed. In addition, we found that the addition of negative phosphate ions to water lowers the boundary lubricating properties of negatively charged hydrophilic PDMS surfaces by 1 order of magnitude to mu approximately 0.01. We consider this to arise from the large hydration sheaths and resulting "ball-bearing" properties of the hydrated phosphate ions, which form a lubricating barrier against asperity contact. These findings offer new insights toward biolubrication processes and suggest that chitosan-hyaluronan polyelectrolyte multilayer films have the potential to be used in (bio-) applications requiring low friction as well as resistance to biofouling and wear.

Optimization of flowrate for expansion of human embryonic stem cells in perfusion microbioreactors

Publisher: Wiley

Date: 22-07-2011

DOI: 10.1002/BIT.23260

Abstract: Microfluidic systems create significant opportunities to establish highly controlled microenvironmental conditions for screening pluripotent stem cell fate. However, since cell fate is crucially dependent on this microenvironment, it remains unclear as to whether continual perfusion of culture medium supports pluripotent stem cell maintenance in feeder-free, chemically defined conditions, and further, whether optimum perfusion conditions exist for subsequent use of human embryonic stem cell (hESCs) in other microfludic systems. To investigate this, we designed microbioreactors based on resistive flow to screen hESCs under a linear range of flowrates. We report that at low rates (conditions where glucose transport is convection-limited with Péclet number 95% of cells over 7 days. For MEL1 hESCs the optimum flowrate also coincided with the time-averaged medium exchange rate in static cultures, which may therefore provide a good first estimate of appropriate perfusion rates. Overall, we demonstrate hESCs can be maintained in microbioreactors under continual flow for up to 7 days, a critical outcome for the future development of microbioreactor-based screening systems and assays for hESC culture.

Tailored integrin-extracellular matrix interactions to direct human mesenchymal stem cell differentiation.

Publisher: Mary Ann Liebert Inc

Date: 09-2012

DOI: 10.1089/SCD.2011.0615

Modelling ischemia-reperfusion injury (IRI) in vitro using metabolically matured induced pluripotent stem cell-derived cardiomyocytes

Publisher: AIP Publishing

Date: 20-03-2018

DOI: 10.1063/1.5000746

Abstract: Coronary intervention following ST-segment elevation myocardial infarction (STEMI) is the treatment of choice for reducing cardiomyocyte death but paradoxically leads to reperfusion injury. Pharmacological post-conditioning is an attractive approach to minimize Ischemia-Reperfusion Injury (IRI), but candidate drugs identified in IRI animal models have performed poorly in human clinical trials, highlighting the need for a human cell-based model of IRI. In this work, we show that when we imposed sequential hypoxia and reoxygenation episodes [mimicking the ischemia (I) and reperfusion (R) events] to immature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), they display significant hypoxia resistance and minimal cell death (∼5%). Metabolic maturation of hPSC-CMs for 8 days substantially increased their sensitivity to changes in oxygen concentration and led to up to ∼30% cell death post-hypoxia and reoxygenation. To mimic the known transient changes in the interstitial tissue microenvironment during an IRI event in vivo, we tested a new in vitro IRI model protocol that required glucose availability and lowering of media pH during the ischemic episode, resulting in a significant increase in cell death in vitro (∼60%). Finally, we confirm that in this new physiologically matched IRI in vitro model, pharmacological post-conditioning reduces reperfusion-induced hPSC-CM cell death by 50%. Our results indicate that in recapitulating key aspects of an in vivo IRI event, our in vitro model can serve as a useful method for the study of IRI and the validation and screening of human specific pharmacological post-conditioning drug candidates.

The effect of time-dependent deformation of viscoelastic hydrogels on myogenic induction and Rac1 activity in mesenchymal stem cells

Publisher: Elsevier BV

Date: 02-2014

DOI: 10.1016/J.BIOMATERIALS.2013.11.023

Abstract: Cell behaviours within tissues are influenced by a broad array of physical and biochemical microenvironmental factors. Whilst 'stiffness' is a recognised physical property of substrates and tissue microenvironments that influences many cellular behaviours, tissues and their extracellular matrices are not purely rigid but 'viscoelastic' materials, composed of both rigid-like (elastic) and dissipative (viscous) elements. This viscoelasticity results in materials displaying increased deformation with time under the imposition of a defined force or stress, a phenomenon referred to as time-dependent deformation or 'creep'. Previously, we compared the behaviour of human mesenchymal stem cells (hMSCs) on hydrogels tailored to have a constant stiffness, but to display varying levels of creep in response to an applied force. Using polyacrylamide as a model material, we showed that on high-creep hydrogels (HCHs), hMSCs displayed increased proliferation, spread area and differentiation towards multiple lineages, compared to their purely stiff analogue, with a particular propensity for differentiation towards a smooth muscle cell (SMC) lineage. In this present study, we investigate the mechanisms behind this phenomenon and show that hMSCs adhered to HCHs have increased expression of SMC induction factors, including soluble factors, ECM proteins and the cell-cell adhesion molecule, N-Cadherin. Further, we identify a key role for Rac1 signalling in mediating this increased N-Cadherin expression. Using a real-time Rac1-FRET biosensor, we confirm increased Rac1 activation on HCHs, an observation that is further supported functionally by observed increases in motility and lamellipodial protrusion rates of hMSCs. Increased Rac1 activity in hMSCs on HCHs provides underlying mechanisms for enhanced commitment towards a SMC lineage and the compensatory increase in spread area (isotonic tension) after a creep-induced loss of cytoskeletal tension on viscoelastic substrates, in contrast to previous studies that have consistently demonstrated up-regulation of RhoA activity with increasing substrate stiffness. Tuning substrate viscoelasticity to introduce varying levels of creep thus equips the biomaterial scientist or engineer with a new tool with which to tune and direct stem cell outcomes.

Microfluidic interferometer

Publisher: AIP Publishing

Date: 03-01-2005

DOI: 10.1063/1.1849415

Abstract: We present a class of compact, fluid-based, interferometric, tunable optical components: the single-beam microfluidic Mach–Zender interferometer. Phase delay is achieved through light propagation across a fluid–air interface (meniscus). The effect of meniscus curvature on the device transmission is considered using the three-dimensional beam propagation method and shown to be an important device parameter. We engineer the meniscus curvature using monomer surface chemistry, rendering it flat, and find that the experimental response corresponds well with simulation. The device has a resonance at 1.3μm with a 25dB extinction ratio the latter can be adjusted by shifting the meniscus position.

Modulation of collagen II fiber formation in 3-D porous scaffold environments

Publisher: Elsevier BV

Date: 07-2011

DOI: 10.1016/J.ACTBIO.2011.03.022

Abstract: Collagen II, a major extracellular matrix component in cartilaginous tissues, undergoes fibrillogenesis under physiological conditions. The present study explored collagen II fiber formation in solution and in two- (coverslip) and three-dimensional (scaffold) environments under different incubation conditions. These conditions include variations in adsorption buffers, the presence of 1-ethyl-3-(3-dimenthylaminopropyl) carbodiimide/N-hydroxysuccinimide crosslinker and the nature of the material surfaces. We extend our observations of collagen II fiber formation in two dimensions to develop an approach for the formation of a fibrillar collagen II network throughout surface-modified polylactide-co-glycolide porous scaffolds. Morphologically, the collagen II network is similar to that present in native articular cartilage. Biological validation of the resultant optimized functional scaffold, using rat bone marrow-derived mesenchymal stem cells, shows appreciable cell infiltration throughout the scaffold with enhanced cell spreading at 24h post-seeding. This economic and versatile approach is thus believed to have significant potential in cartilage tissue engineering applications.

Driving Osteocytogenesis from Mesenchymal Stem Cells in Osteon-like Biomimetic Nanofibrous Scaffolds

Publisher: Cold Spring Harbor Laboratory

Date: 02-07-2022

DOI: 10.1101/2022.06.28.497866

Abstract: The repair of critical-sized bone defects, resulting from tumor resection, skeletal trauma or infection, remains a significant clinical problem. A potential solution is a tissue-engineered approach that utilises the combination of human mesenchymal stem cells (hMSCs) with synthetic biomaterial scaffolds, mimicking many of the biochemical and biophysical cues present within the native bone. Unfortunately, osteocyte cells, the orchestrators of bone maturation and homeostasis, are rarely produced within such MSC-seeded scaffolds, limiting the formation of true mature cortical bone from these synthetic implants. In this contribution, a bone morphogenic protein-6 (BMP6)-presenting osteon-like scaffolds based on electrospun poly(lactic-co-glycolic acid) (PLGA) fibrous scaffolds and poly(ethylene glycol) (PEG) based-hydrogels is reported. BMP6 peptide is shown to drive higher levels of SMAD signalling than the full-length protein counterpart. Osteon-mimetic scaffolds promoted the formation of osteocyte-like cells displaying multi-dendritic morphology and osteocyte-specific marker, E11/gp38 (E11), along with significant production of dentin matrix protein 1 (DMP1), confirming maturation of the ososteocyte-like cells. These results demonstrate that osteon-like scaffolds presenting chemo-topographical cues can drive the formation of mature osteocyte-like cells from hMSCs, without the need for osteogenic factor media supplements, providing a novel ex vivo production platform for osteocyte-like cells from human MSCs in cortical bone mimics.

Arrayed cellular environments for stem cells and regenerative medicine

Publisher: Wiley

Date: 14-08-2012

DOI: 10.1002/BIOT.201200149

Abstract: The behavior and composition of both multipotent and pluripotent stem cell populations are exquisitely controlled by a complex, spatiotemporally variable interplay of physico-chemical, extracellular matrix, cell-cell interaction, and soluble factor cues that collectively define the stem cell niche. The push for stem cell-based regenerative medicine models and therapies has fuelled demands for increasingly accurate cellular environmental control and enhanced experimental throughput, driving an evolution of cell culture platforms away from conventional culture formats toward integrated systems. Arrayed cellular environments typically provide a set of discrete experimental elements with variation of one or several classes of stimuli across elements of the array. These are based on high-content/high-throughput detection, small s le volumes, and multiplexing of environments to increase experimental parameter space, and can be used to address a range of biological processes at the cell population, single-cell, or subcellular level. Arrayed cellular environments have the capability to provide an unprecedented understanding of the molecular and cellular events that underlie expansion and specification of stem cell and therapeutic cell populations, and thus generate successful regenerative medicine outcomes. This review focuses on recent key developments of arrayed cellular environments and their contribution and potential in stem cells and regenerative medicine.

Deformation of a viscoelastic droplet passing through a microfluidic contraction

Publisher: Elsevier BV

Date: 11-2008

DOI: 10.1016/J.JNNFM.2008.05.002

Profiling of immunoglobulins in resting and mechanically stimulated saliva

Publisher: Future Science Ltd

Date: 03-2014

DOI: 10.4155/BIO.14.21

Abstract: Background: Dysregulation of salivary immunoglobulins has been implicated in illnesses ranging from periodontal disease to HIV aids and malignant cancers. Despite these advances there is a lack of agreement among studies with regard to the salivary immunoglobulin levels in healthy controls. Methodology: Resting and mechanically stimulated saliva s les and matching serum s les were collected from healthy in iduals (n = 33 40–55 years of age gender: 23 female, 10 male). A matrix-matched AlphaLISA ® assay was developed to determine the concentrations of IgG1 and IgG4 in serum and saliva s les. Conclusion: Clear relationships were observed in the flow rate and concentration of each immunoglobulin in the two types of saliva. This study affirms the need to establish and standardize collection methods before salivary IgGs are used for diagnostic purposes.

Characterisation of amine functionalised poly(3-hydroxybuturate-co-3-hydroxyvalerate) surfaces

Publisher: Elsevier BV

Date: 07-2011

DOI: 10.1016/J.POLYMER.2011.05.048

Microfluidic Elucidation of the Effects of Interfacial Rheology on Droplet Deformation

Publisher: American Chemical Society (ACS)

Date: 03-10-2011

DOI: 10.1021/IE200631M

Effects of bound versus soluble pentosan polysulphate in PEG/HA-based hydrogels tailored for intervertebral disc regeneration

Publisher: Elsevier BV

Date: 2014

DOI: 10.1016/J.BIOMATERIALS.2013.10.056

Abstract: Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFβ). In this present study, we aimed to assess whether a 'bound' PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in increased matrix formation when compared to the addition of soluble PPS to the hydrogel, or the hydrogel alone. We believe that this new generation injectable, degradable hydrogel, incorporating now a covalently bound-PPS, when combined with MPCs, has the potential to assist cartilage regeneration in a multitude of therapeutic targets, including for intervertebral disc (IVD) degeneration.

