Cell-based Tissue Engineering For Meniscus Repair

The menisci are two pieces of semilunar fibrocartilage between femoral condyle and tibia plateau and aid in shock absorption,load distribution,joint lubrication and stability.Meniscus injury is the most common problem seen in orthopaedic clinics.Meniscus injuries mainly include degenerative tears and traumatic tears.Degenerative meniscal tears are most common findings in middle-aged group,which was considered as a sign of early-stage OA.Recent research has shown that partial meniscectomy has no superiority over non-surgical treatment for degenerative tears.However,the histological features of degenerative meniscus and the change of degenerative meniscal cells phenotype,as well as their regenerative capacity are still not clear.In traumatic tears,longitudinal vertical tears in red zone in young adult patients are proper indications for meniscus repair.Irreparable tears were commonly treated with partial meniscectomy,but the long-term results are not satisfied.For patients develop knee pain several years after APM,meniscal allograft transplantation(MAT)or partial meniscus synthetic scaffold replacement could be compromise options.However,both MAT and meniscal scaffolds offer disadvantages including meniscal extrusion,graft shrinkage,relative high reoperation and failure rates.Nevertheless,the APM rate remains too high even though the robust scientific evidence indicates the value of meniscal preservation over the years.These weaknesses have shifted the research interest towards to novel cell-based tissue engineering approaches for meniscal repair.Part 1: Characterisation of Regional Meniscal Cell Phenotypes and Histological Analysis in Osteoarthritic Donor-Matched TissuesObjective: To investigate the histological features of degenerative meniscus and characterise avascular and vascular meniscal cell phenotypes and their chondrogenic capacity in vitro.Methods: Donor-matched avascular(Avas)and vascular(Vas)meniscal cells(MC)and chondrocytes(Ch)were isolated from the menisci and cartilage of TKR patients(n=10).Meniscus tissue were also sectioned for histological analysis.Cell population doubling time was used to compare their cell proliferation rate.To compare the three cell phenotypes,flow cytometry was used to assess the immunopositivity for cell surface molecules at P0 and P2;PCR was used to evaluate the chondrogenic genes expression level cells.Donor matched Avas,Vas and Ch fractions were assessed for their chondrogenic potential using an established 3D pellet culture system.Results: The histology of degenerative meniscus showed the decreased vascularity in the “tree-like” collagen fibres.Vas showed a significantly higher proliferation rate compared to Avas and Chondrocytes in primary culture.Flow cytometry showed that a significantly greater percentage of Avas were positive for CD49 b and CD49 c compared to Vas and chondrocytes in P0 and P2.The chondrogenic potency analysis showed,chondrocytes produced highest level of GAG.The collagen type I gene expression level of Vas was significantly higher than Avas and Chondrocytes,which matched the immunohistochemistry staining results that Vas pellets had the highest collagen type I staining intensity.Conclusion: We demonstrated the cell surface markers CD49 b CD49c are valuable to distinguish avascular and vascular meniscus cell phenotypes from degenerative meniscus.The “tree-like” collagen fibres from parameniscal tissue was likely to be involved in the meniscus pathological process.Part 2: Phenotypic Characterisation of Regional Human Meniscus Progenitor CellsObjective: To isolate and characterise the human meniscal progenitor cells from avascular and vascular region.Methods: Herein,we extracted the progenitor cell populations from avascular and vascular region of human meniscus and donor-matched cartilage tissue by fibronectin adhesion assay(n=5),as well as their whole mixed population from each region as a comparison.Cell-IQ? was used to measure the colonies formation of progenitor cells.We characterised and compared the cell surface markers by growth kinetics,flow cytometry(P0),gene expression profiles(P2)and 3D pellets chondrogenic capacity in vitro between progenitor and mixed population groups.Results: Avascular progenitors,vascular progenitors and chondroprogenitors all showed colony forming capacity on monolayer culture,whereas their mixed populations were distributed randomly.PVas had a significant lower population doubling times