ORCID Profile
0000-0002-3009-7881
Current Organisations
University of Adelaide
,
Royal Adelaide Hospital
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Publisher: Elsevier BV
Date: 09-2023
Publisher: Springer Science and Business Media LLC
Date: 24-02-2016
Publisher: Cold Spring Harbor Laboratory
Date: 13-09-2021
DOI: 10.1101/2021.09.13.459432
Abstract: Over-activity of transforming growth factor β1 (TGFβ1) in subchondral bone has a direct causal role in rodent models of knee osteoarthritis (OA), which can be blocked by TGFβ1 neutralisation. In this study, we investigated whether the spatially distributed level of active TGFβ1 in human subchondral bone associates with the characteristic structural, cellular and molecular parameters of human knee OA. Subchondral bone s les (35 OA arthroplasty patients, aged 69±9 years) were obtained from regions below either macroscopically present or denuded cartilage. Bone s les were processed to determine the concentration of active TGFβ1 (ELISA) and gene-specific mRNA expression (RT-PCR). Synchrotron micro-CT imaging was utilised to assess the bone microstructure, bone mineralization, the osteocyte lacunar network and bone matrix vascularity. Finally, s les were histologically examined for cartilage OARSI grading, quantification of tartrate resistant acid phosphatase positive cells and bone marrow micro-vasculature. Subchondral bone below severely degenerated/depleted cartilage, characterised by impaired bone matrix quality due to sclerotic microarchitecture, disorganised collagen, high heterogeneity of the mineral distribution, contained increased concentrations of active TGFβ1, compared to adjacent areas with more intact cartilage. In addition, increased levels of active TGFβ1 related directly to increased bone volume while increased OARSI grade associated directly with morphometric characteristics (size, shape and orientation) of osteocyte lacunae. These results indicate that increased active TGFβ1 associates spatially with impaired bone quality and the disease severity of human OA. This study therefore suggests that TGFβ1 could be a therapeutic target to prevent or reduce human disease progression.
Publisher: Wiley
Date: 06-2016
Abstract: Magnetic resonance imaging (MRI) is a non-invasive technique routinely used to investigate pathological changes in knee osteoarthritis (OA) patients. MRI uniquely reveals zones of the most severe change in the subchondral bone (SCB) in OA, called bone marrow lesions (BMLs). BMLs have diagnostic and prognostic significance in OA, but MRI does not provide a molecular understanding of BMLs. Multiple N-glycan structures have been observed to play a pivotal role in the OA disease process. We applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of N-glycans to formalin-fixed paraffin-embedded (FFPE) SCB tissue sections from patients with knee OA, and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was conducted on consecutive sections to structurally characterize and correlate with the N-glycans seen by MALDI-MSI. The application of this novel MALDI-MSI protocol has enabled the first steps to spatially investigate the N-glycome in the SCB of knee OA patients.
Publisher: Springer Science and Business Media LLC
Date: 11-10-2021
DOI: 10.1007/S12024-021-00428-3
Abstract: We report on the use of a DNA staining dye to locate and record nucleated osteocytes and other bone-related cells within sections of archived formalin-fixed and paraffin-embedded human tibia from which informative DNA profiles were obtained. Eleven of these archived tibia s les were sectioned at a thickness of 5 µm. Diamond™ Nucleic Acid Dye was applied to the sections and cells within the matrix of the bone fluoresced so that their location and number of cells could be photographed. DNA was isolated from these 11 s les using a standard extraction process and the yields were quantified by real-time PCR. Complete STR profiles were generated from ten bone extracts where low-level inhibition was recorded with an incomplete STR profile obtained from one s le with higher inhibition. The stained image of this s le showed that few cells were present. There was a significant relationship between the number of DD-stained cells and the number of alleles obtained (p < 0.05). Staining cells to determine the prevalence of bone cell nuclei allows a triage of s les prior to any subsequent DNA profiling.
