In vitro chondrogenesis of rat adipose-derived stem cells combined with sodium alginate hydrogel scaffold induced by cartilage-derived morphogenetic protein-1.

OBJECTIVE: To explore the feasibility of chondrogenic differentiation of rat adipose-derived stem cells (ADSCs) combined with sodium alginate (NaAlg) hydrogel scaffolds induced by cartilage-derived morphogenetic protein 1 (CDMP-1). METHODS: ADSCs were isolated and cultured in vitro from rat inguinal fat pads using type I collagenase digestion, centrifugation and adherent culture methods. Flow cytometry was applied for cell identification, and passage 3 ADSCs were used for subsequent experiments. ① Biocompatibility evaluation of materials: ADSCs were co-cultured with NaAlg hydrogel scaffolds. Cell cytoskeleton staining, cell migration assays and cell proliferation tests were performed to assess the biosafety of the scaffolds. ② In vitro chondrogenesis experiment: According to the addition of CDMP-1 and application of three-dimensional NaAlg culture, the cells were divided into four groups: ADSCs group, ADSCs + NaAlg group, CDMP-1 + ADSCs group, and CDMP-1 + ADSCs + NaAlg group. Cell proliferation activity was detected by Cell Counting Kit-8 (CCK-8) at days 1, 3, 5, 7 and 9 after chondrogenic induction. Toluidine blue staining for sulfated glycosaminoglycans (GAGs) and immunohistochemical staining for collagen II (COL-II) were conducted at days 7 and 14 of induction to observe chondrogenic differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining were adopted to detect the mRNA and protein expression levels of chondrogenic marker genes SOX9, COL2A1 and ACAN at days 7 and 14 after induction. RESULTS: Flow cytometry results showed that ADSCs were positive for surface antigens CD29, CD44 and CD105, and negative for CD31 and CD45. After chondrogenic induction, the cell proliferation curves of each group presented an approximate "S" shape, and there were statistically significant differences among all groups (P < 0.05). ① Material biosafety and biocompatibility assessment: Phalloidin cytoskeleton staining, scratch assay, Transwell assay, CCK-8 assay and live/dead cell staining verified that NaAlg hydrogel scaffolds exerted no adverse effects on cell morphology, migration capacity or proliferation activity. ② In vitro chondrogenesis experiment: Positive results of toluidine blue staining for sulfated GAGs and immunohistochemical staining for COL-II were observed in all groups at days 7 and 14 of induction, and the expression of GAGs and COL-II was the strongest in the CDMP-1 + ADSCs + NaAlg group. At the same time points, the expression levels of chondrogenic markers SOX9, COL-II and ACAN were significantly higher in CDMP-1-treated groups (CDMP-1 + ADSCs group and CDMP-1 + ADSCs + NaAlg group) than those in the control groups (ADSCs group and ADSCs + NaAlg group). Moreover, the expression of SOX9, COL-II and ACAN in NaAlg hydrogel scaffold culture groups (ADSCs + NaAlg group and CDMP-1 + ADSCs + NaAlg group) was remarkably upregulated compared with that in two-dimensional culture groups (ADSCs group and CDMP-1 + ADSCs group). CONCLUSION: NaAlg hydrogel scaffold possesses favorable biosafety as a cell-carrier scaffold. CDMP-1 can effectively induce chondrogenic differentiation of ADSCs seeded on NaAlg hydrogel scaffolds in vitro. This strategy is expected to provide a novel tissue engineering approach for the repair of cartilage defects.