Study On Repairing Articular Cartilage Defects With Acellular Derma Base Material Seeded With Autogenic Adipose-derived Mesenchymal Stem Cells

Hyaline articular cartilage, the load-bearing tissue of the joint, has very limited repair and regeneration capacities once injury occurrence because the extracellular matrix of articular cartilage is distinct from that of other connective tissues. Chondral defects cause pain and functional disability. The lack of efficient treatment modalities for large chondral defects has motivated attempts to engineer cartilage constructs by seeding cells, scaffold materials and environmental factors, including growth factors, signaling molecules, and physical influences. The seeding cell play a important role in cartilage tissue engineering.Adipose-derived mesenchymal stem cells (ADMSCs) as alternative cell source for tissue engineering have recently received widespread attention for its strong proliferation capacity and multilineage differentiation.Objective:1 To investigate the feasibility of isolation and cultivation of adipose-derived mesenchymal stem cells to identify and analyze the biological characteristics of rabbit ADMSCs, in order to provide experiment basis for optimizing and extending seeding cells resources in cartilage tissue engineering. 2 To repaire articular cartilage defects of rabbits by ADMSCs seeded into acellular derma matrix(ADM),we grade the repaired tissues and evaluate repairing effect of defects in order to provide experimental and theoretical basis for clinical application.Methods:Thirty-six Newzealand rabbits were chosen in this experiment. Adipose tissue from rabbit was minced and digested using 0.1%collagenase typeⅠto isolate ADMSCs,which were collected by centrifugation and then incubated in vitro. The expressions of CD44 were assayed by flow cytometry and immunofluorescence technique.We observed morphology of cells with inverted microscopy. Growth curve of primary cells and the third passage ADMSCs was drawn by cytometry. It is demonstrated that multilineage differentiation potential of ADMSCs cultered in specific medium differentiated into adipocytes/ osteocytes and chondrocytes in vitro verified by specific staining. The ADMSCs cultured in DMEM supplemented with 0.10 volume fraction FBS , IBMX, 200μmol/L dexamethasone 1μmol/L indomethacin and 10μg/mL insulin for adipogenic induction.The ADMSCs differentiated into osteocytes were cultured in DMEM supplemented with 0.10 volume fraction FBS, 0.1μmol/L dexamethasone, 50μg/mL ascorbate and10 mmol/Lβ-glycerophosphate . Medium for the differentiations to chondrocytes used DMEM with 10 % FBS,10μg /L transforming growth factor-β1(TGF-β1), 50μg/mL ascorbate and 6.25 mg/L insulin .Multilineage differentiation potential were verified by histologic/immunohistochemical assay of Oil red staining for adipocytes , alkaline phosphatase (ALP) and alizarin red staining for osteocytes and immunocytochemical staining for chondrocytes.ADM were prepared by calf derma for cell carrier. Concentration of suspension of the the third passage of ADMSCs was 5×105/ml.After the ADMSCs were seeded into ADM and cultured for 2 days in vitro, Scanning electron microscopy were carried out on the second day.ADMSCs/ADM were transplantated into defects of the cartilages at intercondylar fossa.Thirty-six healthy Newzealand rabbits were divided into three groups randomly. There were 12 rabbits in each group. The cartilage defects in the intercondylar fossa were filled with ADMSCs/ADM in experimental group(ADMSCs/ADM group),with only ADM in negative control group(ADM control group),and with nothing in blank control group (blank control group). Four rabbits were killed at 6weeks, 9 weeks and 12 weeks after transplantation in each group, repaired tissues in the zones of articular cartilage defects were observed with macroscopic views, histological/histopathologic scores and immunohistochemistrical stains. The data were input SPSS 16.0 software for statistical analysis.Results: 1 ADMSCs were adhered at 24 hours,and shaped with spindle and polygon after separating.Primary ADMSCs proliferated swiftly and 80～90% of cells could coalite at 10-14 days.2 Surface antigen CD44 was positively expressed on ADMSCs by flow cytometry and immunofluorescence technique.3 The growth curve of ADMSCs was like"S"shape.Growth curve manifested that ADMSCs increased gradually at the 2nd d, reached the peak at the 7th d and begun to enter the platform at 9th d.4 Red stained fats characteristic of adipocytes by Oil Red stain in the ADMSCs differentiated into adipocytes group could be observed.Dense black precipitation by alkaline phosphatase (ALP) and calcium nodes by alizarin red staining of ADMSCs differentiated into osteocytes group is positive.There is detection of CollagenⅡby immunohistochemical method in the ADMSCs differentiated into chondrocytes group.5 Electron microscope showed the porous structure of ADM. The porosity was suitable for the implantation and growth of ADMSCs.6 At 9 weeks after transplantation,In the ADMSCs /ADM experimental group repaired tissues integrated with peripheral cartilages without visible ADM, but repaired tissues in ADM group and blank group showed fibrous repair or no repair .Histological grading scale of repaired tissues showed that ADMSCs /ADM experimental group excelled control group and blank control group, differences were statistically significant(p<0.05), but diffrences between ADM control group and blank control group were not significant(p>0.05); Histopathologic grading scale of repaired tissues indicated that ADMSCs /ADM group excelled ADM group and blank group, differences were significant(p<0.05),but diffrences between ADM control group and blank control group were not significant(p>0.05).7 In the ADMSCs /ADM experimental group repaired tissues represented hyaline-like, integrated with peripheral cartilages excellently, but repaired tissues in ADM group and blank group showed fibrous repair or no repair at 12 weeks after transplantation. Histological grading scale of repaired tissues showed that ADMSCs /ADM group excelled control group and blank control group, differences were statistically significant(p<0.05), and diffrences between ADM control group and blank control group were significant(p<0.05); Histopathologic grading scale of repaired tissues indicated that ADMSCs /ADM group excelled ADM group and blank group, differences were statistically significant(p<0.05),and diffrences between ADM control group and blank control group were statistically significant(p<0.05).8 Immunohistochemistrical stains of repaired tissues showed that chondrocytes in the zones of repaired tissues riched in type-II collagen matrix and integrated with adjacent cartilages satisfactorily in the ADMSCs /ADM experimental group.Conclusion:1 The ADMSCs of rabbits isolated in this trial are characterized by stable growth and quick proliferation in vitro.These cells can differentiate into adipocytes / osteocytes and chondrocyte in induction mediums. Multi-directional differentiation demonstrates that these original cells are mesenchymal stem cells derived from adipose.2 ADM can fit for adhesion and proliferation of ADMSCs. It is a kind of ideal scaffold in cartilage tissue engineering. It is feasible to repair articular cartilage defects using adipose-derived mesenchymal stem cells into acellular derma base material.3 After operation, the histological specimen verified by specific staining and the positive expression of collagenⅡconfirm the differentiation ability of ADMSCs induced to chondrocytes.