ADSCs Combined With SDF-1/PLGA Bilayer Scaffold For Cartilage Defects Repair

Articular cartilage play an important role in weight-bearing and movement, however, it is also more easily be damaged in intra-articular fractures. As the cartilage has no vessel and nerve, chondrocytes, particularly hyaline cartilage have limited proliferation. Once the cartilage injury occurs, there were acceleration of chondrocytes apoptosis and formation of local cartilage defects, and osteoarthritis was inevitable, which caused pain, joint deformity, disability, and has a serious impact on the patient’s health and life. Currently, a variety of methods have been applyed in the treatment of cartilage defects regeneration. However; the repair effect particularly long-term effect was unstable and unsatisfactory. Recently, development of tissue engineering technology gives the new possibility to solve this problem. Mesenchymal stem cells derived from kinds of tissues were used for seed cells of tissue engineering, in which adipose-derived mesenchymal stem cells (ADSCs) were widely used because of its wide range of sources, stability, small injury on body, relatively simple culture conditions, and strong capacity of proliferation. Stem cells can migrate to the injury site of the body, and this migration activity was related to a variety of chemokines. The stromal-derived factor-1(SDF-1), also known as CXCL12, is a CXC chemokine which is secreted by bone marrow stromal cells and other related mesothelial cells and epithelial cells, and is considered as one of the most potent chemoattractant of stem cell to date, which plays an important role in stem cell recruitment and homing. The biological functions of SDF-1is complex, which is widely involved in cell growth, regulation of stem cell migration and metastasis of malignant tumors. Recent studies have shown that SDF-1also plays an important role in regulation of chondrocyte differentiation and maturation. The total length of the whole humeri of SDF-1-/-mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1-/-mice.SDF-1regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy. We believe that the SDF-1may have broad application in the regeneration and repair of cartilage defects. In the present study, SDF-1was integrated into PLGA bilayer scaffold to locally release SDF-1in the body, and then combined with ADSCs to repair cartilage defects. SDF-1can make the implanted stem cells and autologous stem cells migrate to the defect sites, thus promoting cartilage regeneration. Meanwhile, we used CM-Dil to label ADSCs to trace the implanted ADSCs. The study was divided into three parts:(1) the culture and identification of rabbit ADSCs and effects of SDF-1on proliferation and differentiation of ADSCs;(2) the fabrication of bilayer PLGA scaffold and effects of SDF-1/PLGA on the proliferation and differentiation of ADSCs;(3) SDF-1/PLGA combined with ADSCs for cartilage defects repair in rabbit model.Part I The obtain and identification of rabbit ADSCs and effects of SDF-1on the proliferation and differentiation of ADSCsObjective:To investigate the effects of SDF-1on proliferation and differentiation of ADSCs and find an optimal concentration of SDF-1.Methods:Adipose tissue was isolated from the groin area of adult New Zealand rabbits by enzyme digestion gradient centrifugation and adhesive culture methods. The third generation of AMSCs was used. The MTT method was used to detect the effect of different concentrations (Oμg/L、1μg/L、10μg/L、100μg/L、200μg/L) of SDF-1on the proliferation of ADSCs. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the CXCR4expressions of ADSCs. Meanwhile, transwell method was used to detect the migration of ADSCs with different concentrations of SDF-1.Results:CD105, CD73, CD90, CD166, CD29, CD44were positive in ADSCs (>90%). ADSCs have the three linage differentiation of bone, cartilage and adipose tissue. SDF-1can promote the proliferation, CXCR4expression and migration of ADSCs. With the increase of concentration of SDF-1, these effects were strengthened. Compared to the same concentration of SDF-1, the proliferation, CXCR4expression and migration of ADSCs was all decreased after ADSCs were treated with