| Objective: A technology based on coaxial electrospinning was adopted to prepare bone tissue engineering scaffolds with poly(3-hydroxybutyrateco-4-hydroxybutyrate)(P(3HB-CO-4HB)) in core and shell construction of gelatin(GA) and polyvinyl alcohol(PVA), by seeding human bone marrow mesenchymal stem cells(h BMSCs) on scaffolds to build cell-scaffold composite in vitro, aiming at researching ectopic bone formation in nude mice with cell-scaffold composite, which was observed in vivo and in vitro experiments. Methods: Marrow extracted from the anterior superior iliac spine to isolate and culture h BMSCs and subculture to the fifth generation. Flow cytometry was used to identify cell surface antigens. Osteogenesis induction medium was replaced for inducing osteoblast lasting 14 days and then to assess osteogenesis differentiation by alkaline phosphatase staining(AKP), alizarin red staining(ARS) and collagen type I immunological histological chemistry staining. GA+PVA mixed aqueous solution(8% w/v) was prepared as shell construction of solution and P(3HB-CO-4HB) solution(6% w/v) was used as core solution for preparing scaffolds in utilizing coaxial electrospinning technique, which was observed by scanning electron microscopy(SEM) and transmission electron microscopy(TEM). At the same time, GA aqueous solution(6% w/v) without PVA was used as shell construction of solution and P(3HB-CO-4HB) solution(6% w/v) was used as the core solution in utilizing coaxial electrospinning technique, which was observed by SEM. GA+PVA mixed aqueous solution(8% w/v) was prepared as shell construction of solution and P(3HB-CO-4HB) solution(6% w/v) was used as core solution for preparing scaffolds in utilizing coaxial electrospinning technique, and h BMSCs were seeded on the surface of prepared scaffolds. The cell-scaffold composites were cultured in vitro, which observed by DAPI staining at the first and seventh day. After osteogenesis induction for 7 and 14 days, growth situation of cells on scaffolds was determined by SEM and DAPI staining. Compounds were divided into experimental group and control group. Osteogenesis induction medium was used in the experimental group, while the control group was cultured by complete medium, which was implanted under subcutaneous layer of nude mice after14 days, respectively. Ectopic bone formation of cell-scaffold composites in vivo was evaluated in the two groups respectively at the eighth and sixteenth week by using HE staining, Vonkossa staining and alizarin red staining(ARS) and collagen type I immunological histological chemistry staining. Results: As primary culture of h BMSCs increasing with time, shape of anchorage-dependent cells was changed from circular to short fusiform. After cultivating for 12 days, cell fusion has reached to more than 90%. While subcultivation of cells up to the fifth generation, h BMSCs changed into long fusiform shape, which arranged in the same direction and growing in spiral and parallel shaped. Results of flow cytometry determination showed expression of CD44, CD73 and CD90, CD105 surface antigen were high without expression of CD34, CD45 and HLA-DR surface antigen. Cells were continued to culture for 14 days after complete medium replaced by osteogenesis induction medium, which showed that morphology of cells changed into short fusiform and polygonal shape. Staining results of AKP, ARS and collagen type I immunological histological chemistry staining were all positive. The morphology of h BMSCs cultured without osteogenesis induction medium was still long fusiform shape with negative bone chemical staining. TEM showed that coaxial electrospinning scaffolds with P(3HB-CO-4HB) in core and shell construction of GA+PVA have obvious core-shell structure, while SEM showed that coaxial electrospinning scaffold with P(3HB-CO-4HB) in core and shell construction of GA there was poor filamentation of electrospinning and lots of droplets, which was found fibers extended on the surface, but failed to form a continuous and complete fiber, and part of fibers fractured; And scaffold with P(3HB-CO-4HB) in core and shell construction of GA+PVA was a three-dimensional network and interconnected structure, with relatively uniform fiber diameter. Fluorescence staining results of cell-scaffold composites cultured in vitro after 1 day showed that relatively uniform distribution of cells on the scaffolds, and the number of cells was increased after 7 days and distributing on scaffolds. SEM showed cells distributed on the surface of scaffolds after osteogenesis induction of complex lasting for 7 days and covering after 14 days, which turned out to be well growth on scaffolds with lots of extracellular matrix secreting to cover the surface of scaffolds. DAPI staining showed that many cells on scaffolds after osteogenesis induction of complex lasting at 7 and 14 days. After cell-scaffold composites implanted, growth of nude mice was in good condition, without systemic or local inflammation and toxic effects. Morphological structures of cell-scaffold composites in the experimental group remained unchanged after eight and sixteen weeks, and with texture hardening gradually; After cell-scaffold composite implanted for eight weeks, the composite volume of the control group did not shrink obviously with soft texture, which almost fully degraded after 16 weeks. Results of HE staining, Vonkossa staining and alizarin red staining(ARS) and collagen type I immunological histological chemistry staining in the experimental group showed bone tissue formed with developing bone cells and bone lacuna. Calcium salt deposition and type I collagen were increased gradually with time and becoming mature gradually. Related bone chemical staining of the control group was negative, without formation of bone cells, osteoblasts and bone lacuna. Conclusion: Scaffolds composite h BMSCs with P3HB-CO-4HB/(Gelatin Polyvinyl alcohol) based on coaxial electrospinning was a good bone tissue engineering scaffolds after osteogenic induction in vitro, with capacity of ectopic construction of tissue-engineered bone in vivo. |
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