THE ROLE OF RESIDENT MAST CELLS IN ISCHAEMIA-REPERFUSION INJURY OF SKELETAL MUSCLE.
Funder
National Health and Medical Research Council
Funding Amount
$226,320.00
Summary
NHMRC 209113 LAY DESCRIPTION Ischaemia reperfusion injury occurs in skeletal muscle when the blood-oxygen supply is cut off (ischaemia) and later restored (reperfusion). If the duration of ischaemia is short some of the muscle survives. However, when blood flow and oxygen are restored the muscle is subjected to more injury, which is thought to be caused by oxygen and-or white blood cells. This type of injury occurs in muscle which has been crushed, limbs that have been broken or traumatized, in ....NHMRC 209113 LAY DESCRIPTION Ischaemia reperfusion injury occurs in skeletal muscle when the blood-oxygen supply is cut off (ischaemia) and later restored (reperfusion). If the duration of ischaemia is short some of the muscle survives. However, when blood flow and oxygen are restored the muscle is subjected to more injury, which is thought to be caused by oxygen and-or white blood cells. This type of injury occurs in muscle which has been crushed, limbs that have been broken or traumatized, in replantation of amputated parts, in transplantation, after some surgical procedures and after microsurgical transfer of muscle. Once established there is no effective treatment. Our experiments show that a particular cell, the mast cell, and a particular molecule, nitric oxide, are involved in causing ischaemia reperfusion injury. However, the extent of their involvement is unknown. In this proposal we will investigate the effect of replacing mast cells into muscles, in a unique variety of mice which normally don t contain mast cells and are resistant to ischaemia reperfusion injury. In one group of mice we will put back normal mast cells and in a second group of mice we will put back mast cells that cannot produce the nitric oxide molecule. These experiments will determine, unambiguously, the extent of involvement of mast cells and mast cell-derived nitric oxide. In the second part of this proposal will carry out a time course study, using pharmacologically induced mast cell degranulation, to determine when the mast cells become injurious to skeletal muscle. These experiments will identify the period during which mast cell behaviour can be modulated in order to protect the muscle from ischaemia reperfusion injury. Determination of the role of mast cells, and an understanding of the timing during which they become injurious would provide a logical basis for optimizing drug therapy in clinical applications of these findings.Read moreRead less
Three Dimensional Ex Vivo Modelling Of Neuromuscular Junction Formation
Funder
National Health and Medical Research Council
Funding Amount
$120,253.00
Summary
Re-establishing functional connections between neurons and muscle is an important step in the recovery process after neuromuscular injury or surgery. In order to study the connection forming process in isolation a biological model of nerve muscle connection formation is required. This study aims to buid a biological model consisting of neurons and muscles in a three dimensional environment and to assess the quality of the functional connections that develop.
GENERATION OF VASCULARISED, BIOENGINEERED SOFT TISSUES
Funder
National Health and Medical Research Council
Funding Amount
$445,045.00
Summary
One of the most exciting areas in reconstructive surgery today is the tissue engineering of body parts, the process by which blood vessels are implanted into skin, muscle, bone, cartilage or even synthetic materials, to build composite living structures. Once a circulation becomes established, the engineered part can be transferred by joining the implanted blood vessels to corresponding ones at the recipient site. We have discovered that new tissue will grow out of a surgically created blood ves ....One of the most exciting areas in reconstructive surgery today is the tissue engineering of body parts, the process by which blood vessels are implanted into skin, muscle, bone, cartilage or even synthetic materials, to build composite living structures. Once a circulation becomes established, the engineered part can be transferred by joining the implanted blood vessels to corresponding ones at the recipient site. We have discovered that new tissue will grow out of a surgically created blood vessel loop placed in a cylindrical plastic chamber filled with a scaffold made of naturally occurring structural molecules. In Part 1 of this project, it is planned to optimise the rate of new vascularised tissue growth through the addition to the growth chamber of various biodegradable scaffolds. In Part 2, we aim to produce fat by 3 possible methods using: (a) cells from the