Microfluidic devices for developing tissue scaffolds

Publisher: Elsevier

Date: 2013

DOI: 10.1533/9780857097040.3.363

Strong through to weak 'sheared' gels

Publisher: Elsevier BV

Date: 12-2004

DOI: 10.1016/J.JNNFM.2004.07.013

The impact of saliva collection and processing methods on CRP, IgE, and Myoglobin immunoassays

Publisher: Wiley

Date: 05-09-2012

DOI: 10.1186/2001-1326-1-19

High-Throughput Routes to Biomaterials Discovery

Publisher: American Chemical Society (ACS)

Date: 02-07-2021

DOI: 10.1021/ACS.CHEMREV.0C01026

Abstract: Many existing clinical treatments are limited in their ability to completely restore decreased or lost tissue and organ function, an unenviable situation only further exacerbated by a globally aging population. As a result, the demand for new medical interventions has increased substantially over the past 20 years, with the burgeoning fields of gene therapy, tissue engineering, and regenerative medicine showing promise to offer solutions for full repair or replacement of damaged or aging tissues. Success in these fields, however, inherently relies on biomaterials that are engendered with the ability to provide the necessary biological cues mimicking native extracellular matrixes that support cell fate. Accelerating the development of such "directive" biomaterials requires a shift in current design practices toward those that enable rapid synthesis and characterization of polymeric materials and the coupling of these processes with techniques that enable similarly rapid quantification and optimization of the interactions between these new material systems and target cells and tissues. This manuscript reviews recent advances in combinatorial and high-throughput (HT) technologies applied to polymeric biomaterial synthesis, fabrication, and chemical, physical, and biological screening with targeted end-point applications in the fields of gene therapy, tissue engineering, and regenerative medicine. Limitations of, and future opportunities for, the further application of these research tools and methodologies are also discussed.

Surface-bound stem cell factor and the promotion of hematopoietic cell expansion

Publisher: Elsevier BV

Date: 09-2009

DOI: 10.1016/J.BIOMATERIALS.2009.04.043

Abstract: In vivo, stem cell factor (SCF) exists in both a bound and soluble isoform. It is believed that the bound form is more potent and fundamentally required for the maintenance of hematopoietic stem cells (HSCs). This theory is supported by the observation that steel-Dickie mice lacking the bound isoform of SCF are unable to maintain hematopoiesis and by the fact that bound SCF displayed on the surface of transgenic cells is better able to maintain c-kit activation than soluble SCF. Further work has shown that recombinant SCF molecules, which include a surface-binding domain, are more potent than their soluble equivalent. It is generally assumed that such an elegant approach is necessary to provide the correct molecular orientation and avoid the pitfalls of random cross-linking or the denaturation associated with the adsorption of proteins to surfaces. However, in this work we demonstrate that SCF physisorbed to tissue culture plastic (TCP) is not only bioactive, but more potent than the soluble equivalent. By contrast, cross-linking of SCF via free amines is shown to compromise its bioactivity. These observations demonstrate that simple surface modification solutions cannot be discounted and with the advent of low-cost pharmaceutical grade proteins, they should not be.

Gelation Kinetics and Viscoelastic Properties of Pluronic and α-Cyclodextrin-Based Pseudopolyrotaxane Hydrogels

Publisher: American Chemical Society (ACS)

Date: 19-09-2013

DOI: 10.1021/BM401168H

Abstract: The results of a systematic investigation into the gelation behavior of α-cyclodextrin (α-CD) and Pluronic (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers) pseudopolyrotaxane (PPR) hydrogels are reported here in terms of the effects of temperature, α-CD concentration, and Pluronic type (Pluronic F68 and Pluronic F127). It was found that α-CD significantly modifies the gelation behavior of Pluronic solutions and that the PPR hydrogels are highly sensitive to changes in the α-CD concentration. In some cases, the addition of α-CD was found to be detrimental to the gelation process, leading to slower gelation kinetics and weaker gels than with Pluronic alone. However, in other cases, the hydrogels formed in the presence of the α-CDs reached higher moduli and showed faster gelation kinetics than with Pluronic alone and in some instances α-CD allowed the formation of hydrogels from Pluronic solutions that would normally not undergo gelation. Depending on composition and ratio of α-CD/Pluronic, these highly viscoelastic hydrogels displayed elastic shear modulus values ranging from 2 kPa to 7 MPa, gelation times ranging from a few seconds to a few hours and self-healing behaviors post failure. Using dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), we probed the resident structure of these systems, and from these insights we have proposed a new molecular mechanism that accounts for the macroscopic properties observed.

A novel saliva-based microRNA biomarker panel to detect head and neck cancers

Publisher: Springer Science and Business Media LLC

Date: 26-08-2014

DOI: 10.1007/S13402-014-0188-2

Abstract: MicroRNAs (miRNAs) are known to play an important role in cancer development by post-transcriptionally affecting the expression of critical genes. The aims of this study were two-fold: (i) to develop a robust method to isolate miRNAs from small volumes of saliva and (ii) to develop a panel of saliva-based diagnostic biomarkers for the detection of head and neck squamous cell carcinoma (HNSCC). Five differentially expressed miRNAs were selected from miScript™ miRNA microarray data generated using saliva from five HNSCC patients and five healthy controls. Their differential expression was subsequently confirmed by RT-qPCR using saliva s les from healthy controls (n = 56) and HNSCC patients (n = 56). These s les were ided into two different cohorts, i.e., a first confirmatory cohort (n = 21) and a second independent validation cohort (n = 35), to narrow down the miRNA diagnostic panel to three miRNAs: miR-9, miR-134 and miR-191. This diagnostic panel was independently validated using HNSCC miRNA expression data from The Cancer Genome Atlas (TCGA), encompassing 334 tumours and 39 adjacent normal tissues. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic capacity of the panel. On average 60 ng/μL miRNA was isolated from 200 μL of saliva. Overall a good correlation was observed between the microarray data and the RT-qPCR data. We found that miR-9 (P <0.0001), miR-134 (P <0.0001) and miR-191 (P <0.001) were differentially expressed between saliva from HNSCC patients and healthy controls, and that these miRNAs provided a good discriminative capacity with area under the curve (AUC) values of 0.85 (P <0.0001), 0.74 (P < 0.001) and 0.98 (P < 0.0001), respectively. In addition, we found that the salivary miRNA data showed a good correlation with the TCGA miRNA data, thereby providing an independent validation. We show that we have developed a reliable method to isolate miRNAs from small volumes of saliva, and that the saliva-derived miRNAs miR-9, miR-134 and miR-191 may serve as novel biomarkers to reliably detect HNSCC.

A biocompatible thermoset polymer binder for Direct Ink Writing of porous titanium scaffolds for bone tissue engineering

Publisher: Elsevier BV

Date: 02-2019

DOI: 10.1016/J.MSEC.2018.10.033

Abstract: There is increasing demand for synthetic bone scaffolds for bone tissue engineering as they can counter issues such as potential harvesting morbidity and restrictions in donor sites which h er autologous bone grafts and address the potential for disease transmission in the case of allografts. Due to their excellent biocompatibility, titanium scaffolds have great potential as bone graft substitutes as they mimic the structure and properties of human cancellous bone. Here we report on a new thermoset bio-polymer which can act as a binder for Direct Ink Writing (DIW) of titanium artificial bone scaffolds. We demonstrate the use of the binder to manufacture porous titanium scaffolds with evenly distributed and highly interconnected porosity ideal for orthopaedic applications. Due to their porous structure, the scaffolds exhibit an effective Young's modulus similar to human cortical bone, alleviating undesirable stress-shielding effects, and possess superior strength. The biocompatibility of the scaffolds was investigated in vitro by cell viability and proliferation assays using human bone-marrow-derived Mesenchymal stem cells (hMSCs). The hMSCs displayed well-spread morphologies, well-organized F-actin and large vinculin complexes confirming their excellent biocompatibility. The vinculin regions had significantly larger Focal Adhesion (FA) area and equivalent FA numbers compared to that of tissue culture plate controls, showing that the scaffolds support cell viability and promote attachment. In conclusion, we have demonstrated the excellent potential of the thermoset bio-polymer as a Direct Ink Writing ready binder for manufacture of porous titanium scaffolds for hard tissue engineering.

Cyclin-dependent–like kinase 5 is required for pain signaling in human sensory neurons and mouse models

Publisher: American Association for the Advancement of Science (AAAS)

Date: 08-07-2020

DOI: 10.1126/SCITRANSLMED.AAX4846

Abstract: CDKL5 controls nociception in human neurons and murine models of CDKL5 deficiency disorder via CaMKII-dependent mechanisms.

Effects of electric fields on human mesenchymal stem cell behaviour and morphology using a novel multichannel device

Publisher: Oxford University Press (OUP)

Date: 19-05-2015

DOI: 10.1039/C4IB00297K

Interactions between meniscal cells and a self assembled biomimetic surface composed of hyaluronic acid, chitosan and meniscal extracellular matrix molecules

Publisher: Elsevier BV

Date: 08-2010

DOI: 10.1016/J.BIOMATERIALS.2010.04.018

Abstract: Menisci are one of the most commonly injured parts of the knee with a limited healing potential. This study focuses on fabrication and characterization of biomimetic surfaces for meniscal tissue engineering. To achieve this, a combination of hyaluronic acid/chitosan (HA/CH) mutilayers with covalently immobilized major extracellular matrix (ECM) components of native meniscus, namely collagen I/II (COL.I/II) and chondroitin-6-sulfate (C6S) was employed. Adsorption of the biomolecules was monitored using a quartz crystal microbalance with dissipation (QCM-D) and fourier transform-surface plasmon resonance (FT-SPR). Immobilization of the biomolecules onto HA/CH mutilayers was achieved by sequential adsorption, with optimum binding at a molar ratio of 1.4:1 (COL.I/II: C6S). After adding COL.I/II, the layers became relatively more rigid and large aggregates were evident. The effects of the modified surfaces on cell proliferation, gene expression and proteoglycan production of rat meniscal cells were examined. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) analysis showed that primary meniscal cells dedifferentiated rapidly after one passage in monolayer culture. This process could be reversed by culturing the cells on C6S surfaces, as indicated by increases in both collagen II and aggrecan gene expression, as well as proteoglycan production. Cells with abundant lipid vacuoles were evident on all the surfaces over an extended culture period. The results demonstrate the feasibility of C6S surfaces to avoid the dedifferentiation that normally occurs during monolayer expansion of meniscal cells.

The use of an electrostatic lens to enhance the efficiency of the electrospinning process

Publisher: Springer Science and Business Media LLC

Date: 28-01-2012

DOI: 10.1007/S00441-011-1318-Z

Abstract: Electrospun scaffolds manufactured using conventional electrospinning configurations have an intrinsic thickness limitation, due to a charge build-up at the collector. To overcome this limitation, an electrostatic lens has been developed that, at the same relative rate of deposition, focuses the polymer jet onto a smaller area of the collector, resulting in the fabrication of thick scaffolds within a shorter period of time. We also observed that a longer deposition time (up to 13 h, without the intervention of the operator) could be achieved when the electrostatic lens was utilised, compared to 9–10 h with a conventional processing set-up and also showed that fibre fusion was less likely to occur in the modified method. This had a significant impact on the mechanical properties, as the scaffolds obtained with the conventional process had a higher elastic modulus and ultimate stress and strain at short times. However, as the thickness of the scaffolds produced by the conventional electrospinning process increased, a 3-fold decrease in the mechanical properties was observed. This was in contrast to the modified method, which showed a continual increase in mechanical properties, with the properties of the scaffold finally having similar mechanical properties to the scaffolds obtained via the conventional process at longer times. This “focusing” device thus enabled the fabrication of thicker 3-dimensional electrospun scaffolds (of thicknesses up to 3.5 mm), representing an important step towards the production of scaffolds for tissue engineering large defect sites in a multitude of tissues.

