Allogeneic Porous Acellular Cartilage Sheets Combined With Microfracture Promotes Cartilage Defect Regeneration In Weight-bearing Region Of Sheep Knee Joint

Objective1.To explore the feasibility of obtaining allogeneic acellular cartilage natural scaffold by acellular treatment of large area intact cartilage.2.To explore the effect of allogeneic porous acellular cartilage sheets(APACS)combined with microfracture(MF)in repairing cartilage defects in the weightbearing region of knee joint in large mammals sheep.Methods1.The complete cylindrical sheep knee cartilage sheets with a diameter of 7 mm and thickness of 1 mm were acellular treated by physical,chemical and biological methods,then APACS were obtained.The degree of decellularization of APACS and retention of cartilage extracellular matrix(ECM)residual components were evaluated by histological staining of APACS,including DAPI,HE,safranin O,toluidine blue and immunohistochemistry of collagen type Ⅱ,as well as DNA residue,glycosaminoglycan(GAG)content and total collagen content assay.The maintenance of the microstructure of APACS and the adhesion of cells to APACS after APACS combined with rat adipose-derived mesenchymal stem cells(ADMSCs)were observed by scanning electron microscope(SEM).After coculture of rat ADMSCs and APACS for one week,the biocompatibility of APACS and the ability to promote cell proliferation were measured by living/dead cell staining and CCK-8.2.Twenty-four sheep were randomly divided into 4 groups with 6 sheep in each group.The cartilage defect(CD)group: full-thickness cartilage defects with a diameter of 7 mm and a thickness of 1 mm were made in the weight-bearing area of the medial and lateral condyle of the bilateral knee joint of sheep(the subchondral bone was retained intact).The microfracture(MF)treatment group:the cartilage defects were treated by microfracture.Allogeneic porous frozen cartilage sheets(APFCS)transplantation combined with MF treatment group(APFCS+MF): the porous cartilage sheets were frozen at-80℃ for 24 h and transplanted into the defect combined with MF to treat the cartilage defect.Allogeneic porous acellular cartilage sheets(APACS)transplantation combined with MF treatment group(APACS+MF): APACS were transplanted into the defect and combined with MF to treat the cartilage defect.At 6 and 12 months after operation,the repair effect of each group was evaluated by gross scoring,biomechanical testing,biochemical examination and histological staining.Results1.The results of DAPI and HE staining of APACS showed that the cell nucleus components were not visible under the microscope with a better acellular effect than the unperforated cartilage sheets.The quantitative of residual DNA in APACS were less than 50 ng/mg,which met the standard of decellularization,while the unperforated cartilage sheets did not meet the standard.Staining results of safranin O,toluidine blue and collagen type Ⅱ showed that APACS retained the GAG and collagen type Ⅱ components in cartilage ECM.The results of GAG and total collagen quantification and biomechanical test showed that the residual amount of GAG and total collagen in APACS was lower than natural cartilage,as well as the elastic modulus.After one week of APACS/ADMSCs co-culture,the living/dead cells staining showed that the cells grew well in APACS,and the results of CCK-8 also showed that APACS had a remarkable effect on the proliferation of ADMSCs.2.Gross scoring: at 6 months after operation,there was no significant difference in the gross scores among the MF group,APFCS+MF group and APACS+MF group.At 12 months after operation,there was no obvious difference between the APFCS+MF group and APACS+MF group,but both groups were higher than the MF group.Biomechanical testing: there was no significant difference in the elasticity modulus among the MF group,APFCS+MF group and APACS+MF group;at 12 months after operation,the elastic modulus of the APACS+MF group was higher than the APFCS+MF group,which was both higher than the MF group.Determination of GAG content: at 6 months after operation,the content of GAG in the APACS+MF group was higher than that in the APFCS+MF group,and both groups were higher than that in the MF group.At 12 months after operation,the comparison results between the four groups was the same as at 6 months.Determination of collagen type Ⅰ content: at 6 months after operation,the content of collagen Ⅰ in the MF group was significantly higher than other groups.There was no significant difference between the APFCS+MF group and APACS+MF group.At 12 months after operation,the comparison results between the four groups were the same as those at 6 months after operation.The determination of collagen type Ⅱ content and histological score had the same results: at 6 months after operation,there was no significant difference between APFCS+MF group and APACS+MF group,and both groups were significantly higher than the MF group.At 12 months after operation,the content of collogen Ⅱ and the histological score in the APACS+MF group were higher than the APFCS+MF group,and both groups were higher than the MF group.The results of the CD group in these detection index were the worst in all groups,while the APACS+MF group showed the best repair effect.Conclusions1.APACS can preserve the GAG and collagen components of cartilage ECM while removing cells with little structural damage,certain mechanical strength,no cytotoxicity,and can support cell adhesion and promote cell proliferation with a outstanding effect.APACS is expected to play a superior role in promoting cartilage regeneration.2.The APFCS+MF group and the APACS+MF group have excellent repair effect on cartilage defect in weight-bearing area of knee joint in sheep.Both these two kinds of materials may promote the differentiation of bone marrow mesenchymal stem cells into chondroblasts,and enhance the repair effect of MF.Moreover,APACS showed better repair effect than APFCS in this study,which is expected to be widely used in future clinical treatment.