Engineering Hydroxyapatite-Gelatin Nanocomposites With MAPC Cells For Calvarium Bone Regeneration

Objectives: Adequate bony support is key to reestablish both esthetics and function in the craniofacial region. 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 approaches. Thus, bioengineering techniques have been developed combining biomaterials and cells in constructs to accelerate bone regeneration. 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. Thus, we investigated in vivo applicability of macro-porous titanium dioxide (TiO₂)-enriched HAP-GEL scaffolds with osteogenically-differentiated MAPC (OD-MAPC) aggregates for bone regeneration. Methods: Cell-free macro-porous TiO₂-enriched HAP-GEL scaffolds and scaffolds loaded with 1.5x10⁵ or 6x10⁵ undifferentiated and dexamethasone-differentiated MAPCs (OD-MAPC) from a novel aggregate culture system were implanted in 8mm critical-size defects (CSD) in rat calvaria. Bone autograft was used as positive control. Collagen and HAP-GEL scaffolds without titanium were implanted for comparison purposes. Pre-implanted scaffolds and calvaria bone from pig were analyzed for ultimate compressive strength with an Instron 4411^® apparatus and for porosity with microcomputerized tomography (microCT). Animals were sacrificed at 4, 8 and 12 weeks post-implantation. Osteointegration and newly formed bone (NFB) were assessed by microCT and histology with Stevenel’s Blue and van Gieson, and quantified by calcium fluorescence labeling. Results: We found that TiO₂-HAP-GEL has a comparable strength relative to natural calvaria bone (13.8±4.5MPa and 24.5±8.3MPa, respectively). Porosity was 1.52±0.8mm and 0.64±0.4mm for TiO₂-HAP-GEL and calvaria bone, respectively. Eight and twelve 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 collagen scaffolds (p<0.05). Conclusions: Tissue-engineered TiO₂-enriched HAP-GEL constructs with OD-MAPC aggregates are a potential useful therapeutic approach for calvarium bone regeneration.