Publisher: Cold Spring Harbor Laboratory
Date: 29-03-2022
DOI: 10.1101/2022.03.28.486155
Abstract: Hip osteoarthritis (HOA) is a degenerative joint disease that leads to the progressive destruction of subchondral bone and cartilage at the hip joint. Development of effective treatments for HOA remains an open problem, primarily due to the lack of knowledge of its pathogenesis and a typically late-stage diagnosis. We describe a novel network analysis methodology for micro-computed tomography (micro-CT) images of human trabecular bone. We explored differences between the trabecular bone microstructure of femoral heads with and without HOA. Large-scale automated extraction of the network formed by trabecular bone revealed significant network properties not previously reported for bone. Profound differences were discovered, particularly in the proximal third of the femoral head, where HOA networks demonstrated elevated numbers of edges, vertices and graph components. When further differentiating healthy joint and HOA networks, the latter showed fewer small-world network properties, due to decreased clustering coefficient and increased characteristic path length. Furthermore, we found that HOA networks had reduced length of edges, indicating the formation of compressed trabecular structures. In order to assess our network approach, we developed a deep learning model for classifying HOA and control cases, and we fed it with two separate inputs: ( i ) micro-CT images of the trabecular bone, and ( ii ) the network extracted from them. The model with plain micro-CT images achieves 74.63% overall accuracy while the trained model with extracted networks attains 96.47% accuracy. We anticipate our findings to be a starting point for a novel description of bone microstructure in HOA, by considering the phenomenon from a graph theory viewpoint.
Publisher: Elsevier BV
Date: 06-2020
Publisher: Oxford University Press (OUP)
Date: 11-2022
DOI: 10.1093/PNASNEXUS/PGAC258
Abstract: Hip osteoarthritis (HOA) is a degenerative joint disease that leads to the progressive destruction of subchondral bone and cartilage at the hip joint. Development of effective treatments for HOA remains an open problem, primarily due to the lack of knowledge of its pathogenesis and a typically late-stage diagnosis. We describe a novel network analysis methodology for microcomputed tomography (micro-CT) images of human trabecular bone. We explored differences between the trabecular bone microstructure of femoral heads with and without HOA. Large-scale automated extraction of the network formed by trabecular bone revealed significant network properties not previously reported for bone. Profound differences were discovered, particularly in the proximal third of the femoral head, where HOA networks demonstrated elevated numbers of edges, vertices, and graph components. When further differentiating healthy joint and HOA networks, the latter showed fewer small-world network properties, due to decreased clustering coefficient and increased characteristic path length. Furthermore, we found that HOA networks had reduced length of edges, indicating the formation of compressed trabecular structures. In order to assess our network approach, we developed a deep learning model for classifying HOA and control cases, and we fed it with two separate inputs: (i) micro-CT images of the trabecular bone, and (ii) the network extracted from them. The model with plain micro-CT images achieves 74.6% overall accuracy while the trained model with extracted networks attains 96.5% accuracy. We anticipate our findings to be a starting point for a novel description of bone microstructure in HOA, by considering the phenomenon from a graph theory viewpoint.
Publisher: Cold Spring Harbor Laboratory
Date: 20-01-2023
DOI: 10.1101/2023.01.18.524641
Abstract: It is unclear if different factors influence osteoarthritis (OA) progression and the changes characterising OA disease in hip and knee. We investigated the difference between hip OA and knee OA at the subchondral bone tissue and cellular level, relative to the degree of cartilage degeneration. Bone s les were collected from 11 patients (aged 70±8 years) undergoing knee arthroplasty and 8 patients (aged 64±12 years) undergoing hip arthroplasty surgery. Bone microstructure, osteocyte-lacunar network and bone matrix vascularity were evaluated using synchrotron micro-CT imaging. S les were additionally examined histologically to determine osteocyte density, viability, and connectivity. After adjustment for donor gender and age, associations between the extent of cartilage degeneration, bone volume fraction [8.7, 95% CI (3.4, 14.1)], trabecular number [1.5, 95% CI (0.8, 2.3)], osteocyte lacunar density [4714.9 95% CI (2079.1, 7350.6)] and trabecular separation [-0.06, 95% CI (0.01, 0.1)] were found in both knee and hip OA. When compared to knee OA, hip OA was characterised by higher trabecular thickness [0.006, 95% CI (-4, 0.01)], larger but less spheric osteocyte lacunae [47.3 95% CI (11.2, 83.4), -0.04 95% CI (-0.6, -0.01), respectively], lower vascular canal density [-22.8 95% CI (-35.4, -10.3)] lower osteocyte density [-84.9 95% CI (-102.4, -67.4)], and less senescent but more apoptotic osteocytes [-2.4 95% CI (-3.6, -1.2), 24.9 95% CI (17.7, 32.1)], respectively. Subchondral bone from hip OA and knee OA exhibits different characteristics at the tissue and cellular levels, suggesting different mechanisms of OA progression between the hip and knee joints.
