The Animal Experiment And Clinical Research Of The Articular Cartilage Lesions And Reconstruction

Objective:①To investigate the feasibility of the articular cartilage defect reparation in the rabbit knee by culture of autogeneic bone marrow derived mesenchymal stem cells(BMSCs) and implantation into Polylatic/glycolic acid copolymer(PLGA) biomaterial scaffold, using the technique of tissue engineering.②To compare and evaluate the biomechanical properties and the apoptosis of repair tissue cells among subchondral bone drilling, autologous osteochondral transplantation (mosaicplasty) and autologous osteochondral mosaicplasty combined with autogeneic bone marrow derived mesenchymal stem cells(BMSCs)-fibrin gel, repairing the large area of full-thickness defects of articular cartilage.To discuss the difference of three methods,the purpose is for proposing the animal experimental basis to the clinical application.③To evaluate the clinical results from the treatment of local chondral or osteochondral lesions on the talus dome with autologous osteochondral transplantation or mosaicplasty, harvested from the articular non-weight bearing surface of ipsilateral knee.Methods:①Bone marrow derived mesenchymal stem cells(BMSCs) were separated from 28 New Zealand white rabbits,then were cultured,enlarged and induced into cartilage cells in vitro,and finally embedded into the Polylatic/glycolic acid copolymer(PLGA) biomaterial scaffold.After implantation of the BMSCs-PLGA to repair the articular cartilage defects of the knees,the reconstructed tissues were observed marcroscopically,optical microscopic view including HE and Toluidine blue staining, type-Ⅱcollagen immunohistochemical staining by SABC method,cotents of proteoglycan (GAG) determined by dimethylmethylene blue method,and Reverse Transcriptase–Polymerase Chain Reaction for type-Ⅱcollagen at 12 and 24 weeks postoperatively.②48 New Zealand white rabbits were devided into 6 groups according to factorial design.About 5mm full-thickness defects of articular cartilage was made in weight-bearing area of the femoral medial condyle. The reconstructions were respectively with subchondral bone drilling,autologous osteochondral mosaicplasty and mosaicplasty combined with BMSCs-fibrin gel.The reconstructed tissues were observed marcroscopically,the countss of apoptosis of chondrocytes used TDT - mediate X - DUTP nick end labeling(TUNEL), the ELISA analysis for caspase-3 proteinase activity in chondrocytes, detection of mean largest tessile strength and strain at break at 12 and 24 weeks postoperatively.All data were statistical analysis of variance.③There were 23 patients with chondral or osteochondral defects of the talus dome in this study,who were 16-49 years old (mean 33) with 15 males and 8 females,including post-traumatic cartilage defects(n=11), osteochondritis dissecans(n=9), and local osteoarthritis(n=3). The chondral defects were Berndt and Harty's typeⅡ—Ⅳ,located on the medial talus dome in 14 cases and on lateral in 9 cases.The focal defectes were full-thickness and mean 1.6 cm~2(range 0.7—3.5cm~2).The position and size of the defect were defined at ankle arthroscopy.The procedures consisted of debriding its edges and base drilled under ankle arthroscopy or arthrotomy, then harvesting osteochondral cylinders from non-weight bearing surface of the ipsilateral knee under arthroscopy, and using the osteochondral autograft transfer system(OATS) to implant the donor graft into the recipient holes of talus cartilaginous defects with press-fit technique. A single donor transplantation or the mosaicplasty was used.The pain degree was evaluated by visual analogue scale pre-and post-operatively.The ankle range of motion(ROM) was detected with the angle instrument pre-and post-operatively.The ankle whole assessment was according to Mazur et al'method pre-and post-operatively(0—100points).The preoperative and postoperative comparison between groups effects of the related factors was assessed with American Orthopaedic Foot and Ankle Society(AOFAS) ankle-foot rating system(0—100points). The whole knee assessment was by the Hospital for Special Surgery(HSS) rating system pre-and post-operatively (0—100points).The clinical outcomes of the ankle and function of the knee were statistical analysis of paired t test pre-and post-operatively, statistical significance as P<0.05. The preoperative and postoperative comparison between groups effects of the related factors were statistical analysis of individual t test,statistical significance as P<0.05.Results:①The result of HE and Toluidine blue staining of samples shown that defects were repaired by hyaline cartilage in BMSCs-PLGA group.The morphology and arrange in order of chondrocytes were similar to normal,and extracellular matrix staining was tend to normal.The reconstruction of cartilage and subchondral bone was good.The defects of control group were repaired by fibrocartilage, and the morphology and arrange in order were abnormal.The joint surface was irregular and surrounding cartilage appeared degeneration.At 24 weeks potoperatively,the result of immunohistochemical staining was that repair tissue cells were circular and arrange in cylider order,which had good integration with surrounding cartilage and subchondral bone.The cytoplasm and extracellular matrix was positive in type-Ⅱcollagen staining,with many yellow-brown drops in BMSCs-PLGA group. However there were not positive cells in control group during all postoperation. The GAG cotents of restored tissue was 8.52±0.64mg/L in BMSCs-PLGA group and 1.46±0.12 mg/L in control group at 12 weeks postoperatively. The GAG cotents of restored tissue was 19.65±1.68mg/L in MSCs-PLGA group and 2.67±0.31 mg/L in control group at 24 weeks postoperatively.The difference between the groups was statistical significance(F=125.33,P<0.001). The GAG cotents in BMSCs-PLGA group were higher than in control group.There were obviously express of type-Ⅱcollagen and strong proteoglycan in BMSCs-PLGA group,but strong type-Ⅰcollagen,weak proteoglycan and very weak type-Ⅱcollagen in control group.It demonstrated hyaline cartilage rpairment in BMSCs-PLGA group and fibroid tissue in control group.All results indicate that the repair tissues with BMSCs-PLGA transplantation involved normal chondrocyte,a great quantity type-Ⅱcollagen and large numbers of GAG,were fitted for hyaline cartilage.