Articular Osteocartilage Defect Repaired With The Composite Of Integrated Scaffolds And Adipose-derived Stromal Cells

Articular osteocartilage have poor healing potential. Basic science and clinical investigation have led to different approaches. However, obtaining autograft would cause additional injury and resource of autograft is limited. Allograft could cause immunological rejection and propagation of disease. Many solutions have been proposed to constructe tissue-engineered bone and articular cartilage respectively, culture pluripotent cells with bioreactor in vitro, and integrate them before transplantation. The procedure of these approaches, unfortunately, is complicated. Guided by new theory of"In vivo bioreactor", in this work, we integrated tissue-engineered BMP-Cell-Scaffold composite, including ADSCs seeded in vitro and in vivo repair the articular osteochondral defect.PartⅠ. Construction of the collagen / chitosan scaffoldObjective: To evaluate effect of concentration, proportion of compositions in scaffold and freezing temperature on microstructure and mechanics of the collagen/chitosan scaffold. Methods: Type I collagen was extracted from rat tail by means of acidolysis and enzymolysis. Quantitative and qualitative analysis was performed as follows: molecular weight was estimated by SDS-PAGE, and absorbtion peak of UV was measured by spectrophotometric analysis and nitrogen composition was determined by micro Kjeldahl method. The scaffolds were constructed by lyopyilization and then were divided into 5 groups according to freezing temperature, concentration and proportion as follows: group A -20℃,2%,1:3;group B -80℃,2%,1:3;group C -20℃,1%,3:1;group D -20℃,1%,1:3;group E -20℃,2%,3:1. The characteristic of scaffolds were evaluated through SEM observation and mechanical test. Results: The collagen was readily available by means of acidolysis and enzymolysis from rat tail. SDS-PAGE results showed that the molecular weight was around 97KD and aborbsion of UV values were at 230nm wavelength, which was typical characteristic of typeⅠcollagen. Micro Kjeldahl method demonstrated the purity of collagen was 96%. The pores of the scaffolds of group A were uniform and abundant. The inter-connected pores were hardly observed between layers of the scaffolds of group B. The walls of the scaffolds of group C and D were tenuity. Membranes were observed in some areas of the scaffolds of group E. There were no significant difference of aperture sizes between group A,C,D and E. The scaffolds of group A performed better in biomechanical test. Conclusion: Rat tail collagen is typeⅠcollagen with high purity. The microstructure and biomechanical properties of the scaffolds of Group A are better than the others.PartⅡ.Crosslinking of collagen/chitosan Scaffolds with GenipinObjective: To investigate the changes of pore sizes, crosslinking index, swelling ratio and degradation rate of genipin crosslinked collagen/chitosan scaffolds affected by the variation of the methods of crosslinking, concentration of cross-linking agent, crosslinking temperature points and crosslinking time.Methods: The scaffolds were divided into 10 groups according to the variation of the methods of crosslinking, concentration of cross-linking agent, crosslinking temperature points and crosslinking time. The mixing-crosslinking groups are group A(0.1%,20℃,24h),group B(0.5%,20℃,24h) and group C(1.0%,20℃,24h). The scaffold-crosslinking groups are group D(0.1%,20℃,24h),group E(0.5%,20℃,24h),group F(1.0%,20℃,24h),group G(0.5%,4℃,24h),group I(0.5%,36℃,24h),group J(0.5%,20℃,12h) and group L(0.5%,20℃,72h). The collagen/chitosan scaffolds without crosslinking were chosen as control group X. The pore sizes, crosslinking index, swelling ratio and degradation rate were measured. Results: The values of pore diameters of the scaffolds in group A, B and C were 113.62±53.44μm,258.36±77.93μm and 384.43±92.14μm. The differences between each two groups are significant. The pore diameter and swelling ratio were similar between each two groups of the scaffold-crosslinking groups. The crosslinking index of group E was 67.6%±3.6%, and the degradation rate of group E was 0.9%±5.9%. They were better than group D,G,(JP<0.01). No significant different were observed between group E and group F, I, L. The degradation