Effects of biomimetic surfaces and oxygen tension on redifferentiation of passaged human fibrochondrocytes in 2D and 3D cultures

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1016/J.BIOMATERIALS.2011.04.033

Abstract: Due to its limited healing potential within the inner avascular region, functional repair of the meniscus remains a significant challenge in orthopaedic surgery. Tissue engineering of a meniscus implant using meniscal cells offers the promise of enhancing the reparative process and achieving functional meniscal repair. In this work, using quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) analysis, we show that human fibrochondrocytes rapidly dedifferentiate during monolayer expansion on standard tissue culture flasks, representing a significant limit to clinical use of this cell population for meniscal repair. Previously, we have characterized and described the feasibility of a tailored biomimetic surface (C6S surface) for reversing dedifferentiation of monolayer-expanded rat meniscal cells. The surface is comprised of major meniscal extracellular matrix (ECM) components in the inner region, namely collagen I/II (at a 2:3 ratio) and chondroitin-6-sulfate. We thus have further evaluated the effects of the C6S surface, alongside a number of other tailored surfaces, on cell adhesion, proliferation, matrix synthesis and relevant marker gene expression (collagen I, -II, aggrecan and Sox-9 etc) of passaged human fibrochondrocytes in 2D (coated glass coverslips) and 3D (surface-modified polymeric scaffolds) environments. We show that the C6S surface is permissive for cell adhesion, proliferation and ECM synthesis, as demonstrated using DNA quantification, 1,9-dimethylmethylene blue (DMMB) assay, histology and immunohistochemistry. More importantly, RT-qPCR analyses corroborate the feasibility of the C6S surface for reversing phenotypic changes, especially the downregulation of collagen II, of dedifferentiated human fibrochondrocytes. Furthermore, human fibrochondrocyte redifferentiation was enhanced by hypoxia in the 3D cultures, independent of hypoxia inducible factor (HIF) transcriptional activity and was shown to potentially involve the transcriptional activation of Sox-9.

The effects of silica fillers on the gelation and vitrification of highly filled epoxy-amine thermosets

Publisher: Wiley

Date: 05-2001

DOI: 10.1002/1521-3900(200105)169:1<171::AID-MASY171>3.0.CO;2-Y

Systematic selection of solvents for the fabrication of 3D combined macro- and microporous polymeric scaffolds for soft tissue engineering

Publisher: Informa UK Limited

Date: 2006

DOI: 10.1163/156856206776374142

Abstract: In this study, we investigate the fabrication of 3D porous poly(lactic-co-glycolic acid) (PLGA) scaffolds using the thermally-induced phase separation technique. The current study focuses on the selection of alternative solvents for this process using a number of criteria, including predicted solubility, toxicity, removability and processability. Solvents were removed via either vacuum freeze-drying or leaching, depending on their physical properties. The residual solvent was tested using gas chromatography-mass spectrometry. A large range of porous, highly interconnected scaffold architectures with tunable pore size and alignment was obtained, including combined macro- and microporous structures and an entirely novel 'porous-fibre' structure. The morphological features of the most promising poly(lactic-co-glycolic acid) scaffolds were analysed via scanning electron microscopy and X-ray micro-computed tomography in both two and three dimensions. The Young's moduli of the scaffolds under conditions of temperature, pH and ionic strength similar to those found in the body were tested and were found to be highly dependent on the architectures.

Long term culture of human embryonic stem cells on recombinant vitronectin in ascorbate free media

Publisher: Elsevier BV

Date: 11-2010

DOI: 10.1016/J.BIOMATERIALS.2010.07.037

Abstract: Human embryonic stem cells (hESC) are expected to provide revolutionary therapeutic applications and drug discovery technologies. In order for this to be achieved a reproducible, defined animal component free culture system is required for the scale-up production of undifferentiated hESC. In this work we have investigated the applicability of a recombinantly produced domain of human vitronectin as an extracellular matrix alternative to the common standards Geltrex or Matrigel. In addition we have validated an ascorbate free media capable of supporting CD30(low) populations of hESC through a multi-factorial analysis of bFGF and Activin A. The recombinant vitronectin domain combined with the ascorbate free media were capable of supporting 3 cell lines, MEL1, MEL2 and hES3 for 10 or more passages while maintaining hESC pluripotency markers and differentiation capacity. The culture method outlined here provides a platform for future investigation into growth factor and extracellular matrix effects on hESC maintenance prior to bioreactor scale-up.

Membrane bioreactors enhance microenvironmental conditioning and tissue development

Publisher: Mary Ann Liebert Inc

Date: 06-2010

DOI: 10.1089/TEN.TEC.2009.0367

Tensile properties and in vitro degradation of P(TMC-co-LLA) elastomers

Publisher: Royal Society of Chemistry (RSC)

Date: 2015

DOI: 10.1039/C5TB00202H

Abstract: The degradation mechanism of P(TMC- co -LLA) films was dependent on the LLA content and found to transition from heterogeneous to homogeneous bulk degradation.

Architecture control of three‐dimensional polymeric scaffolds for soft tissue engineering. I. Establishment and validation of numerical models

Publisher: Wiley

Date: 09-08-2004

DOI: 10.1002/JBM.A.30126

Abstract: One of the most important functions of artificial three-dimensional (3D) polymeric scaffolds is to serve as a physical support to provide tissues with an appropriate architecture for in vitro cell culture as well as in vivo tissue regeneration. The production of three-dimensional (3D) polymeric scaffolds with tailored macroporous architecture is thus a crucial step in promoting controlled vascularization and tissue growth within host environments. In this study, 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds were manufactured by a thermally induced phase-separation (TIPS) technique. By controlling the quenching strategy, 3D interconnected PLGA scaffolds with tunable pore size and alignment were obtained and characterized with the use of scanning electron microscopy (SEM). A series of numerical heat-transfer models were established in an effort to describe the cooling process within the PLGA freezing regime. Among them, a two-dimensional (2D) solidification model has proved to be the most successful in describing the quenching of the polymer solution and has the potential to be used to infer the various 3D macroporous architectures created from different quenching conditions.

The role of biomechanical factors in models of intervertebral disc degeneration across multiple length scales

Publisher: AIP Publishing

Date: 08-05-2023

DOI: 10.1063/5.0137698

Abstract: Low back pain is the leading cause of disability, producing a substantial socio-economic burden on healthcare systems worldwide. Intervertebral disc (IVD) degeneration is a primary cause of lower back pain, and while regenerative therapies aimed at full functional recovery of the disc have been developed in recent years, no commercially available, approved devices or therapies for the regeneration of the IVD currently exist. In the development of these new approaches, numerous models for mechanical stimulation and preclinical assessment, including in vitro cell studies using microfluidics, ex vivo organ studies coupled with bioreactors and mechanical testing rigs, and in vivo testing in a variety of large and small animals, have emerged. These approaches have provided different capabilities, certainly improving the preclinical evaluation of these regenerative therapies, but challenges within the research environment, and compromises relating to non-representative mechanical stimulation and unrealistic test conditions, remain to be resolved. In this review, insights into the ideal characteristics of a disc model for the testing of IVD regenerative approaches are first assessed. Key learnings from in vivo, ex vivo, and in vitro IVD models under mechanical loading stimulation to date are presented alongside the merits and limitations of each model based on the physiological resemblance to the human IVD environment (biological and mechanical) as well as the possible feedback and output measurements for each approach. When moving from simplified in vitro models to ex vivo and in vivo approaches, the complexity increases resulting in less controllable models but providing a better representation of the physiological environment. Although cost, time, and ethical constraints are dependent on each approach, they escalate with the model complexity. These constraints are discussed and weighted as part of the characteristics of each model.

Effects of Varying Heptylamine and Propionaldehyde Plasma Polymerization Parameters on Mesenchymal Stem Cell Attachment

Publisher: Wiley

Date: 29-08-2012

DOI: 10.1002/PPAP.201200009

Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells

Publisher: Elsevier BV

Date: 06-2017

DOI: 10.1016/J.STEMCR.2017.05.019

Isolation of Contractile Cardiomyocytes from Human Pluripotent Stem-Cell-Derived Cardiomyogenic Cultures Using a HumanNCX1-EGFPReporter

Publisher: Mary Ann Liebert Inc

Date: 2015

DOI: 10.1089/SCD.2014.0195

Abstract: The prospective isolation of defined contractile human pluripotent stem cell (hPSC)-derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as NKX2.5 or MYH6, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the SLC8A1 (NCX1) gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation, NCX1cp-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1(+) cardiac stromal cells or CD31/PECAM(+) endothelial cells. Flow-assisted cytometrically sorted EGFP(+) fractions of differentiated cultures were highly enriched in both early (NKX2.5 and TBX5) and late (CTNT/TNNI2, MYH6, MYH7, NPPA, and MYL2) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-cl recordings. We conclude that the use of the cardiac-specific promoter of the human SLC8A1(NCX1) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures.

New Murine Model of Spontaneous Autologous Tissue Engineering, Combining an Arteriovenous Pedicle with Matrix Materials

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 2004

DOI: 10.1097/01.PRS.0000095942.71618.9D

Hydrolytically Degradable Polyrotaxane Hydrogels for Drug and Cell Delivery Applications

Publisher: American Chemical Society (ACS)

Date: 12-12-2014

DOI: 10.1021/BM501615P

Abstract: Self-assembled pseudopolyrotaxane (PPR) hydrogels formed from Pluronic polymers and α-cyclodextrin (α-CD) have been shown to display a wide range of tailorable physical and chemical properties that may see them exploited in a multitude of future biomedical applications. Upon the mixing of both components, these self-assembling hydrogels reach a metastable thermodynamic state that is defined by the concentrations of both components in solution and the temperature. However, at present, their potential is severely limited by the very nature by which they form and hence also disassemble. Even if the temperature is kept constant, PPR hydrogels will dissociate and collapse within a few hours when immersed in a liquid (such as cell culture media) that contains a lower concentrations of, or no, Pluronic or α-CD due to differences in chemical potential driving dissolution. In this article, an enzymatically mediated covalent cross-linking function and branched eight-arm poly(ethylene glycol) (PEG) were thus introduced into the PPR hydrogels to improve their robustness to such environmental changes. The eight-arm PEG also acted as an end-capping group to prevent the dethreading of the α-CD molecules. The covalent cross-linking successfully extended the lifetime of the hydrogels when placed in cell culture media from a few hours to up to 1 week, with the ability to control the degradation rate (now initiated by hydrolysis of the introduced ester bonds and not by dissolution) by changing the amount of eight-arm PEG present in the hydrogels. Highly tunable hydrogels were obtained with an elastic modulus between 20 and 410 kPa and a viscous modulus between 150 Pa and 22 kPa by varying the concentrations of α-CD and eight-arm PEG. Sustained release of a model drug from the hydrogels was achieved, and viability of mouse fibroblasts encapsulated in these hydrogels was assessed. These self-assembling, hydrolytically degradable, and highly tunable hydrogels are seen to have potential applications in tissue engineering relying on controlled drug or cell delivery to sites targeted for repair.

Controlled presentation of recombinant proteins via a zinc-binding peptide-linker in two and three dimensional formats

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1016/J.BIOMATERIALS.2009.08.033

Abstract: The presentation of proteins on surfaces is fundamental to numerous cell culture and tissue engineering applications. While a number of physisorption and cross-linking methods exist to facilitate this process, few avoid denaturation of proteins or allow control over protein orientation, both of which are critical to the functionality of many signal proteins and ligands. Often recombinant protein sequences include a poly-histidine tag to facilitate purification. We utilize this sequence to anchor proteins to biosurfaces via a peptide bonded to the surface which conjugates with the poly-histidine tag in the presence of zinc rather than nickel, which is more traditionally used to conjugate poly-histidine tags to surfaces. We demonstrate that this strategy enables the display of proteins on 2D and 3D surfaces without compromising protein function through direct cross-linking or physisorption.

Effects of fluid-fluid interfacial elasticity on droplet formation in microfluidic devices

Publisher: Wiley

Date: 26-07-2010

DOI: 10.1002/AIC.12382

Polymer concentration dependence of the gel point for chemically modified biopolymer networks using small amplitude oscillatory rheometry

Publisher: Elsevier BV

Date: 04-2001

DOI: 10.1016/S0032-3861(00)00795-3

Effects of Cell Density and Biomacromolecule Addition on the Flow Behavior of Concentrated Mesenchymal Cell Suspensions

Publisher: American Chemical Society (ACS)

Date: 21-11-2013

DOI: 10.1021/BM401335G

Abstract: With the rapidly growing interest in the use of mesenchymal stromal cells (MSCs) for cell therapy and regenerative medicine applications, either alone as an injected suspension, or dispersed within injectable hydrogel delivery systems, greater understanding of the structure-function-property characteristics of suspensions of adhesion-dependent mesenchymal cells is required. In this paper, we present the results of an experimental study into the flow behavior of concentrated suspensions of living cells of mesenchymal origin (fibroblasts) over a wide range of cell concentrations, with and without the addition of hyaluronic acid (HA), a commonly utilized biomolecule in injectable hydrogel formulations. We characterize the change in the shear viscosity as a function of shear stress and shear rate for cell volume fractions varying from 20 to 60%. We show that high volume fraction suspensions of living mesenchymal cells, known to be capable of homotypic interactions, exhibit highly complex but reproducible rheological footprints, including yield stress, shear thinning and shear-induced fracture behaviors. We show that with the addition of HA, we can significantly modify and tailor the rheology of these cell suspensions at all volume fractions. Using FACS and confocal imaging, we show that the observed effect of HA addition is due to a significantly modulation in the formation of cellular aggregates in these suspensions, and thus the resultant volume spanning network. Considering the aggregates as fractal structures, we show that by taking into account the changes in volume fractions with shear, we are able to plot a master curve for the range of conditions investigated and extract from it the average adhesion force between in idual cells, across a population of millions of cells. The outcomes of this study not only provide new insight into the complexity of the flow behaviors of concentrated, adhesive mesenchymal cell suspensions, and their sensitivity to associative biomacromolecule addition, but also a novel, rapid method by which to measure the average adhesion force between in idual cells, and the impacts of biomacromolecules on this important parameter.

