Molecular and functional characterisation of tissue-engineered skeletal muscle constructs.
Lead Research Organisation:
University College London
Department Name: UNLISTED
Abstract
Diseases that affect skeletal muscle are devastating. Such conditions include children who are born with diseases such as Duchenne muscular dystrophy and those born with a blind-ending food-pipe who are unable to feed properly. Adults may also succumb to the effects of skeletal muscle loss (following conditions such as trauma, or cancer that may, for example, result in amputation or require an operation to remove a cancerous food-pipe) with equally devastating functional effects.
Tissue-engineering aims to grow tissues in the laboratory by seeding stem cells on to a scaffold. These tissues can then be used to replace diseased ones. Often, the patient‘s own stem cells are used, so the tissue is not rejected by the immune system.
Our aim is to tissue-engineer skeletal muscle by growing immature muscle cells in the laboratory and seeding them on to a scaffold. We have had encouraging results with tissue-engineering human wind-pipes in adults and a child. Our research will aim to tissue-engineer a food-pipe that contains skeletal muscle in its upper portion.
Important extensions of the project include the development of methods which might help treat diseases of other hollow organs that possess muscle, including diseases of the bowel, bladder and larynx.
Tissue-engineering aims to grow tissues in the laboratory by seeding stem cells on to a scaffold. These tissues can then be used to replace diseased ones. Often, the patient‘s own stem cells are used, so the tissue is not rejected by the immune system.
Our aim is to tissue-engineer skeletal muscle by growing immature muscle cells in the laboratory and seeding them on to a scaffold. We have had encouraging results with tissue-engineering human wind-pipes in adults and a child. Our research will aim to tissue-engineer a food-pipe that contains skeletal muscle in its upper portion.
Important extensions of the project include the development of methods which might help treat diseases of other hollow organs that possess muscle, including diseases of the bowel, bladder and larynx.
Technical Summary
Aims
The over-riding goal of this proposal is to develop an in vitro tissue-engineered skeletal muscle replacement.
Objectives
1. Expansion and characterisation of skeletal muscle precursor cells in tissue culture.
2. Functional assessment of cells.
3. Preparation and characterisation of a decellularised scaffold.
4. Seeding of cells to the scaffold.
5. Functional status of the tissue-engineered constructs in vitro.
6. In vivo studies.
Design and Methodology
Expansion and characterisation of skeletal muscle precursor cells in tissue culture
Rodent skeletal muscle precursor cells will be isolated, expanded in cell culture and characterised using immunofluoresence and semi-quantitative RT-PCR for known myocyte markers. Cells will be compared to differentiated adult myocytes and C2C12 myoblasts. Use of GFP mice will enable cells to be tracked.
Functional assessment of cells
Contractile status of the cells will be measured with a collagen lattice contraction assay and time-lapse microscopy.
Preparation and characterisation of a decellularised scaffold
Rat oesophagus will be decellularised using detergent-enzymatic treatments (sodium deoxycholate and DNase) and the scaffold characterised. The decellularised scaffold will be assessed with histology, immunohistology (for muscle, connective tissue and HLA-components), DNA and collagen assays, western blotting.
Tensile stress studies will be conducted and its mechanical properties will be compared with control oesophagus (stress/strain relationship). The optimal number of cycles of detergent-enzymatic treatments will be determined (trade-off between loss of immunogenicity with increasing cycles vs. loss of mechanical and structural properties).
Seeding of cells to the scaffold
Cells will be seeded to the decellularised scaffold with a rotating bioreactor. Seeded constructs will be rigorously assessed with histology, immunohistology, scanning electron microscopy.
Functional status of the tissue-engineered constructs in vitro
Physiological organ bath studies evaluating the contraction of muscle tissue upon pharmacological or electrical stimulation. Tissue-engineered constructs will be compared with control rat oesophagus.
In vivo studies
A further research proposal is envisaged to continue the experimentation in vivo, implanting constructs into rodents.
Scientific and medical opportunities
Tissue-engineered solutions have the major advantage of not requiring immunosuppression, but are presently limited to static organs and tissues, such as skin and trachea, or those which can function through passive movement alone, such as heart valves, blood vessels and bladder. The ability to produce functioning muscles would hugely extend the possible applications of regenerative medicine.
The over-riding goal of this proposal is to develop an in vitro tissue-engineered skeletal muscle replacement.
Objectives
1. Expansion and characterisation of skeletal muscle precursor cells in tissue culture.
2. Functional assessment of cells.
3. Preparation and characterisation of a decellularised scaffold.
4. Seeding of cells to the scaffold.
5. Functional status of the tissue-engineered constructs in vitro.
6. In vivo studies.
Design and Methodology
Expansion and characterisation of skeletal muscle precursor cells in tissue culture
Rodent skeletal muscle precursor cells will be isolated, expanded in cell culture and characterised using immunofluoresence and semi-quantitative RT-PCR for known myocyte markers. Cells will be compared to differentiated adult myocytes and C2C12 myoblasts. Use of GFP mice will enable cells to be tracked.
Functional assessment of cells
Contractile status of the cells will be measured with a collagen lattice contraction assay and time-lapse microscopy.
Preparation and characterisation of a decellularised scaffold
Rat oesophagus will be decellularised using detergent-enzymatic treatments (sodium deoxycholate and DNase) and the scaffold characterised. The decellularised scaffold will be assessed with histology, immunohistology (for muscle, connective tissue and HLA-components), DNA and collagen assays, western blotting.
Tensile stress studies will be conducted and its mechanical properties will be compared with control oesophagus (stress/strain relationship). The optimal number of cycles of detergent-enzymatic treatments will be determined (trade-off between loss of immunogenicity with increasing cycles vs. loss of mechanical and structural properties).
Seeding of cells to the scaffold
Cells will be seeded to the decellularised scaffold with a rotating bioreactor. Seeded constructs will be rigorously assessed with histology, immunohistology, scanning electron microscopy.
Functional status of the tissue-engineered constructs in vitro
Physiological organ bath studies evaluating the contraction of muscle tissue upon pharmacological or electrical stimulation. Tissue-engineered constructs will be compared with control rat oesophagus.
In vivo studies
A further research proposal is envisaged to continue the experimentation in vivo, implanting constructs into rodents.
Scientific and medical opportunities
Tissue-engineered solutions have the major advantage of not requiring immunosuppression, but are presently limited to static organs and tissues, such as skin and trachea, or those which can function through passive movement alone, such as heart valves, blood vessels and bladder. The ability to produce functioning muscles would hugely extend the possible applications of regenerative medicine.