Acellular mineral deposition within injectable, dual-gelling hydrogels for bone tissue engineering

This study sought to characterize the composition and morphology of acellular mineralization occurring in thermally and chemically gelable hydrogels comprising copolymers of hydrophobic N-isopropylacrylamide as a function of hydrogel hydrophobicity and culture medium formulation. The deposition of calcium phosphate (CaP) mineral was hypothesized to occur with increasing hydrogel hydrophobicity and presence of serum proteins in the culture medium. Two hydrogel compositions with a solid content of 15 and 20 wt % were examined in serum-containing and nonserum-containing media for 0, 14, 28, and 56 days. Using biochemical assays, calcium, but not phosphate content, was found to significantly increase over time in hydrophobic hydrogels soaked in cell culture medium with fetal bovine serum. Significant increases in the calcium to phosphate ratio were observed within these hydrogels from day 0 to 56, with mineralization indicated by von Kossa histological staining. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) were used to analyze CaP mineral characteristics. No crystalline apatitic reflection peaks were observed using XRD, which was supported by the lack of observable mineral deposits as observed using SEM/EDX. However, FTIR showed the presence of new absorption peaks in the serum-containing samples at 28 and 56 days which suggested the formation of an immature apatitic-like mineral. The ability to undergo hydrophobicity-dependent and protein-mediated mineralization demonstrates the potential of these dual-gelling hydrogels as acellular self-mineralizing materials for bone tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 110-117, 2017.