rate and swelling ratio of each crosslinking group were lower than control group X(P<0.01).Conclusion: The swelling ratio and degradation rate of the collagen/chitosan scaffolds were significantly decreased by genipin without any obvious change of pore sizes. The increase of concentration of cross-linking agent, crosslinking temperature points and crosslinking time caused higher crosslinking index and lower degradation rate in scaffold-crosslinking groups. The crosslinking method used in Group E was most effective.PartⅢ. Evaluation of Biocompatibility of crosslinked scaffoldsObjective: Evaluate the biocompatibility of the crosslinked scaffolds and select more suitable cell for the scaffolds. In vivo, observe the inter-react of cell-scaffold complex in vivo. Methods: The growth rates of BMSCs and ADSCs seeded onto the crosslinked scaffolds and noncrosslinked scaffolds were tested by means of MTT colorimetric microassay. The attachment of ADSCs on crosslinked scaffolds was observed by SEM. In vivo bioreactions of the scaffolds and the cell-scaffold complexes were assessed by histological examination. Results: The growth rate of ADSCs was higher than that of BMSCs. Few cells were attached to the crosslinked scaffolds. No obvious inflammatory reaction was observed. High density ADSCs well-distributed in the cell-scaffold complexes constructed with diphase seeding technique and the ADSCs survivaled in 4 weeks. Conclusion: ADSCs were suitable for the crosslinked scaffolds. These scaffolds had good biocompatibility. Diphase seeding technique was effective.PartⅣ. Construction of the composite scaffolds with isolation layer and repair of the articular osteochondral defects in rabbits with the BMP-ADSCs-scaffold complexes through diphase seeding techniqueObjective: To construct the integrative scaffolds with isolation layer and repair articular osteochondral defects in rabbits with the BMP-ADSCs-scaffold composite constructed with diphase seeding technique. Methods: Cancellous bone scaffolds were constructed with the methods of degreasing, deproteinization and hybridization with collagen. 2% collagen solution was placed on the cancellous bone scaffolds in 2mm thick. After the layer dry, construct collagen/chitosan scaffold on the layer with the method used in group A of the partⅠand crosslinked them with the method used in group E of the partⅡ. BMP-ADSCs-scaffold compositions were constructed with diphase seeding technique. 12 BMP-ADSCs-scaffold compositions were implanted into articular osteochondral defects of rabbits, 12 cell-free compositions were implanted into articular osteochondral defects as control. 8 articular osteochondral defects without treatment were set as blank. Reparative profile of the defect was assessed by histological examination and Pineda scoring system at regular intervals of 8th and 12th week. Results: At 8th week, the defects implanted BMP-ADSCs-scaffold compositions were filled with cartilage-like tissue. There was no evidence of chondrogenesis and subchondral osteogenesis indicated by H&E and saffron"O"staining. The defects of control group were filled with cell-free complexes. At 12th week, the cartilage and subchondral bone of the defects of the experimental group were fully regenerated, and the borderline were filled with cartilage like tissue at the surface the defects in control group. The defects in the blank group were filled with fibrous like tissue. Pineda score revealed statistical difference between experimental group and other two groups. Pineda score of experimental group was significantly higher at 12th week than that at 8th week. Conclusion: Guided by the theory of"in vivo bioreactor", the BMP-ADSCs-scaffold composite constructed with diphase seeding technique has the ability to repair osteochondral defect in rabbits.In this research, we constructed BMP-ADSCs-scaffold composite. Its component were similar to extracellar matrix and structure were designed to adapt the vivo condition of oxy pressure, mechanical stimulation ect. Guided by the new theory of"In vivo bioreactor", we implanted the composite into the articular osteochondral defect in vivo and found that its two phase could separately differentiate into bone and cartilage in different environment. The results showed the composite could repair osteochondral defect.