rat scrotum, (b) skeletal muscle separated from its blood supply for 24 hours, or (c) bone marrow-derived stem cells, as well as bone from stem cells of the same source. In Part 3, vascularised bone, fat and connective tissue, as produced in Part 2, will be microsurgically transferred to another site in the body to study the short-term (4 weeks) and long-term (12 weeks) survival and changes (if any) in these tissues. These unique methods are currently being patented. This technology introduces the possibility of producing tailor-made tissues of specific composition to suit the repair of a particular tissue type, for example, (1) myocutaneous flaps to replace tissue loss following traumatic injury, (2) bone for nose, digit or joint repair, and (3) fat to provide a bulky flap as required in contour defects of the face and neck. The development of new growth chambers of appropriate body shapes (eg. ears, noses, etc) has significant commercial implications.Read moreRead less
Optimizing Implanted Cell Survival Using A Tissue Engineering Model
Funder
National Health and Medical Research Council
Funding Amount
$589,175.00
Summary
Cell therapy and tissue engineering involve the insertion of specific cells into damaged tissues or into a bioraector in a patient's body to generate new replacement tissues. This project seeks to improve two factors associated with inserting cells : 1. The innate survival characteristics of the cells being inserted, and 2. The blood vessel supply at the site of insertion. These techniques will greatly improve the survival of inserted cells.
Engineering Tissues And Organs In Vivo From Stem Cells
Funder
National Health and Medical Research Council
Funding Amount
$549,480.00
Summary
Tissue engineering is an exciting new area of medical research. We have developed a unique animal model of tissue engineering where new tissue spontaneously sprouts from the surface of a vascular loop enclosed inside a plastic chamber. The tissue thus created has its own blood supply. By adding cultured cells or altering the environment of the chamber we have been able to grow new specific tissues such as fat and muscle. This technology potentially allows the generation of spare body parts to re ....Tissue engineering is an exciting new area of medical research. We have developed a unique animal model of tissue engineering where new tissue spontaneously sprouts from the surface of a vascular loop enclosed inside a plastic chamber. The tissue thus created has its own blood supply. By adding cultured cells or altering the environment of the chamber we have been able to grow new specific tissues such as fat and muscle. This technology potentially allows the generation of spare body parts to replace lost or worn out organs and tissues. We have recently reproduced this model in the mouse to be able to screen a range of mouse and human stem cells. These cells have the ability to change (i.e. differentiate) into many different types of cell depending on how they are stimulated. In Part 1 of this project we will determine in the mouse chamber the growth characteristics and survival rates of these stem cells. A chamber encapsulating a flowing blood vessel will be implanted subcutaneously in each groin. In one chamber we will inject fluorescently labelled stem cells in a growth medium and in the other growth medium alone. Tissue will be analysed at 1, 2 and 4 weeks. In Part 2 we will inject a variety of Rosa26 labelled mouse stem cells obtained from several different tissues. Through the aid of naturally occurring growth and differentiation factors they will differentiate into one of several different tissues including fat, cartilage, bone, neural tissue, blood vessels, liver, etc, which will be identified by histology and cell culture. In one experiment we will genetically alter cells injected into the chamber so that they produce only skeletal muscle. In Part 3 we will grow new human tissues by injecting human stem cells into the same tissue engineering chambers in mice which will tolerate cells from other mammals (these are known as SCID mice). Success in novel method would be the precursor for the production of new human tissues to repair specific defects.Read moreRead less
In Vivo Tissue Engineering Of Adipose Tissue For Reconstructive Surgery
Funder
National Health and Medical Research Council
Funding Amount
$713,545.00
Summary
