The Mechanism Of The Osteogenic Activities Of Amino Acid Substituted Polyphosphazenes

Bone defect is a serious clinical problem, and tissue engineering acts as a promising method in solving this problem. One of the most important aspects of tissue engineering is the cell scaffold. Biodegradable polyphosphazenes are attractive candidates for scaffolding materials due to their biocompatibility, non-toxic degradation products with a near-neutral pH, synthesis tunability and adjustable mechanical property and degradation behavior. Moreover, A series of studies have shown that amino acid ester substituted polyphosphazenes can stimulate osteogenic differentiation of stem cells and induce the deposition of hydroxyapatite. However, the mechanism for the osteogenic activity of these polyphosphazenes is still unclear.In order to explore the reason that how the amino acid ester substituted polyphosphazenes can enhance osteogenic differentiation, we designed and synthesized three kinds of amino acid ester substituted polyphosphazenes with different degradation rates, including two glycine ethyl ester (G) and alanine ethyl ester (B) co-substituted polyphosphazene (G7B3, G3B7), and one glycine ethyl ester (G) and phenylalanine ethyl ester (BB) co-substituted polyphosphazene. Their hydrolysis rates were in the order of G7B3> G3B7> G3BB7.On one hand, the osteogenic activities of the fully degraded polyphosphazene products were investigated by culturing rat bone mesenchymal stromal cells (rBMSCs) with the quantitative additions of corresponding degradation products, using inorganic phosphate ion (Pi) as comparison. On the other hand, rBMSCs were cultured in the presence of polyphosphazene materials by being seeded onto the materials directly or using transwell culture chamber, i.e. the cell and the material did not contact directly. Proliferation and osteogenic differentiation were determined to evaluate their correlations with the chemical composition and contact effect of polyphosphazene substrates. Some results were obtained and listed as below.(1) The addition amounts of both Pi and degradation products into the culture medium can greatly affect the proliferation and differentiation of rBMSCs. At higher concentration, rBMSCs would grow slower, while express significantly higher osteogenic differentiation related genes and proteins.(2) As the concentration of phosphate ions in the case of degradation products being used was adjusted to the same level of Pi, it was found the osteogenic differentiation of rBMSCs were significantly enhanced in the former case. It indicated that those degradation products derived from amino acid ester side groups of polyphosphazene had played a synergistic promoting effect in enhancing the osteogenic differentiation.(3) By culturing rBMSCs using the transwell chamber in the presence of various amino acid ester substituted polyphosphazenes. Among the three polyphosphazenes, it was found G7B3 showed the strongest ability in promoting the osteogenic differentiation. The reason was ascribed to its fastest degradation rate, which meant most amount of degradation products being released from G7B3 in comparison with G3B7 and G7BB3 at the same time points.(4) when rBMSCs were cultured directly on the surfaces of these amino acid ester substituted polyphosphazenes, however, it was found G7BB3 demonstrated the highest promotion in osteogenic differentiation. Moreover, the enhancement was much significant in this case than the case of transwell culture.To summarize, the degradation products including phosphate and amino acid could enhance osteogenic differentiation of rBMSCs, and the polyphosphazene with faster degradation rate would have stronger ability in enhancing differentiation in this consideration. However, the contact effect of polyphosphazene on cell biological behaviors was more efficient than soluble degradation ingredients, because the degradation products would be continuously diluted with culture medium refreshing. Since the surface feature and degradation rate of polyphosphazene depended on their chemical compositions; therefore, biodegradable amino acid ester substituted polyphosphazenes were good substrates for bone tissue engineering via adjusting their side groups.