Multilayered Microspheres for the Controlled Release of Growth Factors in Tissue Engineering

Publisher: American Chemical Society (ACS)

Date: 17-03-2011

DOI: 10.1021/BM1014574

Abstract: Tissue regeneration may be stimulated by growth factors but to be effective, this delivery must be sustained and requires delivery vehicles that overcome the short half-life of these molecules in vivo. One promising approach is to couple growth factors to the biomaterial surface so that they are readily bioavailable. Here the layer-by-layer process was used to construct a multilayered polyelectrolyte delivery system on the surface of poly(lactic-co-glycolic) acid constructs. The system was first optimized on a planar surface before translation to a 3D microsphere system. The layers incorporated heparin to facilitate the loading of basic fibroblast growth factor and increase growth factor stability. Cross-linked capping layers also reduced any burst release. The model growth factor was released in a sustained manner and stimulated significantly higher cell proliferation in vitro on release compared with the addition of the growth factor heparin complex free in solution, demonstrating the promise of this approach.

Use of Two-Step Grafting to Fabricate Dual-Functional Films and Site-Specific Functionalized Scaffolds

Publisher: American Chemical Society (ACS)

Date: 29-01-2015

DOI: 10.1021/LA504629H

Abstract: Polycaprolactone (PCL) is a widely utilized bioresorbable polymer in tissue engineering applications. However, the absence of intrinsic functional groups in the polymer backbone necessitates the incorporation of functional chemistries to enable the further addition of bioactive molecules to PCL-based surfaces and scaffolds. The current study aimed to incorporate two different functional groups, amine and carboxylate, first on two-dimensional (2D) spin-coated PCL films and, thereafter, throughout all surfaces within three-dimensional (3D) porous PCL-based scaffolds, produced using the thermally induced phase separation (TIPS) method, but in a spatially separated manner. Specifically, gamma irradiation induced grafting of acrylic acid (AA) and 2-aminoethyl methacrylate hydrochloride (AEMA) onto PCL was performed in selected solvents and the resulting substrates were characterized using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and contact angle measurements to determine the surface free energy. Results demonstrated that stepwise graft copolymerization of AEMA and AA allows the fabrication of dual-functional surfaces, with chemistry depending on the order of grafting of the two monomers. In addition, 3D scaffolds could be decorated exclusively with carboxylate groups in the interior, while the outer surface displayed dual-functionality. This simple surface modification methodology, with the ability to create spatially separated surface functional groups throughout 3D porous scaffolds post their fabrication, has the potential to be applied to many current and future scaffold systems being investigated in the field of tissue engineering.

Nanoscale presentation of cell adhesive molecules via block copolymer self-assembly

Publisher: Elsevier BV

Date: 09-2009

DOI: 10.1016/J.BIOMATERIALS.2009.05.039

Abstract: Precise control over the nanoscale presentation of adhesion molecules and other biological factors represents a new frontier for biomaterials science. Recently, the control of integrin spacing and cellular shape has been shown to affect fundamental biological processes, such as differentiation and apoptosis. Here, we present the self-assembly of maleimide functionalised polystyrene-block-poly (ethylene oxide) copolymers as a simple, yet highly precise method for controlling the position of cellular adhesion molecules. By manipulating the phase separation of the functional PS-PEO block copolymer used in this study, via a simple blending technique, we alter the nanoscale (on PEO domains of 8-14 nm in size) presentation of the adhesion peptide, GRGDS, decreasing lateral spacing from 62 nm to 44 nm and increasing the number density from approximately 450 to approximately 900 islands per microm2. The results indicate that the spreading of NIH-3T3 fibroblasts increases as the spacing between domains of RGD binding peptides decreases. Further, the same functional PS-PEO surfaces have been utilised to immobilise, via a zinc chelating peptide sequence, poly-histidine tagged proteins and extracellular matrix (ECM) fragments. This method is seen as an ideal platform for investigations into the role of spatial arrangements of cell adhesion molecules and ECM molecules on cell function and, in particular, control of cell phenotype.

Substrate stiffness facilitates improved induced pluripotent stem cell production through modulation of both early and late phases of cell reprogramming

Publisher: Cold Spring Harbor Laboratory

Date: 03-2023

DOI: 10.1101/2023.02.27.530366

Abstract: Cell reprogramming involves time-intensive, costly processes that ultimately produce low numbers of reprogrammed cells of variable quality. By screening a range of polyacrylamide hydrogels (pAAm gels) of varying stiffness (1 kPA – 1.3 MPa) we found that a gel of medium stiffness significantly increases the overall number of reprogrammed cells by up to ten-fold with accelerated reprogramming kinetics, as compared to the standard Tissue Culture PolyStyrene (TCPS)-based protocol. We observe that though the gel improves both early and late phases of reprogramming, improvement in the late (reprogramming prone population maturation) phase is more pronounced and produces iPSCs having different characteristics and lower remnant transgene expression than those produced on TCPS. Comparative RNA-Seq analyses coupled with experimental validation reveals that modulation of Bone Morphogenic Protein (BMP) signalling by a novel reprogramming regulator, Phactr3, upregulated in the gel at an earliest time-point without the influence of transcription factors used for reprogramming, plays a crucial role in the improvement in the early reprogramming kinetics and overall reprogramming outcomes. This study provides new insights into the mechanism via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes, opening new avenues for producing higher numbers of quality iPSCs or other reprogrammed cells at shorter timescales.

Modelling oxygen diffusion and cell growth in a porous, vascularising scaffold for soft tissue engineering applications

Publisher: Elsevier BV

Date: 09-2005

DOI: 10.1016/J.CES.2005.03.051

Engineering tissue tubes using novel multilayered scaffolds in the rat peritoneal cavity

Publisher: Wiley

Date: 15-01-2008

DOI: 10.1002/JBM.A.31781

Abstract: Our aim was to develop novel scaffolds to engineer tissue tubes of smooth muscle-like cells for autologous grafting. Small diameter tubular poly(lactic acid) scaffolds with randomly distributed, interconnected pores up to 100 mum were produced using a thermally induced phase separation method. The scaffolds were surface modified using various biomolecules via a layer-by-layer deposition technique, and implanted in the peritoneal cavities of rats. Histological analysis of scaffolds 3 weeks after implantation showed fully-developed tissue capsules on their outer surfaces, with macrophage-like cells present throughout the internal spaces. Surfaces coated in Matrigel supported the strongest cellular response whereas multilayer coatings with elastin, collagen I, collagen III, or chitosan outermost showed the lowest levels of cellular interaction. Although differences in capsule thickness and the presence or absence of cellularized layers on the inside and outside surfaces of the scaffolds were observed, none of these biomolecule coatings was able to overcome the foreign body response within the peritoneal cavity, even in the presence of a nonadsorptive HA undercoat.

Cross-linked poly(trimethylene carbonate- co - L -lactide) as a biodegradable, elastomeric scaffold for vascular engineering applications

Publisher: American Chemical Society (ACS)

Date: 27-10-2011

DOI: 10.1021/BM201291E

Abstract: A series of copolymers of trimethylene carbonate (TMC) and L-lactide (LLA) were synthesized and evaluated as scaffolds for the production of artificial blood vessels. The polymers were end-functionalized with acrylate, cast into films, and cross-linked using UV light. The mechanical, degradation, and biocompatibility properties were evaluated. High TMC polymers showed mechanical properties comparable to human arteries (Young's moduli of 1.2-1.8 MPa and high elasticity with repeated cycling at 10% strain). Over 84 days degradation in PBS, the modulus and material strength decreased gradually. The polymers were nontoxic and showed good cell adhesion and proliferation over 7 days using human mesenchymal stem cells. When implanted into the rat peritoneal cavity, the polymers elicited formation of tissue capsules composed of myofibroblasts, resembling immature vascular smooth muscle cells. Thus, these polymers showed properties which were tunable and favorable for vascular tissue engineering, specifically, the growth of artificial blood vessels in vivo.

Electrospinning and mechanical properties of P(TMC-co-LLA) elastomers

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7TB00137A

Abstract: P(TMC- co -LLA) elastomers have shown great potential for various biomaterial and tissue engineering applications.

Tumor-suppressor Gene Promoter Hypermethylation in Saliva of Head and Neck Cancer Patients

Publisher: Elsevier BV

Date: 10-2012

DOI: 10.1593/TLO.12232

Lateral spacing of adhesion peptides influences human mesenchymal stem cell behaviour.

Publisher: The Company of Biologists

Date: 15-01-2012

DOI: 10.1242/JCS.087916

Abstract: Mesenchymal stem cells (MSCs) have attracted great interest in recent years for tissue engineering and regenerative medicine applications due to their ease of isolation and multipotent differentiation capacity. In the past, MSC research has focussed on the effects of soluble cues, such as growth factors and cytokines however, there is now increasing interest in understanding how parameters such as substrate modulus, specific extracellular matrix (ECM) components and the ways in which these are presented to the cell can influence MSC properties. Here we use surfaces of self-assembled maleimide-functionalized polystyrene-block-poly(ethylene oxide) copolymers (PS-PEO-Ma) to investigate how the spatial arrangement of cell adhesion ligands affects MSC behaviour. By changing the ratio of PS-PEO-Ma in mixtures of block copolymer and polystyrene homopolymer, we can create surfaces with lateral spacing of the PEO-Ma domains ranging from 34 to 62 nm. Through subsequent binding of cysteine–GRGDS peptides to the maleimide-terminated end of the PEO chains in each of these domains, we are able to present tailored surfaces of controlled lateral spacing of RGD (arginine-glycine-aspartic acid) peptides to MSCs. We demonstrate that adhesion of MSCs to the RGD-functionalized block-copolymer surfaces is through specific attachment to the presented RGD motif and that this is mediated by α5, αV, β1 and β3 integrins. We show that as the lateral spacing of the peptides is increased, the ability of the MSCs to spread is diminished and that the morphology changes from well-spread cells with normal fibroblastic morphology and defined stress-fibres, to less-spread cells with numerous cell protrusions and few stress fibres. In addition, the ability of MSCs to form mature focal adhesions is reduced on substrates with increased lateral spacing. Finally, we investigate differentiation and use qRT-PCR determination of gene expression levels and a quantitative alkaline phosphatase assay to show that MSC osteogenesis is reduced on surfaces with increased lateral spacing while adipogenic differentiation is increased. We show here, for the first time, that the lateral spacing of adhesion peptides affects human MSC (hMSC) properties and might therefore be a useful parameter with which to modify hMSC behaviour in future tissue engineering strategies.

Transgenic human ES and iPS reporter cell lines for identification and selection of pluripotent stem cells in vitro

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.SCR.2014.05.006

Abstract: Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency, and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3, H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers, such as OCT4 (POU5F1), TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors.

Adhesion measurements between alginate gel surfaces via texture analysis

Publisher: Elsevier BV

Date: 2008

DOI: 10.1016/J.FOODHYD.2007.05.005

Tailorable cell culture platforms from enzymatically cross-linked multifunctional poly(ethylene glycol)-based hydrogels

Publisher: American Chemical Society (ACS)

Date: 15-01-2013

DOI: 10.1021/BM301652Q

Abstract: As stem-cell-based therapies rapidly advance toward clinical applications, there is a need for cheap, easily manufactured, injectable gels that can be tailored to carry stem cells and impart function to such cells. Herein we describe a process for making hydrogels composed of hydroxyphenyl propionic acid (HPA) conjugated, branched poly(ethylene glycol) (PEG) via an enzyme mediated, oxidative cross-linking method. Functionalization of the branched PEG with HPA at varying degrees of substitution was confirmed via attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and (1)H NMR. The versatility of this hydrogel system was exemplified through variations in the degree of HPA substitution, polymer concentration, and the concentration of cross-linking reagents (horseradish peroxidase and H(2)O(2)), which resulted in a range of mechanical properties and gelation kinetics for these gels. Cross-linking of the PEG-HPA conjugate with a recombinantly produced Fibronectin fragment (Type III domains 7-10) encouraged attachment and spreading of human mesenchymal stem cells (hMSCs) when assessed in both two-dimensional and three-dimensional formats. Interestingly, when encapsulated in both nonfunctionalized and functionalized cross-linked PEG-HPA gels, MSCs showed good viability over all time periods assessed. With tunable gelation kinetics and mechanical properties, these hydrogels provide a flexible in vitro cell culture platform that will likely have significant utility in tissue engineering as an injectable delivery platform for cells to sites of tissue damage.

