The use of nanosized hydroxyapatite on silk fibroin hydrogels to improve osteointegration

摘要

Introduction: The development of scaffolds to repair bone defects is the main purpose in bone tissue engineering. The use of hydrogels as synthetic extracellular matrix is drawing a great attention due to their high water content and consequent viscoelastic and diffusive transport properties. In this context, a hydrogel, that will act as a temporary matrix must be cytocompatible and have a number of biological functions actively encouraging cells ingrowth. Silk fibroin (SF) is a natural polymer which possesses important properties for bone tissue engineering, including biocompatibility, biodegradability, high permeability to oxygen and water vapor, and low-costW. This work aimed to investigate the influence of the addition of nanosized hydroxyapatite (nanoHA) on SF hydrogels, and to evaluate cell-matrix surface interactions. Materials and Methods: The cocoons of Bombyx mori were degummed with Na_2CO_3 solution to remove the sericin and the SF fibers were dissolved in a ternary solvent of CaCl_2:CH_3CH_2OH:H_2O. Composite hydrogels were obtained using an innovative technique where the dry powder of nanoHA aggregates was first mixed with ethanol at 70 vol% and then slowly mixed with the SF aqueous solution, prior to hydrogel formation. The hydrogel structure was investigated by SEM, XRD and FTIR. For the biological characterization, human Bone Marrow Stromal Cells (hBMSCs) were seeded on the top of each hydrogel and cultured up to 21 days. The cells-seeded hydrogels were assessed for cell viability and proliferation, and observed by CLSM. Results and Discussion: Morphological study revealed that the composite hydrogels presented both macro- and microporous structures, providing good conditions for cell proliferation and diffusion of nutrients and metabolites. The nanoHA particles were embedded throughout the SF matrix (Figure 1 A). Both XRD and FTIR spectra showed that composite hydrogels simultaneously exhibited characteristic peaks from nanoHA and a predominant structure of silk Ⅱ. Concerning the biological properties, the increase of metabolic activity and proliferation of hBMSCs on hydrogels over time indicates the cytocompatible and non-toxic nature. The higher metabolic activity and proliferation was achieved when the hBMSCs were cultured on composite hydrogels with nanoHA, indicating that the nanoHA may play a major role in metabolic cell activities and proliferation. These results were well correlated with confocal images that showed extensive cell spreading and proliferation on the hydrogels, and at day 21 cells completely covered the surface of SFanoH A hydrogels with a dense cell layer (Figure 1B). Conclusion: In the present work it was shown that the addition of nanoHA on SF hydrogels allowed optimizing biological properties, improving hBMSCs attachment, viability and proliferation. This combination of macro- and microporous material and biological properties provide a promising hydrogel for bone tissue engineering for temporary supporting the formation of new tissue.