| Co-culturing scaffolds with seeded cells in vitro is an indispensable process for construction of engineered tissues. It is essential to understand what effects the constituent particles and substrata surface of scaffold might have on seeded cells. Bone marrow derived mesenchymal stem cells (MSCs) are a well-known system for bone tissue engineering. They have self-renew capability and multilineage differentiation potential the same as embryonic stem cells but are free of ethics argument and risk of tumorigeness. Compared with osteoblasts, MSCs could be obtained from human body with less injury caused, and have much greater extracorporal expansive capabilityHydroxyapatite (HAP) is a well documented biomaterial for bone tissue substitution during the past decades because of its chemical similarity with natural bone. This similarity leads to excellent biocompatibility and biomimicry of HAP. However, conventional hydroxyapatite (cHAP) has quite different crystal size from that of natural one, which is characterized by oriented arrangement of nano-sized particles. Better supermicrostructural biomimicry and osteoconductivity can be achieved if nano-sized HAP is employed as biomaterial of bone tissue engineering scaffold.In this study, we investigated the effects of nano-sized hydroxyapatite (nHAP) on proliferation and osteogenic differentiation of bone marrow derived mesenchymal stem cells (MSCs).First, nHAP particles were co-cultured with MSCs separated from rabbit. Cellular effects of particles were determined by cell counts, vonkossa stains, and RT-PCR examinations. Results showed that nHAP particles could promote the MSCs growth when particle concentrations were lower than 20μg /10~4 cells. This effect disappeared when particles and cells were co-cultured in serum free media. Higher particle concentrations would significantly inhibit cell growth. In standard culture condition, the only effect of nHAP particles on osteogenic differentiation of MSCs was to enhance the expression of collagen I. In osteogenic inductive culture condition, nHAP particles could inhibit mineralization of cells but promote their osteogenic differentiation. Those cellular effects of particles still existed when particles and cells were cultured in indirect co-culture system. nHAP particles could decrease calcium and phosphate concentrations of culture media, which possibly contributed to the cellular effects of nHAP particles.Then, BMSCs were cultured on HAP coated films and glass films. Osteogenic differentiation of the cells on two different substrata was evaluated by determining the expression of alkaline phosphatase, collagen I, osteopontin and osteocalcin of the cells. Results showed that, when cultured with standard culture media, expression of alkaline phosphatase, collagen I, and osteocalcin were significantly enhanced in the cells on the HAP surface. Osteopontin was not expressed in the cells on both HAP films and glass films. When cultured with osteogenic inductive culture media, alkaline phosphatase, collagen I, osteopontin and osteocalcin expression of the cells on the HAP films were significantly enhanced. Besides, the conditioned media also significantly enhanced the expression of alkaline phosphatase, collagen I, osteopontin and osteocalcin. Those results indicated that HAP surface could enhance the osteogenic differentiation of BMSCs. This effect might be mediated by the soluble factors released into culture media. It is possible that artificial bones constructed by loading BMSCs on HAP scaffolds exhibit excellent bone forming capability after implanting into injured bone tissue.Finally, whether nHAP as cell growth substrata could give better support for attachment, proliferation and osteogenic differentiation of BMSCs than cHAP was determined. nHAP and cHAP films were prepared as the substrata for the cell growth. BMSCs were seeded on the films. Attachment, proliferation and osteogenic differentiation of BMSCs on the two kinds of films were evaluated. Results: Cell attachment ratio on nHAP films was significantly higher than that on cHAP films. Doubling time on nHAP films was significantly shorter than that on cHAP films. Amount of total proteins detected from cells cultured on nHAP films was significantly higher than that on cHAP films. However, alkaline phosphatase activity and osteocalcin content of the two groups showed no significant difference. nHAP films favored cell attachment and proliferation but not osteogenic differentiation of BMSCs compared with cHAP films.To investigate whether decreased calcium and phosphate conconcentrations were responsible for the cellular effects of nHAP, the effects of calcium and phosphate conconcentrations on growth and differentiation of MSCs were observed. The results showed that decreased calcium and phosphate would inhibit proliferation, mineralization, and osteogenic differentiation of MSCs, instead of increasing them. Increased calcium conconcentration would enhance mineralization of MSCs, but inhibit their osteogenic differentiation, with no effect on their proliferation. Increased phosphate concentration would inhibit cell proliferation but have no significant effect on their proliferation and osteogenic differentiation. Decreased calcium and phosphate conconcentrations in culture media caused by nHAP might be responsible for the inhibited proliferation and mineralization of MSCs, but might not contribute to the enhanced osteogenic differentiation.
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