The influence of substrate creep on mesenchymal stem cell behaviour and phenotype

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.BIOMATERIALS.2011.04.003

Abstract: Human mesenchymal stem cells (hMSCs) are capable of probing and responding to the mechanical properties of their substrate. Although most biological and synthetic matrices are viscoelastic materials, previous studies have primarily focused upon substrate compressive modulus (rigidity), neglecting the relative contributions that the storage (elastic) and loss (viscous) moduli make to the summed compressive modulus. In this study we aimed to isolate and identify the effects of the viscous component of a substrate on hMSC behaviour. Using a polyacrlyamide gel system with constant compressive modulus and varying loss modulus we determined that changes to substrate loss modulus substantially affected hMSC morphology, proliferation and differentiation potential. In addition, we showed that the effect of substrate loss modulus on hMSC behaviour is due to a reduction in both passive and actively generated isometric cytoskeletal tension caused by the inherent creep of substrates with a high loss modulus. These findings highlight substrate creep, or more explicitly substrate loss modulus, as an important mechanical property of a biomaterial system that can be tailored to encourage the growth and differentiation of specific cell types.

Effects of synthetic biomacromolecule addition on the flow behavior of concentrated mesenchymal cell suspensions

Publisher: American Chemical Society (ACS)

Date: 12-12-2015

DOI: 10.1021/BM501481Y

Translational control of enzyme scavenger expression with toxin-induced micro RNA switches

Publisher: Springer Science and Business Media LLC

Date: 28-01-2021

DOI: 10.1038/S41598-021-81679-6

Abstract: Biological computation requires in vivo control of molecular behavior to progress development of autonomous devices. miRNA switches represent excellent, easily engineerable synthetic biology tools to achieve user-defined gene regulation. Here we present the construction of a synthetic network to implement detoxification functionality. We employed a modular design strategy by engineering toxin-induced control of an enzyme scavenger. Our miRNA switch results show moderate synthetic expression control over a biologically active detoxification enzyme molecule, using an established design protocol. However, following a new design approach, we demonstrated an evolutionarily designed miRNA switch to more effectively activate enzyme activity than synthetically designed versions, allowing markedly improved extrinsic user-defined control with a toxin as inducer. Our straightforward new design approach is simple to implement and uses easily accessible web-based databases and prediction tools. The ability to exert control of toxicity demonstrates potential for modular detoxification systems that provide a pathway to new therapeutic and biocomputing applications.

The generation of definitive endoderm from human embryonic stem cells on 3D biodegradable poly(lactic-co-glycolic acid) scaffolds and its comparison to those generated on 2D monolayer cultures

Publisher: Bentham Science Publishers Ltd.

Date: 17-03-2011

DOI: 10.2174/1876893801103010023

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2

Publisher: Cold Spring Harbor Laboratory

Date: 12-06-2023

DOI: 10.1101/2023.06.12.544552

Abstract: Why in iduals with Down Syndrome (DS, trisomy 21) are particularly susceptible to SARS CoV-2 induced neuropathology remains largely unclear. Since the choroid plexus (CP) performs important barrier and immune-interface functions, secretes the cerebrospinal fluid and strongly expresses the ACE2 receptor and the chromosome 21 encoded TMPRSS2 protease, we hypothesized that the CP could play a role in establishing SARS-CoV-2 infection in the brain. To investigate the role of the choroid plexus in SARS-CoV-2 central nervous system infection in DS, we established a new type of brain organoid from DS and isogenic euploid control iPSC that consists of a core of appropriately patterned functional cortical neuronal cell types that is surrounded by a patent and functional choroid plexus (CPCOs). Remarkably, DS-CPCOs not only recapitulated abnormal features of DS cortical development but also revealed defects in ciliogenesis and epithelial cell polarity of the developing choroid plexus. We next demonstrate that the choroid plexus layer facilitates SARS-CoV-2 replication and infection of cortical neuronal cells, and that this is increased in DS-CPCOs. We further show that inhibition of TMPRSS2 and Furin activity inhibits SARS-CoV-2 replication in DS CPCOs to the level observed in euploid organoids. We conclude that CPCOs are a useful model for dissecting the role of the choroid plexus in euploid and DS forebrain development and enables screening for therapeutics that can inhibit SARS-CoV-2 induced neuro-pathogenesis.

Long-Term Stability of Adipose Tissue Generated from a Vascularized Pedicled Fat Flap inside a Chamber

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 06-2011

DOI: 10.1097/PRS.0B013E3182131C3E

Self-assembling polystyrene-block-poly(ethylene oxide) copolymer surface coatings: Resistance to protein and cell adhesion

Publisher: Elsevier BV

Date: 05-2009

DOI: 10.1016/J.BIOMATERIALS.2009.01.012

Abstract: In this paper we report a method for biomaterial surface modification that utilizes the self-assembly of block copolymers of poly(styrene-block-ethylene oxide) (PS-PEO) to generate micro-phase separated surfaces with varying density PEO domains. These PS-PEO self-assembled surfaces showed a significant reduction in protein adsorption compared to control polystyrene surfaces. The adhesion of NIH-3T3 fibroblast cells was shown to be significantly affected by the surface coverage of PEO nano-domains formed by copolymer self-assembly. These nano-domains, when presented at high number density (almost 1000 domains per square micron), were shown to completely prevent cellular attachment, even though small amounts of protein were able to bind to the surface.

Attachment of poly(acrylic acid) to 3-aminopropyltriethoxysilane surface-modified hydroxyapatite

Publisher: Royal Society of Chemistry (RSC)

Date: 2013

DOI: 10.1039/C3TB21110J

NT-ProBNP Levels in Saliva and Its Clinical Relevance to Heart Failure

Publisher: Public Library of Science (PLoS)

Date: 31-10-2012

DOI: 10.1371/JOURNAL.PONE.0048452

Effect of geometric challenges on cell migration.

Publisher: Mary Ann Liebert Inc

Date: 10-2011

DOI: 10.1089/TEN.TEC.2011.0138

Combinatorial presentation of cartilage-inspired peptides on nanopatterned surfaces enables directed differentiation of human mesenchymal stem cells towards distinct articular chondrogenic phenotypes

Publisher: Elsevier BV

Date: 07-2019

DOI: 10.1016/J.BIOMATERIALS.2019.04.003

From scrawny to brawny: the quest for neomusculogenesis; smart surfaces and scaffolds for muscle tissue engineering

Publisher: Informa UK Limited

Date: 09-2007

DOI: 10.1586/17434440.4.5.709

Abstract: The successful generation of functional muscle tissues requires both an in-depth knowledge of muscle tissue physiology and advanced engineering practices. The inherent contractile functionality of muscle is a result of its high-level cellular and matrix organization over a multitude of length scales. While there have been many attempts to produce artificial muscle, a method to fabricate a highly organized construct, comprised of multiple cell types and capable of delivering contractile strengths similar to that of native smooth, skeletal or cardiac muscle has remained elusive. This is largely due to a lack of control over phenotype and spatial organization of cells. This paper covers state-of-the-art approaches to generating both 2D and 3D substrates that provide some form of higher level organization or multiple biochemical, mechanical or electrical cues to cells in order to successfully manipulate their behavior, in a manner that is conducive to the production of contractile muscle tissue. These so-called 'smart surfaces' and 'smart scaffolds' represent vital steps towards surface-engineered substrates for the engineering of muscle tissues, showing confidently that cellular behavior can be effectively and reproducibly manipulated through the design of the physical, chemical and electrical properties of the substrates on which cells are grown. However, many challenges remain to be overcome prior to reaching the ultimate goal of fully functional 3D vascularized engineered muscle.

Five Piconewtons: The Difference between Osteogenic and Adipogenic Fate Choice in Human Mesenchymal Stem Cells.

Publisher: American Chemical Society (ACS)

Date: 03-10-2019

DOI: 10.1021/ACSNANO.9B03914

Abstract: The ability of mesenchymal stem cells to sense nanoscale variations in extracellular matrix (ECM) compositions in their local microenvironment is crucial to their survival and their fate however, the underlying molecular mechanisms defining how such fates are temporally modulated remain poorly understood. In this work, we have utilized self-assembled block copolymer surfaces to present nanodomains of an adhesive peptide found in many ECM proteins at different lateral spacings (from 30 to 60 nm) and studied the temporal response (2 h to 14 days) of human mesenchymal stem cells (hMSCs) using a panel of real-time localization and activity biosensors. Our findings revealed that within the first 4 to 24 h postadhesion and spreading, hMSCs on smaller nanodomain spacings recruit more activated FAK and Src proteins to produce larger, longer-lived, and increased numbers of focal adhesions (FAs). The adhesions formed on smaller nanospacings rapidly recruit higher amounts of nonmuscle myosin IIA and vinculin and experience tension forces (by >5 pN/FA) significantly higher than those observed on larger nanodomain spacings. The transmission of higher levels of tension into the cytoskeleton at short times was accompanied by higher Rac1, cytosolic β-catenin, and nuclear localization of YAP/TAZ and RUNX2, which together biased the commitment of hMSCs to an osteogenic fate. This investigation provides mechanistic insights to confirm that smaller lateral spacings of adhesive nanodomains alter hMSC mechanosensing and biases mechanotransduction at short times

Development of Myocardial Constructs Using Modulus-Matched Acrylated Polypropylene Glycol Triol Substrate and Different Nonmyocyte Cell Populations

Publisher: Mary Ann Liebert Inc

Date: 09-2011

DOI: 10.1089/TEN.TEA.2010.0743

Simulations of viscoelastic droplet deformation through a microfluidic contraction

Publisher: WIT Press

Date: 19-04-2006

DOI: 10.2495/AFM06008

Rapamycin mitigates Valproic Acid-induced teratogenicity in human and animal models by suppressing AP-1-mediated senescence

Publisher: Cold Spring Harbor Laboratory

Date: 31-08-2023

DOI: 10.1101/2023.08.29.555421

Sintering and biocompatibility of blended elemental Ti-xNb alloys

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1016/J.JMBBM.2020.103691

Dispersion of hydroxyapatite nanoparticles in solution and in polycaprolactone composite scaffolds

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C5TB02255J

Abstract: Structure–property–performance in TIPS fabricated nanocomposite scaffolds: influence of polymer–solvent interaction and phase-separation process on the dispersion and surface distribution of particles.

Full factorial screening of human embryonic stem cell maintenance with multiplexed microbioreactor arrays

Publisher: Wiley

Date: 07-2013

DOI: 10.1002/BIOT.201200375

Abstract: Use of human pluripotent stem cells (hPSCs) in regenerative medicine applications relies on control of cell fate decisions by exogenous factors. This control can be hindered by the use of undefined culture components, poorly understood autocrine aracrine effects, spatiotemporal variations in microenvironmental composition inherent to static culture formats, and signal cross-talk between multiple factors. We recently described microbioreactor arrays that provide a full factorial spectrum of exogenous factors, and allow gradual accumulation of paracrine factors through serial culture chambers. We combined these with defined biochemical conditions, and in situ reporter gene- and immunofluorescence-based readouts to create an hPSC screening platform with enhanced data throughput and microenvironmental control. HES3-EOS-C(3+)-EiP reporter hESCs were screened against FGF-2, TGF-β1, and retinoic acid in a modified mTeSR-1 medium background. Differential pluripotency marker expression reflected mTeSR-1's maintenance capacity, and differentiation in response to removal of maintenance factors or addition of retinoic acid. Interestingly, pluripotency marker expression was downregulated progressively through serial chambers. Since downstream chambers are exposed to greater levels of paracrine factors under continuous flow, this effect is thought to result from secreted factors that negatively influence pluripotency. The microbioreactor array platform decodes factor interplay, and has a broad application in deciphering microenvironmental control of cell fate.

