Osteogenic differentiation of follicular stem cells on nano-Saghez scaffold containing BMP2.

BACKGROUND: Bone tissue is one of the tissues that are capable of self-regeneration. However, bone self-regeneration is defeated in the case of broad lesion of bone structure. Isolated stem cells from wisdom tooth follicles can potentially differentiate into ectodermal and mesodermal cells. Saghez is a natural substance that has been extracted from Pistacia terebinthus with unique features, such as high temperature and mechanical stability, adhesive structure, biocompatibility, and anti-neoplastic properties. METHODS: In this study, Saghez-encapsulated BMP2 was applied as a scaffold for wisdom tooth follicle stem cell differentiation into the osteocyte. A total of three wisdom tooth follicles were obtained for stem cell isolation. For verification of differentiation of the isolated stem cells into osteocyte and adipocyte, Oil Red and Alizarin staining were applied, respectively. Moreover, mesenchymal stem cells were distinguished by profiling their cell surface markers, includingCD73, CD90, CD44, and CD105, by flow cytometry. Saghez scaffold loaded with BMP2 factor was prepared using sol-gel method. Four experimental groups were considered in this study: cells seeded on BMP2 encapsulated in Saghez scaffold, Saghez scaffold, osteogenic medium, and DMEM medium. RESULTS: Mechanical properties of Saghez scaffold, including tensile Young's modulus, ultimate tensile stress, compression Young's modulus, and complex shear modulus, were 19 MPa, 32 MPa, 0.42 MPa, and 0.9 MPa, respectively. The porosity of the scaffold was 70-140 µm, and the percentage of porosity was 75-98%. The results of flow cytometry studies indicated that CD44, CD73, CD90, and CD105 were positively expressed on the membrane of the tooth follicles' stem cell. The results indicated that the rate of differentiation of the follicle stem cells into osteocyte was the highest in the Saghez-BMP2 scaffold containing differentiation medium groups. These findings were verified by morphological studies, osteoblast and osteocalcin gene and protein expression investigations, and alkaline phosphatase activity measurement. The highest osteopontin and osteocalcin genes expression levels (1.7 and 1.9) were seen in positive control, followed by DMEM + differentiation factor (1.5 and 1.6), scaffold + BMP2 (1.2 and 1.4), DMEM + stem cell (1 and 1) and scaffold (0.4 and 0.5), and negative control respectively. CONCLUSION: This study provides a novel system for differentiation of the stem cell into osteocytes. The results of this study suggest that loaded BMP2 in Saghez scaffold possibly acts as an osteocyte differentiator factor.

Protein engineering of recombinant human bone morphogenetic protein 2 with higher interaction with Ca phosphate based scaffold used for osteogenesis.

The aim of the present study was to assess the recombinant bonemorphogenetic protein 2 (RHBMP-2) with higher substantively and solubility for use in calcium phosphate scaffolds for better release in differentiation of mesenchymal stem cells to osteoblast cells. Using bioinformatics tools, two mutations (p. L10D and p. S12E) were chosen and applied in BMP2 CDS sequence to increase interaction with calcium derived composite. The new recombinant mutated sequence (BMP2mut ) was synthesized and then subcloned to expression vector pBV220. Experimental data regarded functional protein expression in E. coli. Since no modification was made in the active sites of proteins namely ß-sheets and α-helixes, not only was there any change in the specific activity occurred in the specific activity of the enzyme in comparison to its commercial counterpart, but also mesenchymal osteogenesis occurred more efficient on biphasic CaP scaffold model. As we hypothesized, use of negatively charged amino acids such as aspartate and glutamate in protein loops increased the interactions of BMP2-Ca2+ and resulted in its slower and more sustained released from CaP scaffolds compare to commercial RHBMP2. Our data suggested that new BMP2mut have greater osteoinductive capacity than RHBMP2 in the same time and dose than RHBMP2. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2799-2805, 2017.