anti-CXCR4, which were still higher than control.Conclusions:SDF-1can promote the proliferation and migration of ADSCs, and with the increase of concentration, the effect was enhanced. SDF-1plays a key role in promoting the proliferation and migration of ADSCs in vitro, probably by upregulating the expression of CXCR4. Mechanisms of SDF-1in promoting the proliferation and migration of ADSCs still needs further study.Part Ⅱ The fabrication of bilayer PLGA scaffold and effects of SDF-1/PLGA on the proliferation and differentiation of ADSCsObjective:To observe the release efficiency of SDF-1released from SDF-1/PLGA scaffold and its effects on proliferation and differentiation of ADSCs in3D environment.Methods:We used porogen leaching technology and gelatin particles as porogen to fabricated bilayer PLGA scaffolds. A solution of SDF-1(100ng/ml)30μl was added into PLGA scaffolds to fabricated SDF-1/PLGA scaffolds. SDF-1release at different time points were detected by Elisa kit. The cumulative release efficiency was calculated. At the same time, ADSCs was seeded in the SDF-1/PLGA scaffold, the release of SDF-1on the CXCR4gene expression of ADSCs in the3D environment was investigated, using RT-PCR.Results:The PLGA scaffold has a dense layer and a loose layer. The dense layer has large micropore (diameter was400μm) and the loose layer has small micropore (diameter was150μm). ADSCs grew well in micropores of the scaffold. The laser confocal microscopy showed that ADSCs had good activity in the scaffold. SDF-1can be slowly released from SDF-1/PLGA scaffold. On the first day, about60%of the SDF-1was released from SDF-1/PLGA scaffold. And it can maintain a stable rate of release, in thirtieth days, SDF-1released from SDF-1/PLGA scaffold was still be detected. The slow released SDF-1can promote the CXCR4gene expression of ADSCs. Compared with the plane culture, CXCR4gene expression of ADSCs in the3D environment was significantly increased. Conclusions:ADSCs grew well in the SDF-1/PLGA scaffold. SDF-1can be effectively released from the SDF-1/PLGA scaffold. Compared to2D, the released SDF-1can promote the CXCR4gene expression of ADSCs in SDF-1/PLGA scaffold, which will further promote ADSCs migration and homing, owing a broad application space in the regeneration and repair of cartilage defectPart III ADSCs loaded SDF-1/PLGA for cartilage defects repair on rabbit modelObjective:We aimed to explore whether ADSCs combined with SDF-1-releasing SDF-1/PLGA scaffold can synergistically promote the repair of rabbit articular cartilage regeneration and delay the degeneration of regenerated cartilage. Moreover; the survival of transplantation of allogeneic ADSCs in vivo were investigated.Methods:48New Zealand white rabbits were chosen, and96articular cartilage defects in the femoral condyle patellofemoral joints were careated. The defects were randomly divided into3groups(n=16):PLGA group, ADSCs/PLGA group, SDF-1/ADSCs/PLGA group. CM-Dil labeled ADSCs were used to trace the implanted ADSCs. After six and twelve weeks, the restored tissues were retrieved for morphological and histological observation. At12weeks later, RT-qPCR were used to the gene expressions of Aggrecan, SOX-9, Collagen type Ⅰ, Ⅱ and X.Results:Compared with PLGA group and ADSCs/PLGA group, the GAG content, the ICRS scores of the tissue in the SDF-1/ADSCs/PLGA group was significantly higher than the other two groups (P<0.05). In further, the related gene expressions of Aggrecan, SOX9, Collagen type Ⅱ in the SDF-1/ADSCs/PLGA group were significantly higher than the other two groups (P<0.05). The related gene expressions of type I and X in the SDF-1/ADSCs/PLGA group were significantly lower than the other two groups (P<0.05). The implanted ADSCs can be detected by small animal in vivo fluorescence imaging system and confocal observation.Conclusions:The ADSCs combined with SDF-1/PLGA scaffold can effectively promote regeneration of articular cartilage defects and interface integration. The implanted allogeneic ADSCs in cartilage defect can survive and participate in the cartilage repair. Such SDF-1based tissue engineering strategies by promoting for stem cell migration and homing might have good prospects in cartilage repair.