Aligned poly(L‐lactic‐co‐e‐caprolactone) electrospun microfibers and knitted structure: A novel composite scaffold for ligament tissue engineering

Publisher: Wiley

Date: 02-08-2010

DOI: 10.1002/JBM.A.32801

Abstract: We developed a novel technique involving knitting and electrospinning to fabricate a composite scaffold for ligament tissue engineering. Knitted structures were coated with poly(L-lactic-co-e-caprolactone) (PLCL) and then placed onto a rotating cylinder and a PLCL solution was electrospun onto the structure. Highly aligned 2-microm-diameter microfibers covered the space between the stitches and adhered to the knitted scaffolds. The stress-strain tensile curves exhibited an initial toe region similar to the tensile behavior of ligaments. Composite scaffolds had an elastic modulus (150 +/- 14 MPa) similar to the modulus of human ligaments. Biological evaluation showed that cells proliferated on the composite scaffolds and they spontaneously orientated along the direction of microfiber alignment. The microfiber architecture also induced a high level of extracellular matrix secretion, which was characterized by immunostaining. We found that cells produced collagen type I and type III, two main components found in ligaments. After 14 days of culture, collagen type III started to form a fibrous network. We fabricated a composite scaffold having the mechanical properties of the knitted structure and the morphological properties of the aligned microfibers. It is difficult to seed a highly macroporous structure with cells, however the technique we developed enabled an easy cell seeding due to presence of the microfiber layer. Therefore, these scaffolds presented attractive properties for a future use in bioreactors for ligament tissue engineering.

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Publisher: Elsevier BV

Date: 12-2012

DOI: 10.1016/J.JCHROMB.2012.10.023

Abstract: Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high ersity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid s le preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme ersity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva.

Experimental and analytical study of the effect of contact angle on liquid convective heat transfer in microchannels

Publisher: Elsevier BV

Date: 10-2006

DOI: 10.1016/J.IJHEATMASSTRANSFER.2006.02.057

Pushing the Limits of High Throughput PET-RAFT Polymerization

Publisher: American Chemical Society (ACS)

Date: 20-09-2018

DOI: 10.1021/ACS.MACROMOL.8B01600

Scaffolds, stem cells, and tissue engineering: A potent combination!

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/CH05145

Abstract: Stem cells, either from embryonic or adult sources, have demonstrated the potential to differentiate into a wide range of tissues depending on culture conditions. This makes them prime candidates for use in tissue engineering applications. Current technology allows us to process biocompatible and biodegradable polymers into three-dimensional (3D) configurations, either as solid porous scaffolds or hydrogels, with controlled macro and/or micro spatial geometry and surface chemistry. Such control provides us with the ability to present highly controlled microenvironments to a chosen cell type. However, the precise microenvironments required for optimal expansion and/or differentiation of stem cells are only now being elucidated, and hence the controlled use of stem cells in tissue engineering remains a very young field. We present here a brief review of the current literature detailing interactions between stem cells and 3D scaffolds of varying morphology and chemical properties, concluding with remaining challenges for those interested in tissue engineering using tailored scaffolds and stem cells.

Controllable Surface Modification of Poly(lactic-co-glycolic acid) (PLGA) by Hydrolysis or Aminolysis I:  Physical, Chemical, and Theoretical Aspects

Publisher: American Chemical Society (ACS)

Date: 09-01-2004

DOI: 10.1021/BM0343040

Abstract: While biodegradable, biocompatible polyesters such as poly (lactic-co-glycolic acid) (PLGA) are popular materials for the manufacture of tissue engineering scaffolds, their surface properties are not particularly suitable for directed tissue growth. Although a number of approaches to chemically modify the PLGA surface have been reported, their applicability to soft tissue scaffolds, which combine large volumes, complex shapes, and extremely fine structures, is questionable. In this paper, we describe two wet-chemical methods, base hydrolysis and aminolysis, to introduce useful levels of carboxylic acid or primary and secondary amine groups, respectively, onto the surface of PLGA with minimal degradation. The effects of temperature, concentration, pH, and solvent type on the kinetics of these reactions are studied by following changes in the wettability of the PLGA using contact angle measurements. In addition, the treated surfaces are studied using X-ray photoelectron spectroscopy (XPS) to determine the effect on the surface chemical structure. Furthermore, we show using XPS analysis that these carboxyl and amine groups are readily activated to allow the covalent attachment of biological macromolecules.

Modeling the adhesion of human embryonic stem cells to poly(lactic-co- glycolic acid) surfaces in a 3D environment

Publisher: Wiley

Date: 26-02-2010

DOI: 10.1002/JBM.A.32401

Abstract: Human embryonic stem cells (hESCs) have previously been cultured on three dimensional (3D) biodegradable polymer scaffolds. Although complex structures were formed from the hESCs, very little is known about the mechanism of adhesion of these cells to the surfaces of the scaffolds. In this study, we achieved the efficient adhesion of pluripotent hESCs to 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds based on our data from a novel two dimensional (2D) model that imitates the surface properties of the scaffolds. In the 2D model, single cell preparations of pluripotent hESCs adhered efficiently and predominantly to PLGA surfaces coated with laminin in comparison to collagen I, collagen IV, or fibronectin-coated surfaces. Flow cytometry analysis revealed that almost all of the pluripotent single cells expressed the integrin alpha 6, with a small percentage also expressing alpha 3ss1, which facilitates adhesion to laminin. This data was then translated into the 3D environment, with the efficient binding of single pluripotent hESCs to PLGA scaffolds coated with laminin. The utility of this system was shown by the directed differentiation of single hESCs seeded within laminin-coated scaffolds toward the endoderm lineage.

Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments

Publisher: Springer Science and Business Media LLC

Date: 09-12-2018

DOI: 10.1007/S11060-018-03049-Z

Abstract: The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.

Self-Organizing 3D Human Choroid Plexus-Ventricle-Cortical Organoids

Publisher: Cold Spring Harbor Laboratory

Date: 10-2020

DOI: 10.1101/2020.09.30.321554

Abstract: Both the choroid plexus (CP) and the cortex are derived from the rostral neural tube during early embryonic development. In addition to producing CSF, the CP secretes essential factors that orchestrate cortical development and later neurogenesis. Previous brain modeling efforts with human pluripotent stem cells (hPSCs) generated either cortical or CP tissues in 3D culture. Here, we used hPSC-derived neuroectodermal cells, the building blocks of the anterior body, to simultaneously generate CP that forms ventricles and cortical cells in organoids (CVCOs), which can be maintained as 3D organoid cultures. Large scale culture revealed reproducibility of the protocol independent of cell lines, clones or batches. CVCOs contain mature and functional CP that projects multiple cilia into the ventricle-like fluid filled cysts and is in direct contact with appropriately patterned cortical cells. CVCOs thus recapitulate key features of developing forebrain structures observed in in vivo and constitute a useful for dissecting the role of CP in human forebrain development in health and disease.

A cell migration device that maintains a defined surface with no cellular damage during wound edge generation

Publisher: Royal Society of Chemistry (RSC)

Date: 2009

DOI: 10.1039/B900791A

Abstract: Studying the rate of cell migration provides insight into fundamental cell biology as well as a tool to assess the functionality of synthetic surfaces and soluble environments used in tissue engineering. The traditional tools used to study cell migration include the fence and wound healing assays. In this paper we describe the development of a microchannel based device for the study of cell migration on defined surfaces. We demonstrate that this device provides a superior tool, relative to the previously mentioned assays, for assessing the propagation rate of cell wave fronts. The significant advantage provided by this technology is the ability to maintain a virgin surface prior to the commencement of the cell migration assay. Here, the device is used to assess rates of mouse fibroblasts (NIH 3T3) and human osteosarcoma (SaOS2) cell migration on surfaces functionalized with various extracellular matrix proteins as a demonstration that confining cell migration within a microchannel produces consistent and robust data. The device design enables rapid and simplistic assessment of multiple repeats on a single chip, where surfaces have not been previously exposed to cells or cellular secretions.

Differential response of patient-derived primary glioblastoma cells to environmental stiffness

Publisher: Springer Science and Business Media LLC

Date: 21-03-2016

DOI: 10.1038/SREP23353

Abstract: The ability of cancer cells to sense external mechanical forces has emerged as a significant factor in the promotion of cancer invasion. Currently there are conflicting reports in the literature with regard to whether glioblastoma (GBM) brain cancer cell migration and invasion is rigidity-sensitive. In order to address this question we have compared the rigidity-response of primary patient-derived GBM lines. Cells were plated on polyacrylamide gels of defined rigidity that reflect the ersity of the brain tissue mechanical environment and cell morphology and migration were analysed by time-lapse microscopy. Invasiveness was assessed in multicellular spheroids embedded in 3D matrigel cultures. Our data reveal a range of rigidity-dependent responses between the patient-derived cell lines, from reduced migration on the most compliant tissue stiffness to those that are insensitive to substrate rigidity and are equally migratory irrespective of the underlying substrate stiffness. Notably, the rigidity-insensitive GBM cells show the greatest invasive capacity in soft 3D matrigel cultures. Collectively our data confirm both rigidity-dependent and independent behaviour in primary GBM patient-derived cells.

Fabrication of nanopatterned, porous microspheres using a glass capillary microfluidic device

Publisher: Royal Society of Chemistry (RSC)

Date: 2012

DOI: 10.1039/C2SM25950H

Metabolic profiling and flux analysis of MEL-2 human embryonic stem cells during exponential growth at physiological and atmospheric oxygen concentrations.

Publisher: Public Library of Science (PLoS)

Date: 20-11-2014

DOI: 10.1371/JOURNAL.PONE.0112757

Development of Defined Culture Conditions for Expansion of Human Mesenchymal Stromal Cells for Clinical Applications

Publisher: Springer Netherlands

Date: 2012

DOI: 10.1007/978-94-007-4798-2_2

Microbioreactor Arrays for Full Factorial Screening of Exogenous and Paracrine Factors in Human Embryonic Stem Cell Differentiation

Publisher: Public Library of Science (PLoS)

Date: 26-12-2012

DOI: 10.1371/JOURNAL.PONE.0052405

Microbioreactor Array Screening of Wnt Modulators and Microenvironmental Factors in Osteogenic Differentiation of Mesenchymal Progenitor Cells

Publisher: Public Library of Science (PLoS)

Date: 23-12-2013

DOI: 10.1371/JOURNAL.PONE.0082931

Microfluidic Screening Reveals Heparan Sulfate Enhances Human Mesenchymal Stem Cell Growth by Modulating Fibroblast Growth Factor-2 Transport

Publisher: Oxford University Press (OUP)

Date: 16-02-2017

DOI: 10.1002/SCTM.16-0343

Abstract: Cost-effective expansion of human mesenchymal stem/stromal cells (hMSCs) remains a key challenge for their widespread clinical deployment. Fibroblast growth factor-2 (FGF-2) is a key hMSC mitogen often supplemented to increase hMSC growth rates. However, hMSCs also produce endogenous FGF-2, which critically interacts with cell surface heparan sulfate (HS). We assessed the interplay of FGF-2 with a heparan sulfate variant (HS8) engineered to bind FGF-2 and potentiate its activity. Bone marrow-derived hMSCs were screened in perfused microbioreactor arrays (MBAs), showing that HS8 (50 μg/ml) increased hMSC proliferation and cell number after 3 days, with an effect equivalent to FGF-2 (50 ng/ml). In combination, the effects of HS8 and FGF-2 were additive. Differential cell responses, from upstream to downstream culture chambers under constant flow of media in the MBA, provided insights into modulation of FGF-2 transport by HS8. HS8 treatment induced proliferation mainly in the downstream chambers, suggesting a requirement for endogenous FGF-2 accumulation, whereas responses to FGF-2 occurred primarily in the upstream chambers. Adding HS8 along with FGF-2, however, maximized the range of FGF-2 effectiveness. Measurements of FGF-2 in static cultures then revealed that this was because HS8 caused increased endogenous FGF-2 production and liberated FGF-2 from the cell surface into the supernatant. HS8 also sustained levels of supplemented FGF-2 available over 3 days. These results suggest HS8 enhances hMSC proliferation and expansion by leveraging endogenous FGF-2 production and maximizing the effect of supplemented FGF-2. This is an exciting strategy for cost-effective expansion of hMSCs.

Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB

Publisher: Springer Science and Business Media LLC

Date: 30-09-2016

DOI: 10.1038/NCOMMS12976

Abstract: Axonal retrograde transport of signalling endosomes from the nerve terminal to the soma underpins survival. As each signalling endosome carries a quantal amount of activated receptors, we hypothesized that it is the frequency of endosomes reaching the soma that determines the scale of the trophic signal. Here we show that upregulating synaptic activity markedly increased the flux of plasma membrane-derived retrograde endosomes (labelled using cholera toxin subunit-B: CTB) in hippoc al neurons cultured in microfluidic devices, and live Drosophila larval motor neurons. Electron and super-resolution microscopy analyses revealed that the fast-moving sub-diffraction-limited CTB carriers contained the TrkB neurotrophin receptor, transiently activated by synaptic activity in a BDNF-independent manner. Pharmacological and genetic inhibition of TrkB activation selectively prevented the coupling between synaptic activity and the retrograde flux of signalling endosomes. TrkB activity therefore controls the encoding of synaptic activity experienced by nerve terminals, digitalized as the flux of retrogradely transported signalling endosomes.