compare to vascular mixed population.PVas also proliferated faster than PAvas and PChs based on colony forming assay.Progenitor cells showed significantly higher positivity for CD49 b and CD49 c compared with their mixed population.Collagen type X was significantly downregulated in progenitor pellets.GAG/DNA analysis demonstrated that progenitor cells generally produced more GAG than mixed population cells in all three cell types.The pellets histology staining showed progenitors formed better chondrogenic pellets.Conclusion: This study demonstrates that the human meniscus contains meniscal progenitor populations that retains stromal cell-like phenotypes in both the avascular and vascular regions.Meniscal progenitors derived from the vascular region exhibit stromal cell characteristics which likely associate with the better meniscal healing potential in the vascular region.Part 3: The Influence of Fibrin Gel on the Delivery of Meniscal Cells in Synthetic Scaffold in Ovine Meniscus Explant.Objective: To investigate the feasibility of using fibrin gel to deliver autologous meniscal cells in a polyurethane scaffold for meniscus regeneration.Methods: Medial and Lateral meniscus were harvested from 5 welsh sheep.Medial menisci were used to isolate avascular meniscal cells,while lateral menisci were created with punch defects(3mm).The same size cylinder polyurethane scaffolds were inserted into defects.The cultured avascular meniscal cells were seeded into scaffold with or without fibrin gel.The experiment was set in three groups:(i)negative controls,(ii)cells and(iii)fibrin gel with cells,and the sheep explants were cultured in vitro for 28 days.After that,GAGs and DNA production and histological staining were tested to compare the matrix formation between groups.Results: The live/dead staining showed that fibrin gel group had a higher cell density and viability compare to cell only group and scaffold group.The GAG and DNA analysis both showed cells delivered by fibrin gel had a higher production compare to cells only group.The histology evaluation of collagen type I further confirmed more collagen formation in fibrin gel group.Conclusion: We proved the feasibility of using fibrin gel to deliver autologous avascular meniscal cells in a clinical grade meniscus substitute in vitro,which built the fundamental base to achieve one-step cell-based meniscus tissue engineering.Cells delivered by fibrin gel showed an increased matrix production within scaffold compared to cells directly seeded into scaffold.Part 4: Developing a Collagen/Alginate based IPN Hydrogel and its Biological Application.Objective: To investigate the substitute cells carrier using collagen and alginate hydrogel.Methods: Collagen was mixed with 10mg/ml,20 mg/ml,30 mg/ml alginate solution to make the hydrogel scaffolds to test their biomechanical properties.Rabbit meniscal cells were then seeded onto theses scaffolds for in vitro culture.Live/dead staining,cells proliferation assay and PCR were used to test the cells biocompatibility in different concentration alginate scaffolds.Results: Infrared and circular dichroic spectroscopy analysis showed alginate and collagen did not interact with each other,but still retained their own biological features.The elasticity modulus of scaffold increased with the rise of alginate concentration.However,the hybrid scaffold with 20mg/ml alginate showed the best cell morphology and cell density.In addition,the gene expression levels of aggrecan,SOX-9 and collagen type II were also highest in 20mg/ml.But no significant difference was found in cell proliferation rate between different alginate concentration scaffolds.Conclusion: We developed a novel alginate/collagen interpenetrating polymer network hydrogel as a cell carrier which aids to cell proliferation and extracellular matrix formation.Our results showed the hybrid scaffold retained the biochemical features of each component and the mechanical property of hydrogel scaffold was positively correlated with alginate concentration,while 20mg/ml alginate maintained the best cell morphology and facilitated a higher gene expression.In conclusion,we presented a comprehensive investigation in meniscus-related research,including meniscus degeneration,meniscal progenitors and cell-based meniscus tissue engineering.This study filled in the gaps of current meniscal biology and pathology,as well as providing a novel cell-based meniscus tissue regeneration strategy as a future treatment option in the clinic.