Publisher: Springer Science and Business Media LLC
Date: 19-11-2022
DOI: 10.1007/S12024-022-00559-1
Abstract: We report on a process to record the presence and the location of osteocyte nuclei using two nucleic staining dyes, Diamond™ Nucleic Acids Dye (DD) and DAPI (4',6-diamidino-2-phenylindole). Knowledge of the presence and number of osteocytes is key to any success in subsequent DNA profiling. Osteocytes are most numerous cells and thus the main source of DNA in bone s les, which can be preserved for histological analyses. Archived s les are either fixed in formalin or preserved in ethanol prior to embedding in resin. These resin-embedded s les are potentially used as ante mortem reference s les. Cases of a missing person investigation are one ex le where this type of preserved reference material may be of value. When resin is required for s le preservation it represents a problem for subsequent DNA profiling, if needed as a reference s le in human identification. It is essential therefore to remove the resin prior to DNA analyses as resin is a known inhibitor of DNA profiling. Current methods of resin removal are lengthy and require toxic chemicals. This report describes a simplified process to remove resin and visualise the location of nucleated osteocytes. Eight sections of bone s les at 5-µm thickness were stained with DD and DAPI. A further three s les were processed using a formalin-fixed method and three additional s les treated following an ethanol-preserved method (11 s les for both the formalin-fixed and 11 for the ethanol-preserved with eight in common). The location and number of nuclei could be recorded clearly due to the fluorescence created by the dye binding to DNA. The number of stained nuclei correlated with the mass of DNA isolated from the sections (r = 0.873, p = 1.21 × 10
Publisher: Wiley
Date: 24-03-2022
Abstract: Bone cells are a suitable substrate for DNA analysis if required to identify the person from whom a s le was taken. Osteocytes, the most abundant cell type in bone, are embedded within mineralized bone matrix. To release DNA from osteocytes for subsequent analyses, either demineralization of the mineral matrix or an overnight incubation is routinely carried out. In this study, we report on a simplified and rapid approach to analyze preserved bone s les that omits this lengthy decalcification process. Nine tibial bone s les were processed to release matrix‐free bone cells after fragmentation without the use of liquid nitrogen. Cell morphology was assessed by microscopy at 220× magnification following staining with Diamond ™ Nucleic Acid Dye. Based on the presence of stained nuclei, s les were processed either using a DNA extraction process or by a semi‐direct PCR process. The analysis of the quantity and quality of DNA isolated by both methods was carried out by real‐time PCR and STR profiling to assess inhibition of PCR and DNA degradation. All s les resulted in informative STR profiles with minimal indication of inhibitors. These results demonstrate a potential approach of STR profiling from matrix‐free bone cells within 8 hours without decalcification and DNA extraction.
Publisher: Elsevier BV
Date: 11-2019
DOI: 10.1016/J.JOCA.2019.07.004
Abstract: The aim of this study was to investigate how bone microstructure within bone marrow lesions (BMLs) relates to the bone and cartilage across the whole human tibial plateau. Thirty-two tibial plateaus from patients with osteoarthritis (OA) at total knee arthroplasty and eleven age-matched non-OA controls, were scanned ex vivo by MRI to identify BMLs and by micro CT to quantitate the subchondral (plate and trabecular) bone microstructure. For cartilage evaluation, specimens were processed histologically. BMLs were detected in 75% of the OA s les (OA-BML), located predominantly in the anterior-medial (AM) region. In contrast to non-OA control and OA-no BML, in OA-BML differences in microstructure were significantly more evident between subregions. In OA-BML, the AM region contained the most prominent structural alterations. Between-group comparisons showed that the AM region of the OA-BML group had significantly higher histological degeneration (OARSI grade) (P < .0001, P < .05), thicker subchondral plate (P < .05, P < .05), trabeculae that are more anisotropic (P < .0001, P < .05), well connected (P < .05, P = n.s), and more plate-like (P < 0.05, P < 0.05), compared to controls and OA-no BML at this site. Compared to controls, OA-no BML had significantly higher OARSI grade (P < .0001), and lower trabecular number (P < .05). In established knee OA, both the extent of cartilage damage and microstructural degeneration of the subchondral bone were dependent on the presence of a BML. In OA-no BML, bone microstructural alterations are consistent with a bone attrition phase of the disease. Thus, the use of BMLs as MRI image-based biomarkers appear to inform on the degenerative state within the osteochondral unit.