Derivation of mesenchymal stromal cells from canine induced pluripotent stem cells by inhibition of the tgfβ/activin signaling pathway

Publisher: Mary Ann Liebert Inc

Date: 15-12-2014

DOI: 10.1089/SCD.2013.0634

CD146-assisted isolation and multi-omics characterisation of human pluripotent stem cell derived cranial Schwann cell precursor-like cells.

Publisher: Research Square Platform LLC

Date: 19-09-2023

DOI: 10.21203/RS.3.RS-3324198/V1

Concise review: new frontiers in microRNA-based tissue regeneration

Publisher: Oxford University Press (OUP)

Date: 29-05-2014

DOI: 10.5966/SCTM.2014-0032

Abstract: In recent years, the role of miRNAs in post-transcriptional gene regulation has come to the fore with strong evidence to indicate an important role for microRNAs (miRNAs) in the regulation of a wide range of fundamental biological processes. Notably, this includes the regulation of both endogenous tissue repair mechanisms and the growth and differentiation of stem cells (both adult and pluripotent). As a result, manipulation of miRNA signaling holds great promise for regenerative medicine, which aims to harness either endogenous or implanted cells to promote tissue repair. However, to fully realize this potential, it will be necessary to combine advances in our biological understanding with new technologies that allow precise spatiotemporal modulation of specific miRNA candidates. In this review, we highlight the role of miRNAs in tissue regeneration, discuss key challenges in translating this knowledge to the clinic, and outline recent technological advances that aim to address these issues. By combining a comprehensive knowledge of miRNA biology with cutting-edge delivery technologies, it is clear that miRNAs hold significant promise for tissue regenerative therapies in the future.

A synthetic elastomer based on acrylated polypropylene glycol triol with tunable modulus for tissue engineering applications.

Publisher: Elsevier BV

Date: 11-2010

DOI: 10.1016/J.BIOMATERIALS.2010.07.007

Abstract: As strategies for manipulating cellular behaviour in vitro and in vivo become more sophisticated, synthetic biomaterial substrates capable of reproducing critical biochemical and biophysical properties (or cues) of tissue micro-environments will be required. Cytoskeletal tension has been shown to be highly deterministic of cell fate decisions, yet few synthetic biomaterials are capable of modulating cytoskeletal tension of adhered cells through variations in stiffness, at least in the ranges applicable to tissue properties (e.g., 1-100 kPa), whilst also possessing other required properties, such as biodegradability, biocompatibility and processability. In this paper we describe a non-cytotoxic polymer system based on acrylated polypropylene glycol triol (aPPGT). This new elastomer system has tunable elastic moduli, is degradable, can be easily surface modified and can be manufactured into porous three dimensional scaffolds or micropatterned substrates. We demonstrate that the PPGT substrates can modulate hMSC morphology, growth, and differentiation, and that they can produce similar outcomes as observed for a non-degradable polyacrylamide substrate, confirming their utility as a degradable elastomer for tissue engineering and other biomedical applications.

Poly(glycerol–sebacate) bioelastomers—kinetics of step‐growth reactions using Fourier Transform (FT)‐Raman spectroscopy

Publisher: Wiley

Date: 24-05-2012

DOI: 10.1002/APP.37719

The focal adhesion targeting domain of p130Cas confers a mechanosensing function

Publisher: The Company of Biologists

Date: 2017

DOI: 10.1242/JCS.192930

Abstract: The Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain we compared wildtype exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain is exchanged for the p130Cas FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower 2D migration. No differences were detected in cell stiffness measured with Atomic Force Microscopy (AFM) and cell adhesion forces measured with a magnetic tweezer device. Thus the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent tyrosine phosphorylation of the NEDD9 substrate domain. This in turn reduced post-translational cleavage of NEDD9 which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers mechanosensing function.

Mode of heparin attachment to nanocrystalline hydroxyapatite affects its interaction with bone morphogenetic protein-2

Publisher: American Vacuum Society

Date: 16-10-2015

DOI: 10.1116/1.4933109

Abstract: Heparin has a high affinity for bone morphogenetic protein-2 (BMP-2), which is a key growth factor in bone regeneration. The aim of this study was to investigate how the rate of release of BMP-2 was affected when adsorbed to nanosized hydroxyapatite (HAP) particles functionalized with heparin by different methods. Heparin was attached to the surface of HAP, either via adsorption or covalent coupling, via a 3-aminopropyltriethoxysilane (APTES) layer. The chemical composition of the particles was evaluated using X-ray photoelectron spectroscopy and elemental microanalysis, revealing that the heparin grafting densities achieved were dependent on the curing temperature used in the fabrication of APTES-modified HAP. Comparable amounts of heparin were attached via both covalent coupling and adsorption to the APTES-modified particles, but characterization of the particle surfaces by zeta potential and Brunauer–Emmett–Teller measurements indicated that the conformation of the heparin on the surface was dependent on the method of attachment, which in turn affected the stability of heparin on the surface. The release of BMP-2 from the particles after 7 days in phosphate-buffered saline found that 31% of the loaded BMP-2 was released from the APTES-modified particles with heparin covalently attached, compared to 16% from the APTES-modified particles with the heparin adsorbed. Moreover, when heparin was adsorbed onto pure HAP, it was found that the BMP-2 released after 7 days was 5% (similar to that from unmodified HAP). This illustrates that by altering the mode of attachment of heparin to HAP the release profile and total release of BMP-2 can be manipulated. Importantly, the BMP-2 released from all the heparin particle types was found by the SMAD 1/5/8 phosphorylation assay to be biologically active.

A spectroscopic study of polyelectrolyte solutions under shear

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1016/S0032-3861(01)00201-4

Single-Cell Transcriptome Profiling Reveals Multicellular Ecosystem of Nucleus Pulposus during Degeneration Progression.

Publisher: Wiley

Date: 26-11-2021

DOI: 10.1002/ADVS.202103631

Abstract: Although degeneration of the nucleus pulposus (NP) is a major contributor to intervertebral disc degeneration (IVDD) and low back pain, the underlying molecular complexity and cellular heterogeneity remain poorly understood. Here, a comprehensive single‐cell resolution transcript landscape of human NP is reported. Six novel human NP cells (NPCs) populations are identified by their distinct molecular signatures. The potential functional differences among NPC subpopulations are analyzed. Predictive transcripts, transcriptional factors, and signal pathways with respect to degeneration grades are explored. It is reported that fibroNPCs is the subpopulation for end‐stage degeneration. CD90+NPCs are observed to be progenitor cells in degenerative NP tissues. NP‐infiltrating immune cells comprise a previously unrecognized ersity of cell types, including granulocytic myeloid‐derived suppressor cells (G‐MDSCs). Integrin α M (CD11b) and oxidized low density lipoprotein receptor 1 (OLR1) as surface markers of NP‐derived G‐MDSCs are uncovered. The G‐MDSCs are found to be enriched in mildly degenerated (grade II and III) NP tissues compared to severely degenerated (grade IV and V) NP tissues. Their immunosuppressive function and alleviation effects on NPCs’ matrix degradation are revealed in vitro. Collectively, this study reveals the NPC‐type complexity and phenotypic characteristics in NP, thereby providing new insights and clues for IVDD treatment.

Spatial control of bone formation using a porous polymer scaffold co-delivering anabolic rhBMP-2 and anti-resorptive agents

Publisher: European Cells and Materials

Date: 31-01-2014

DOI: 10.22203/ECM.V027A08

Abstract: Current clinical delivery of recombinant human bone morphogenetic proteins (rhBMPs) utilises freeze-dried collagen. Despite effective new bone generation, rhBMP via collagen can be limited by significant complications due to inflammation and uncontrolled bone formation. This study aimed to produce an alternative rhBMP local delivery system to permit more controllable and superior rhBMP-induced bone formation. Cylindrical porous poly(lactic-co-glycolic acid) (PLGA) scaffolds were manufactured by thermally-induced phase separation. Scaffolds were encapsulated with anabolic rhBMP-2 (20 µg) ± anti-resorptive agents: zoledronic acid (5 µg ZA), ZA pre-adsorbed onto hydroxyapatite microparticles, (5 µg ZA/2% HA) or IkappaB kinase (IKK) inhibitor (10 µg PS-1145). Scaffolds were inserted in a 6-mm critical-sized femoral defect in Wistar rats, and compared against rhBMP-2 via collagen. The regenerate region was examined at 6 weeks by 3D microCT and descriptive histology. MicroCT and histology revealed rhBMP-induced bone was more restricted in the PLGA scaffolds than collagen scaffolds (-92.3% TV, p < 0.01). The regenerate formed by PLGA + rhBMP-2/ZA/HA showed comparable bone volume to rhBMP-2 via collagen, and bone mineral density was +9.1% higher (p < 0.01). Local adjunct ZA/HA or PS-1145 significantly enhanced PLGA + rhBMP-induced bone formation by +78.2% and +52.0%, respectively (p ≤ 0.01). Mechanistically, MG-63 human osteoblast-like cells showed cellular invasion and proliferation within PLGA scaffolds. In conclusion, PLGA scaffolds enabled superior spatial control of rhBMP-induced bone formation over clinically-used collagen. The PLGA scaffold has the potential to avoid uncontrollable bone formation-related safety issues and to customise bone shape by scaffold design. Moreover, local treatment with anti-resorptive agents incorporated within the scaffold further augmented rhBMP-induced bone formation.

Biomimetic cues from poly(lactic-: Co-glycolic acid)/hydroxyapatite nano-fibrous scaffolds drive osteogenic commitment in human mesenchymal stem cells in the absence of osteogenic factor supplements

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0BM00946F

Abstract: Culturing of hBMSCs on aligned electrospun nanocomposite fibers, composed of PLGA modified with rod-shape n-HA.

Deconstructing replicative senescence heterogeneity of human mesenchymal stem cells at single cell resolution reveals therapeutically targetable senescent cell sub-populations

Publisher: Cold Spring Harbor Laboratory

Date: 27-01-2022

DOI: 10.1101/2022.01.24.476823

Abstract: Cellular senescence is characterised by a state of permanent cell cycle arrest. It is accompanied by often variable release of the so-called senescence-associated secretory phenotype (SASP) factors, and occurs in response to a variety of triggers such as persistent DNA damage, telomere dysfunction, or oncogene activation. While cellular senescence is a recognised driver of organismal ageing, the extent of heterogeneity within and between different senescent cell populations remains largely unclear. Elucidating the drivers and extent of variability in cellular senescence states is important for discovering novel targeted seno-therapeutics and for overcoming cell expansion constraints in the cell therapy industry. Here we combine cell biological and single cell RNA-sequencing approaches to investigate heterogeneity of replicative senescence in human ESC-derived mesenchymal stem cells (esMSCs) as MSCs are the cell type of choice for the majority of current stem cell therapies and senescence of MSC is a recognized driver of organismal ageing. Our data identify three senescent subpopulations in the senescing esMSC population that differ in SASP, oncogene expression, and escape from senescence. Uncovering and defining this heterogeneity of senescence states in cultured human esMSCs allowed us to identify potential drug targets that may delay the emergence of senescent MSCs in vitro and perhaps in vivo in the future.