②The degeneration information of the restoring tissue with the three methods.The reconstruction tissues were the more high number of the apoptosis and caspase-3 activity of chondrocytes,the more degeneration of cartilage.At 12 weeks postoperatively, the count of apoptosis chondrocytes was 4.26±0.92/high-multiple visual field in subchondral bone drilling group(A),2.73±0.67/high-multiple visual field in autologous osteochondral mosaicplasty group(B) and 1.64±0.58/high-multiple visual field in mosaicplasty combined with BMSCs-fibrin gel (C) group. The caspase-3 activity of chondrocytes was 39.58±1.25% in A group,24.48±1.03% in B group and 18.86±0.94% in C group.At 24 weeks postoperatively, the count of apoptosis chondrocytes was 11.08±2.02 in A group, 6.35±1.65 in B group and 4.11±0.97 in C group. The caspase-3 activity was 83.64±4.50% in A group, 56.66±3.18% in B group and 35.72±2.23% in C group.These results displaied that the number sequence of three groups was the drilling group>mosaicplasty group>mosaicplasty combined with BMSCs-fibrin gel group in various phases.The difference between three groups was statistical significance(P<0.01). The stress-strain curve of reconstruction tissue in C group were assumed non-linear index relation,with littler instantaneous strain under lower stress and bigger instantaneous strain under higher stress. The stress-strain curve of reconstruction tissue in B group were assumed non-typical non-linear index relation,with instantaneous strain smaller than C group. The stress-strain curve of reconstruction tissue in A group were assumed low level shape similar linear,with with instantaneous strain much smaller than C group and B group. The reconstruction tissues were the more high largest tessile strength and strain at break,the more high mechanical property of cartilage. The largest tessile strength was 1.12±0.11Mpa in A group, 2.52±0.08Mpa in B group and 3.34±0.26Mpa in C group at 12 weeks postoperatively. It was 1.36±0.08Mpa in A group, 2.89±0.03Mpa in B group and 4.02±0.18Mpa in C group at 24 weeks postoperatively. The largest strain at break was 2.52±0.17% in A group, 3.56±0.14% in B group and 4.15±0.22% in C group at 12 weeks postoperatively. It was 2.47±0.06%in A group, 3.84±0.18% in B group and 4.51±0.05% in C group at 24 weeks postoperatively. These data displaied that the number sequence of three groups was the mosaicplasty combined with BMSCs-fibrin gel group>mosaicplasty group>drilling group in various phases.The difference between the groups was statistical significance(P<0.001).To compare with statistics analysis of variance,there was statistical significance among three methods in biomechanics and chondrocyte apoptosis.The result was that the mosaicplasty combined with BMSCs-fibrin gel was better than pure mosaicplasty and mosaicplasty was better than drilling.The best effect of this experiment was the mosaicplasty combined with BMSCs-fibrin gel transplantation at 24weeks postoperatively after statistics analysis of factorial design.The synthetic outcome the mosaicplasty combined with BMSCs-fibrin gel transplantation was suitable for large osteochondral defects,whereas subchondral drilling and mosaicplasty were unsuitable.③All the patients were followed-up for 15 to 30 months (mean, 22.6±8.4months). The mean pain intensity measured by standard visual analogue scale (VAS) reduced from 4.9±1.21 to 0.8±0.13 at 2 years postoperatively,with statistical significance(t = 19.53,P<0.001). The ankle joints flexion and extension degree of the angle were measured to indicate that the range of motion (ROM) improved significantly from 44°±5.8°to 65°±11.2°compared with preoperative status, with statistical significance (t=15.46,P<0.001). Finally, the overall results of the ankle were validated by the score described by Mazur et al. The scores improved from 51±8.6 points to 92±6.3 points (t=27.05,P<0.001). The grafts showed excellent bony integration with surrounding tissues and congruity of the articular surface through the ankle MRI.The preoperative and postoperative comparison of related factors was assessed by AOFAS rating system,with alteration was 38.26±1.45 points in≥35 years old and 42.50±0.27 points in<35, with statistical significance (t=3.02,P<0.01). The alteration about defect area was 39.71±1.11 points in cases≥1.5c㎡ and 42.88±0.84 points in cases<1.5c㎡, with statistical significance (t=3.54,P<0.01).They demonstrated that there was obviously correlation between autologous osteochondral transplantation and patient's age,defect area.After similar statistical analysis, there was not obviously correlation between autologous osteochondral transplantation and patient's sex,cause factor,with no statistical significance (P>0.05). The whole knee assessment was by HSS,with mean 94.3±5.2 points preoperatively and 93.1±6.7 points at the time of the lastest follow-up, with no statistical significance (t=1.84,P>0.05).It was shown that harvesting osteochondral cylinder from the ipsilateral knee was no influence for whole knee joint.Conclusion:①The articular cartilage defects reconstruction of the rabbit knee were the hyaline cartilage formed from BMSCs implantation into PLGA.②Compared with autologous osteochondral mosaicplasty and subchondral bone drilling,the mosaicplasty combined with BMSCs-fibrin gel transplantation was gained satisfied treatment effects.③Autogenous osteochondral transplantation of the talus using ipsilateral knee osteochondral grafts is a surgical procedure of true curative effects on treating local cartilage or osteochondral lesions of the talus.