Publisher: Elsevier BV
Date: 03-2018
DOI: 10.1016/J.BONE.2018.01.012
Abstract: Bone marrow lesions (BMLs) in the subchondral bone in osteoarthritis (OA) are suggested to be multifactorial, although the pathogenic mechanisms are unknown. Bone metabolism and cardiovascular risk factors associate with BML in epidemiologic studies. However, there are no studies at the tissue level investigating the relationship between these processes and BML. The aim of this study was to investigate the relationship between BMLs in the tibial plateau (TP) of knee OA and bone matrix microdamage, osteocyte density and vascular changes. TP were obtained from 73 patients at total knee replacement surgery and BMLs were identified ex vivo in TP tissue using MRI. Comparator 'No BML' tissue was from matched anatomical sites to the BMLs. Quantitative assessment was made of subchondral bone microdamage, bone resorption indices, osteocyte cellularity, and vascular features. Several key parameters were different between BML and No BML tissue. These included increased microcrack burden (p = .01, p = .0001), which associated positively with bone resorption and negatively with cartilage volume, and greater osteocyte numerical density (p = .02, p = .01), in the subchondral bone plate and subchondral trabeculae, respectively. The marrow tissue within BML zones contained increased arteriolar density (p = .04, p = .0006), and altered vascular characteristics, in particular increased wall thickness (p = .007) and wall:lumen ratio (wall thickness over internal lumen area) (p = .001), compared with No BML bone. Increased bone matrix microdamage and altered vasculature in the subchondral bone of BMLs is consistent with overloading and vascular contributions to the formation of these lesions. Given the important role of BMLs in knee OA, these contributing factors offer potential targets for the treatment and prevention of knee OA.
Publisher: Elsevier BV
Date: 06-2022
DOI: 10.1016/J.JOCA.2022.03.004
Abstract: The association between the spatially distributed level of active TGFβ1 in human subchondral bone, and the characteristic structural and cellular parameters of human knee OA, was assessed. Paired subchondral bone s les from 35 OA arthroplasty patients, (15 men and 20 women, aged 69 ± 9 years) were obtained from beneath macroscopically present (CA+) or denuded cartilage (CA-) to determine the concentration of active TGFβ1 (ELISA) and its relationship to bone quality (synchrotron micro-CT), cellularity, and vascularization (histology). Bone s les beneath (CA-) regions had significantly increased concentrations of active TGFβ1 protein (mean difference: 26.4 95% CI: [3.2, 49.7]), when compared to bone in CA + regions. Trabecular Bone below (CA-) regions had increased bone volume (median difference: 4.3 96.49% CI: [-1.7, 17.8]), increased trabecular number (1.5 [0.006, 2.6], decreased trabecular separation (-0.05 [-0.1,-0.005]), and increased bone mineral density (394.5 [65.7, 723.3]) comparing to (CA+) regions. Further, (CA-) bone regions showed increased osteocyte density (0.012 [0.006, 0.018]), with larger osteocyte lacunae (39.8 [7.8, 71.7]) that were less spherical (-0.02 [-0.04, -0.003]), and increased bone matrix vascularity (12.4 [0.3, 24.5]) compared to (CA+). In addition, increased levels of active TGFβ1 related to increased bone volume (0.04 [-0.11, 0.9]), while increased OARSI grade associated with lacunar volume (-44.1 [-71.1, -17.2]), and orientation (2.7 [0.8, 4.6]). Increased concentration of active TGFβ1 in the subchondral bone of human knee OA associates spatially with impaired bone quality and disease severity, suggesting that TGFβ1 is a potential therapeutic target to prevent or reduce human OA disease progression.
Publisher: Elsevier BV
Date: 08-2021
No related grants have been discovered for Dzenita Muratovic.