Modulation of Stem Cell Adhesion and Morphology via Facile Control over Surface Presentation of Cell Adhesion Molecules

Publisher: American Chemical Society (ACS)

Date: 03-12-2013

DOI: 10.1021/BM4012562

Abstract: To encourage cell adhesion on biomaterial surfaces in a more facile, safe, and low-cost fashion, we have demonstrated a noncovalent approach to spatially conjugate β-cyclodextrin (β-CD) modified peptide sequences onto self-assembled adamantane-terminated polystyrene-b-poly(ethylene oxide) (PS-PEO-Ada) films through inclusion complexing interactions between β-CDs and adamantane. By simply blending various ratios of unmodified PS-PEO with a newly synthesized PS-PEO-Ada, we produced PS polymer films that displayed well-organized adamantine-decorated cylindrical PEO domains with varying average interdomain spacings ranging from 29 to 47 nm. The presence of the adamantane moiety at the terminal end of the PEO chain permitted rapid, and importantly, oriented attachment of β-CD functionalized peptides onto these surfaces. This one-step process not only converted these proven nonadherent PS-PEO surfaces into adherent surfaces, but also permitted precisely controlled presentation and surface distribution of the conjugated peptides. The utility of these surfaces as cell culture substrates was confirmed with human mesenchymal stem cells (hMSCs). We observed that with increasing PS-PEO-Ada content in the PEO cylindrical domains, these novel polymer films displayed improved cell attachment and spreading, with notable differences in hMSC morphology. We further confirmed that this novel PS-PEO-Ada surface provides a flexible platform for facile conjugation of mixtures of β-CDs functionalized with different peptides, specifically RGD and IKVAV peptides. The cell adhesion and spreading assays on these surfaces indicated that the morphologies of hMSCs can be easily manipulated, while no significant changes in cell attachment were observed. The lock-and-key peptide conjugation technique presented in this work is applicable to any substrate that incorporates a moiety capable of forming inclusion complexes with α-, β-, and γ-CDs, providing a facile and flexible method by which to construct peptide-conjugated biomaterial substrates for a multitude of applications in fields ranging from cell bioprocessing and regenerative medicine to cell-based assays.

Conference Scene: A bright future for tissue engineering and regenerative medicine in the Asia Pacific region

Publisher: Future Medicine Ltd

Date: 03-2011

DOI: 10.2217/RME.11.7

Abstract: The Tissue Engineering and Regenerative Medicine International Society – Asia Pacific (TERMIS-AP) annual meeting was held in Sydney, Australia from 15–17 September 2010 and highlighted the latest developments in tissue engineering and regenerative medicine in the Asia–Pacific region. Several of the plenary lectures focused on the vascularization of tissue engineering constructs, an issue that is critical for the success of larger tissue engineered constructs and was central to the meeting overall. In addition, a wide range of research also presented developments in tissue engineering for a range of body tissues (including cardiac, neural, bone, cartilage, tendon, skeletal muscle and skin), as well as advances in technologies (high-throughput screening and microfluidics). Looking more broadly, the meeting incorporated developments covering the spectrum of fundamental research through to clinical studies, with discussions on how best to direct the scientific advances being made into realistic therapies that could be made widely available in the future. Overall, the meeting highlighted the promise of early strategies, which are now showing promising results in clinical trials, and the development of a strong foundation of research from which future therapies will no doubt be developed.

The fabrication and characterization of biodegradable HA/PHBV nanoparticle–polymer composite scaffolds

Publisher: Elsevier BV

Date: 09-2009

DOI: 10.1016/J.ACTBIO.2009.03.017

Abstract: This study reports the fabrication and characterization of nano-sized hydroxyapatite (HA) oly(hydroxyabutyrate-co-hydroxyvalerate) (PHBV) polymer composite scaffolds with high porosity and controlled pore architectures. These scaffolds were prepared using a modified thermally induced phase-separation technique. This investigation focuses on the effect of fabrication conditions on the overall pore architecture of the scaffolds and the dispersion of HA nanocrystals within the composite scaffolds. The morphologies, mechanical properties and in vitro bioactivity of the composite scaffolds were investigated. It was noted that the pore architectures could be manipulated by varying phase-separation parameters. The HA particles were dispersed in the pore walls of the scaffolds and were well bonded to the polymer. The introduction of HA greatly increased the stiffness and strength, and improved the in vitro bioactivity of the scaffolds. The results suggest these newly developed nano-HA/PHBV composite scaffolds may serve as an effective three-dimensional substrate in bone tissue engineering.

Development of an in-process UV-crosslinked, electrospun PCL/aPLA-co-TMC composite polymer for tubular tissue engineering applications

Publisher: Elsevier BV

Date: 05-2016

DOI: 10.1016/J.ACTBIO.2016.03.013

A double chamber rotating bioreactor for enhanced tubular tissue generation from human mesenchymal stem cells: a promising tool for vascular tissue regeneration

Publisher: Hindawi Limited

Date: 05-04-2017

DOI: 10.1002/TERM.2341

Abstract: Cardiovascular diseases represent a major global health burden, with high rates of mortality and morbidity. Autologous grafts are commonly used to replace damaged or failing blood vessels however, such approaches are h ered by the scarcity of suitable graft tissue, donor site morbidity and poor long-term stability. Tissue engineering has been investigated as a means by which exogenous vessel grafts can be produced, with varying levels of success to date, a result of mismatched mechanical properties of these vessel substitutes and inadequate ex vivo vessel tissue genesis. In this work, we describe the development of a novel multifunctional dual-phase (air/aqueous) bioreactor, designed to both rotate and perfuse small-diameter tubular scaffolds and encourage enhanced tissue genesis throughout such scaffolds. Within this novel dynamic culture system, an elastomeric nanofibrous, microporous composite tubular scaffold, composed of poly(caprolactone) and acrylated poly(lactide-co-trimethylene-carbonate) and with mechanical properties approaching those of native vessels, was seeded with human mesenchymal stem cells (hMSCs) and cultured for up to 14 days in inductive (smooth muscle) media. This scaffold/bioreactor combination provided a dynamic culture environment that enhanced (compared with static controls) scaffold colonization, cell growth, extracellular matrix deposition and in situ differentiation of the hMSCs into mature smooth muscle cells, representing a concrete step towards our goal of creating a mature ex vivo vascular tissue for implantation. Copyright © 2016 John Wiley & Sons, Ltd.

Quantitative real time sensing reveals enhanced sensitivity of polar dendrimer thin films for plastic explosive taggants

Publisher: Royal Society of Chemistry (RSC)

Date: 2015

DOI: 10.1039/C5TC01967B

Abstract: A method of introducing pulses of analyte vapours has been developed to study the interactions of nitro-containing analytes with fluorescent sensing films.

A Blank Slate? Layer-by-Layer Deposition of Hyaluronic Acid and Chitosan onto Various Surfaces

Publisher: American Chemical Society (ACS)

Date: 19-04-2006

DOI: 10.1021/BM060044L

Abstract: Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign-body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often-utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular-level interactions defined by the modified surface properties. A promising concept is the so-called "blank slate": essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein-resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM.

Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures

Publisher: SAGE Publications

Date: 2010

DOI: 10.3727/096368909X478560

Abstract: Chondrogenesis of mesenchymal stem cells (MSCs) is typically induced when they are condensed into a single aggregate and exposed to transforming growth factor-β (TGF-β). Hypoxia, like aggregation and TGF-β delivery, may be crucial for complete chondrogenesis. However, the pellet dimensions and associated self-induced oxygen gradients of current chondrogenic methods may limit the effectiveness of in vitro differentiation and subsequent therapeutic uses. Here we describe the use of embryoid body-forming technology to produce microscopic aggregates of human bone marrow MSCs (BM-MSCs) for chondrogenesis. The use of micropellets reduces the formation of gradients within the aggregates, resulting in a more homogeneous and controlled microenvironment. These micropellet cultures (~170 cells/micropellet) as well as conventional pellet cultures (~2 × 10 5 cells ellet) were chondrogenically induced under 20% and 2% oxygen environments for 14 days. Compared to conventional pellets under both environments, micropellets differentiated under 2% O 2 showed significantly increased sulfated glycosaminoglycan (sGAG) production and more homogeneous distribution of proteoglycans and collagen II. Aggrecan and collagen II gene expressions were increased in pellet cultures differentiated under 2% O 2 relative to 20% O 2 pellets but 2% O 2 micropellets showed even greater increases in these genes, as well as increased SOX9. These results suggest a more advanced stage of chondrogenesis in the micropellets accompanied by more homogeneous differentiation. Thus, we present a new method for enhancing MSC chondrogenesis that reveals a unique relationship between oxygen tension and aggregate size. The inherent advantages of chondrogenic micropellets over a single macroscopic aggregate should allow for easy integration with a variety of cartilage engineering strategies.

One-step homogeneous C-reactive protein assay for saliva

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1016/J.JIM.2011.07.013

Abstract: Cardiovascular disease is the leading cause of death in the world. Human C-reactive protein (CRP) has been used in the risk assessment of coronary events. Human saliva mirrors the body's health and well-being and is non-invasive, easy to collect and ideal for third world countries as well as for large patient screening. The aim was to establish a saliva CRP reference range and to demonstrate the clinical utility of salivary CRP levels in assessing the coronary events in a primary health care setting. We have used a homogeneous bead based assay to detect CRP levels in human saliva. We have developed a rapid 15 min (vs 90 min), sequential, one-step assay to detect CRP in saliva. Saliva was collected from healthy volunteers (n=55, ages 20-70 years) as well as from cardiac patients (n=28, ages 43-86 years). The assay incubation time was optimised from 90 min to 15 min and generated a positive correlation (n=29, range 10-2189 pg/mL, r2=0.94 Passing Bablok slope 0.885, Intercept 0, p>0.10), meaning we could decrease the incubation time and produce equivalent results with confidence. The mean CRP level in the saliva of healthy human volunteers was 285 pg/mL and in cardiac patients was 1680 pg/mL (p<0.01). Analysis of CRP concentrations in paired serum and saliva s les from cardiac patients gave a positive correlation (r2=0.84, p<0.001) and the salivary CRP concentration capable of distinguishing healthy from diseased patients. The results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events.

Non-fibrillar Components of Amyloid Deposits Mediate the Self-association and Tangling of Amyloid Fibrils

Publisher: Elsevier BV

Date: 05-2004

DOI: 10.1074/JBC.M314008200

Defined high protein content surfaces for stem cell culture

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1016/J.BIOMATERIALS.2010.03.015

Abstract: Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter, technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple, effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent (N-hydroxysulfosuccinimide/N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple, but important step results in remarkable protein deposition efficiencies often exceeding 50%, whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays, and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions, as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies.

Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration

Publisher: Elsevier BV

Date: 06-2011

DOI: 10.1016/J.ACTBIO.2011.02.036

Abstract: This study investigates the use of patterned collectors to increase the pore size of electrospun scaffolds for enhanced cell infiltration. The morphology of the patterned scaffolds was investigated by scanning electron microscopy, which showed that the collector pattern was accurately mimicked by the electrospun fibres. We observed an enlargement in the pore size and in the pore size distribution compared with conventional electrospinning. Mechanical testing revealed that the mechanical properties could be tailored, to some extent, according to the patterning and that the patterned scaffolds were softer than standard electrospun scaffolds. When NIH 3T3 fibroblasts were seeded onto patterned collectors improved cell infiltration was observed. Cells were able to penetrate up to 250 μm into the scaffolds, compared with 30 μm for the standard scaffolds. This increase in the depth of infiltration occurred as early as 24 h post-seeding and remained constant over 7 days.

Discovery Projects - Grant ID: DP0557990

Start Date: 2005

End Date: 12-2007

Amount: $460,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0562505

Start Date: 10-2005

End Date: 10-2008

Amount: $590,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0558016

Start Date: 2005

End Date: 06-2010

Amount: $965,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0882371

Start Date: 09-2009

End Date: 09-2012

Amount: $308,854.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0986619

Start Date: 2009

End Date: 04-2010

Amount: $140,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP210103827

Start Date: 07-2021

End Date: 06-2024

Amount: $472,248.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP160100707

Start Date: 06-2017

End Date: 12-2022

Amount: $412,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110104446

Start Date: 2011

End Date: 02-2014

Amount: $390,000.00

Funder: Australian Research Council

Linkage - CSIRO - Grant ID: LC0348317

Start Date: 10-2003

End Date: 10-2006

Amount: $193,035.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0209940

Start Date: 03-2004

End Date: 12-2004

Amount: $271,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0775592

Start Date: 02-2007

End Date: 02-2008

Amount: $388,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE180100200

Start Date: 01-2018

End Date: 12-2019

Amount: $270,427.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0346891

Start Date: 2002

End Date: 12-2003

Amount: $200,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354797

Start Date: 01-2004

End Date: 03-2005

Amount: $20,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP190101969

Start Date: 03-2019

End Date: 03-2022

Amount: $477,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1095429

Start Date: 2010

End Date: 12-2012

Amount: $400,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882357

Start Date: 2008

End Date: 01-2009

Amount: $500,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0776963

Start Date: 06-2008

End Date: 03-2011

Amount: $240,000.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC190100020

Start Date: 08-2020

End Date: 08-2025

Amount: $3,998,796.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0560716

Start Date: 05-2005

End Date: 12-2006

Amount: $864,610.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP140104217

Start Date: 2014

End Date: 12-2016

Amount: $365,000.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR1101002

Start Date: 07-2011

End Date: 12-2019

Amount: $21,000,000.00

Funder: Australian Research Council