Engineering hydroxyapatite-gelatin nanocomposites with MAPC cells for calvarium bone regeneration

Abstract

Bones of the craniofacial skeleton provide the foundation upon which other complex facial organs are built. Adequate bony support is key to reestablish both esthetics and function. Autologous bone grafting has been the gold standard for the challenging repair of large bone defects; however, availability and donor site complications have led to alternative substitutes such as allogeneic or synthetic grafts despite their inconsistent performance. To overcome these challenges, bioengineering techniques have been developed combining biomaterials and cells in constructs to accelerate bone regeneration restoring normal function. Thus, we proposed to develop a bioengineering approach combining a novel hydroxyapatite-gelatin biomaterial (HAP-GEL) and multipotent adult progenitor cells (MAPCs) for craniofacial bone regeneration. In these dissertation studies, we: (1) designed an injectable HAP-GEL scaffold with appropriate mechanical and biocompatibility properties upon adding aminosilane cross-linker (enTMOS) and titanium dioxide (TiO ₂) by determining setting time, and performing compression and cell viability testing with pre-osteoblast cultures; (2) determined factors, specifically dexamethasone and TiO₂, regulating in vitro MAPC osteogenesis in monolayer and aggregate culture systems, by assessing the bone-like phenotype in MAPC matrices differentiated with different concentrations of dexamethasone and in TiO₂ surfaces; and (3) investigated in vivo applicability of macro-porous TiO₂-enriched HAP-GEL scaffolds with dexamethasone-differentiated MAPC aggregates for bone regeneration by implanting scaffolds in critical-size defects (CSD) in rat calvaria and determining the newly formed bone (NFB) after microcomputerized tomography, histology, and calcium fluorescence labeling. We found that TiO₂-HAP-GEL were formable organoceramics with an optimal acceptable strength (70MPa) after addition of enTMOS 11-19wt.%. The TiO₂ increased the biocompatibility of HAP-GEL and its osteo-inductive properties in pre-osteoblast and MAPC cultures with dexamethasone at 10⁻⁷M. _{In vitro}, osteogenic dexamethasone-differentiated MAPC (OD-MAPCs) aggregates generated a bone-like matrix with abundant mature collagen and mineralization. Ten days post-implantation in calvaria CSD, OD-MAPC aggregates were retained and growing on TiO ₂-HAP-GEL carrier macro-pores, after an adeno-associated virus with fluorescent protein vector was transduced to track these aggregates. Eight weeks post-implantation, osteointegration and NFB was demonstrated to be significantly greater in tissue-engineered TiO₂-enriched HAP-GEL constructs with OD-MAPCs, when compared to MAPC-loaded constructs, cell-free HAP-GEL with and without titanium, and other commonly used polymer-based scaffolds.