Degradation and the effect of nano particles of calcium sulfate on bone morphogenetic protein
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Calcium sulfate has been used as an alloplastic bone grafting material with a long clinical history and many advantages. It is an osteoconductive material and has also been used as a vehicle for releasing preadsorbed growth factors. In our experiments we used nano-particles of calcium sulfate fabricated by a cryo-vacuum process previously developed by our group. The aims of our study were: (a) to test the degradation of the nano calcium sulfate (ncs); (b) its function as a carrier for growth factors such as bone morphogenetic protein (BMP2); (c) its ability to induce differentiation of human osteoblastic cells using both in-vivo and in-vitro assays. Material and methods. The in-vivo experiment was conducted with Sprague Dawley rats which received dorsal subcutaneous implants including: medical grade sized calcium sulfate, ncs, ncs/alginate (alg)10% and ncs/alg 50%. Each implant had been premixed with 45 Ca as a tracer for calcium remaining in the samples. Three rats were euthanized at 2 weeks, 3 weeks and 4 weeks. The samples were collected for histological analysis and the radioactive calcium remaining was measured using an automated scintillation counter. For the in-vitro studies, disks were made with the dimensions of 5 mm diameter and 2 mm thickness using prefabricated molds. Human osteoblasts were used in these experiments as testing cells. Two different concentrations of BMP-2 used were (B1) 1μg/ml and (B2) 0.01 μg/ml. For the samples mixed with alginate, only 10% alginate, 90% ncs was used in the in-vitro experiment. To check the different characteristics of the ncs, 45 Ca tracing was again used Alkaline phosphatase was measured, using a biochemical assay to assess osteoblastic cell differentiation. The MTT assay was used to assess osteoblastic cell viability and BMP2 released from preloaded ncs was measured with a immunoassay at different time points extending from 0 hours to 7 days. Results. The in-vivo study showed that a degradation of material was occurred between 0 and 2 weeks. Ncs had a higher degradation rate than the calcium sulfate with ncs/alginate10% having the highest degradation rate. Histological analysis showed that remnants of the grafting material were the least in the ncs group. The in-vitro group, the 45 Ca tracing of the ncs revealed no significant association of the calcium with the attached osteoblasts. The MTT assay showed no significant difference between the controls without ncs and the two concentrations of ncs (1 μg/ml and 10 μg/ml). In the alkaline phosphatase experiment, no significant difference was shown in monolayers of cells exposed to released materials from the ncs or the cells grown on the ncs disks for the different concentrations of BMP2 at different time points. The BMP immunoassay showed that the amount of BMP released was significantly higher in the ncs/alginate10% group while the medical grade calcium sulfate only showing a minimal release over a 7 days period. However, all groups released only a small percentage of the loaded growth factor. Conclusion. These experiments showed that ncs is safe and biocompatible. It has properties that make it suitable for use as a grafting material with more rapid degradation characteristics than medical grade calcium sulfate and for the release of growth factors especially when the material is mixed with a porogen such as alginate. However, additional studies are needed with respect to the binding and release of different forms of BMPs and other growth factors under conditions that are specific for different types of in vivo microenvironments.