We are able to grow vascularised tissue in implanted plastic chambers to a predetermined size and shape in the rat and mouse (NHMRC Project Grant 01-03; #145782; CIA Morrison). The basis of this growth is blood vessel sprouting from the surface of the vessel bundle or loop, followed by synthesis of structural molecules and the migration of surrounding cells into the vascularised network to form a stable tissue. Unlike other in vivo models of tissue engineering, the tissue grows spontaneously and ....We are able to grow vascularised tissue in implanted plastic chambers to a predetermined size and shape in the rat and mouse (NHMRC Project Grant 01-03; #145782; CIA Morrison). The basis of this growth is blood vessel sprouting from the surface of the vessel bundle or loop, followed by synthesis of structural molecules and the migration of surrounding cells into the vascularised network to form a stable tissue. Unlike other in vivo models of tissue engineering, the tissue grows spontaneously and is densely vascularised, enabling continuous growth and surgically transfer to another part of the body, or to another animal. In this renewal application of the above NHMRC grant, we propose to direct these findings towards the development of vascularised fat tissue which would be ideal for reconstructive surgery as a stable, inert tissue filler. Our efforts to grow fat tissue in vivo to date have identified 4 major requirements: a fat precursor cell source; an instructive basement membrane matrix (which may include growth-differentiation factors); space into which the tissue can grow; a stable blood supply. We will focus here on optimising the precursor cell source and instructive matrix to generate vascularised fat tissue around the blood supply we can engender in our tissue engineering chamber. We have found Matrigel, a mouse tumor-derived matrix rich in basement membrane components, to be instructive for growing fat, and will also build on preliminary observations that either muscle or fat tissue can provide the appropriate precursor cells for this process. Finally we propose to adapt and upsize the vascularised fat tissue chamber to the pig, in a step towards human use, and assess its transplantability and longevity. The clinical application of our work is to produce breast reconstruction tissue and lipo filling for contour deformities resulting from trauma, congenital deformity, ageing and cancer surgery, particularly breast reconstruction.Read moreRead less
Optimising Islet Transplantation With Vascularized Tissue Engineering Chambers
Funder
National Health and Medical Research Council
Funding Amount
$451,651.00
Summary
Diabetics have high blood sugar levels because cells in the pancreas known as islets produce too little of the hormone insulin. Most diabetics need daily insulin injections to maintain normal blood sugar levels. Transplanting islets is the most promising way to treat type 1 diabetes, but, apart from the obvious difficulty of rejection of foreign islets, several major problems remain: (1) there are insufficient pancreata (and therefore islets) for transplantation; and (2) the efficiency of delive ....Diabetics have high blood sugar levels because cells in the pancreas known as islets produce too little of the hormone insulin. Most diabetics need daily insulin injections to maintain normal blood sugar levels. Transplanting islets is the most promising way to treat type 1 diabetes, but, apart from the obvious difficulty of rejection of foreign islets, several major problems remain: (1) there are insufficient pancreata (and therefore islets) for transplantation; and (2) the efficiency of delivery of surviving islet transplants is too low. In pilot studies we have grown a new living pancreatic organ in mice by inserting islets from genetically-related mice together with a structural protein matrix, growth factors and blood vessels inside a plastic chamber. The blood vessels maintain nutrition to the islet cells and simultaneously allow insulin to be released into the bloodstream, thus normalising the high blood sugar in diabetics. In Aim 1 of these experiments we will find the optimal way to grow mature islets in blood vessel-containing chambers in diabetic mice, focusing on (a) the best time to add islets to the chamber - 0, 1 or 2 weeks after establishment, (b) the minimum number of islets to effectively normalise blood sugar and (c) how long we can keep islets alive and functional in chambers, examining periods up to 12 months. In Aim 2 we will test the ability of islet stem cells (provided by our co-investigators at Walter and Eliza Hall Institute, Melbourne) to survive in the chambers and to produce sufficient insulin to effectively lower blood sugar levels to normal in diabetic mice. In Aim 3 we will grow human islets in chambers in special diabetic mice that do not reject foreign tissue, in order to confirm similar behaviour of human islets in this controlled environment. Using this data, we hope to create a research model of functioning islets, that is accessible, retrievable and manipulable, for the further study of diabetes and transplantation.Read moreRead less