Stromal Cell-derived Factor-1 In Tissue Engineering To Repair Cartilage Damage Mesenchymal Stem Cell Recruitment And To Promote The Role Of Differentiation

Background:Because the articular cartilage has no vascular supply and chondrocytes are embraced by extracellular matrix components, cartilage injury is usually an irreversible pathological process, resulting functional joint deficits. Hyaline cartilage has different structural, composition, and mechanical properties compared with normal cartilage. At present, articular cartilage repair after injury is still a challenging issue. Current approaches to cartilage repair including microfracture and autologous and allogeneic transplantation have some limitations and fail to achieve satisfactory outcomes. Application of tissue engineered cartilage and bone cartilage complexes is recognized as a promising strategy for the treatment of cartilage lesions. Tissue engineering generally involves three components:seeding cells, scaffold materials, and signal factors. Bone marrow stromal stem cells (BMSCs) have multi-lineage differentiation potentials and are capable of differentiating into cartilage under specific conditions. However, cultured BMSCs are prone to de-differentiation in vitro. Mobilization of autologous BMSCs is believed to be an important approach to repair cartilage damage. Stromal cell-derived factor 1 (SDF-1), abundantly secreted by bone marrow stromal cells, is a kind of chemokines. The action of SDF-1 depends on its interaction with specific receptor C-X-C chemokine receptor type 4 (CXCR-4) that is distributed on a variety of cells. This chemokine is implicated in many biological processes including neural development, angiogenesis, haematopoiesis, immune response, and tumor progression. In-vivo studies show that bone marrow homing of hematopoietic stem cells is dependent on their expression levels of CXCR4 as well as the release of SDF-1 by bone marrow stromal cells and endothelial cells. SDF-1 can promote cell proliferation through regulation of the cell cycle. The biological function of SDF-1 is cellular context-dependent; however, the underlying mechanism remains to be defined. This study attempted to determine whether SDF-1 could recruit BMSCs and induce their chondrogenic differentiation.In cartilage tissue engineering, scaffold materials not only play a supporting role, but also affect the property and function of embedded cells. At present, many kinds of scaffolds have been used for cartilage tissue engineering. Among those, Polyactic acid (PLA), Polyglycolic acid (PGA), Poly (lactic-co-glycolic acid) (PLGA), Collagen, and Chitosan are the most commonly applied. PLGA sponge scaffold has already been approved by the Food and Drug Administration of USA as a clinical material. This material has a three-dimensional structure and provides a vast living environment for cells. Seeded cells are thus allowed to move in a three-dimensional space, which overcomes cell contact inhibition in a monolayer. The three-dimensional structure can also facilitate intercellular information transferring, nutrients transport, and metabolites diffusion. Additionally, cells and their extracellular matrix components are enriched in the three-dimensional structure, which may be beneficial for structural organization of new formations.In summary, the present experiment used SDF-1-loaded PLGA sponge scaffold to enrich autologous BMSCs and promote BMSCs to differentiate into cartilage. It included:first, in vitro and in vivo validation of the chemotactic effect of SDF-1 on BMSCs; second, application of SDF-1 to promote the expression of cartilage-specific proteins (typeⅡcollagen and glycosaminoglycan) in BMSCs; and third, the application of SDF-1-loaded PLGA scaffold to repair of cartilage defects, in an attempt to provide a new idea of the treatment of articular cartilage injury.Methods:(1) Isolation, culture, and flow cytometric identification of BMSCs; (2) Verification of SDF-1 receptor expression in BMSCs through immunocytochemistry; (3) Checking the chemotactic effect of SDF-1 on BMSCs using an in vitro Transwell assay; (4) In vitro labeling BMSCs with BrdU; (5) Implantation of SDF-1-loaded PLGA scaffold to animals with cartilage defects and validation of the chemotactic effect of SDF-1 on BMSCs; (6) Addition of different concentrations of SDF-1 to BMSCs in vitro and assessment of the expression of typeⅡcollagen and glycosaminoglycans by Real-time PCR, Enzyme-Linked Immunosorbnent Assay (ELISA), and Western blotting analysis; (7) Application of SDF-1-loaded PLGA scaffold to repair of articular cartilage defects in a model of rabbit. A total of 60 adult male New Zealand rabbits were randomly divided into 4 groups:Group D (defect control group), Group P (PLGA scaffold), Group SP (SDF-1+PLGA scaffold), and Group AP (AMD+PLGA scaffold). A double knee femoral trochlear cartilage defect model (4 mm in diameter,3 mm in depth) was generated. At 4, 8, and 12 weeks after operation, animals were subjected to International Cartilage Repair Society (ICRS) gross and histological grading, histological staining (HE staining and toluidine blue staining), and immunohistochemistry for typeⅡcollagen. The data of histologic scores are expressed as medians±interquartile range (M±QR) and analyzed by the nonparametric Kruskal-Wallis test, followed by Bonferroni post hoc test. Other data are displayed as means±standard deviations and analyzed by one-way analysis of variance (ANOVA) post hoc test of Tukey's method. The statistical significance level of nonparametric Kruskal-Wallis test and one-way ANOVA was set at P<0.008 and P<0.05, respectively. Results:Flow cytometry revealed that isolated BMSCs showed positive for CD90 and CD44 but negative for CD14 and CD45, two markers specific for blood lineage cells. Immunohistochemistry showed SDF-1 specific receptors distributed on BMSCs surface. In vitro Transwell chemotactic experiments demonstrated that, as the SDF-1 concentration increased, chemotactic cell number gradually increased. When the concentration of SDF-1 was 50 ng/ml, the number of migrating cells reached a peak. In order to verify the in vivo recruiting effect of SDF-1, BrdU-labeled BMSCs were intravenously injected to animals with cartilage lesions. When the SDF-1 concentration was 50 ng/ml, a maximal recruitment of BrdU-labeled BMSCs was achieved. Moreover, the expression levels of both type II collagen and glycosaminoglycan in BMSCs were greatly higher in the presence of 50 ng/ml SDF-1 compared to the control group.Animal studies showed that after 12 weeks cartilage defects were repaired in the Group SP. The repaired cartilage had a smooth surface with an organized cell distribution. Superficial cells of the cartilage were parallel to the surface, and deep cells were found in a columnar arrangement. Tide lines in the cartilage were distinctly visible. The boundaries between the repaired and normal cartilage disappeared. The repaired cartilage was positively stained by toluidine blue staining and antibodies against type II collagen. At 4 and 8 weeks after operation, the Group SP did not significantly differ from the Group P in the ICRS histological score (P 0.05). However, at 12 week after operation, the ICRS histological score was significantly better in the Group SP than in the Group P (P=0.001). Additionally, the Group SP had a significantly greater ICRS score compared to the Group D (P<0.008).Conclusions:(1) SDF-1 in vitro and in vivo can recruit BMSCs.(2) The addition of SDF-1 (50 ng/ml) to BMSCs can promote the production of type II collagen and glycosaminoglycan, which contributes to the differentiation of BMSCs to chondrocytes.(3) SDF-1-loaded PLGA scaffold can profoundly facilitate the repair of cartilage lesions through induction of hyaline cartilage-like structures; 12 weeks postoperation, the repaired cartilage show similar morphology and structure to normal cartilage. This study provides experimental and theoretical foundation for the repair of articular cartilage injury by BMSCs-based tissue engineering.