Content-Dependent Osteogenic Response of Nanohydroxyapatite: An in Vitro and in Vivo Assessment within Collagen-Based Scaffolds

The use of collagen-based scaffolds in orthopedic applications has been limited due to poor mechanical properties, but this may be overcome by the introduction of a stiffer supporting phase. Thus, we developed a synthesis technique to produce nonaggregating, stable nanohydroxyapatite (nHA) particles, permitting the fabrication of biomimetic-inspired scaffolds through the combination of nanosized HA with collagen, as found in native bone. This study evaluates the mechanical and biological impact of incorporating increasing concentrations of these nanoparticles into porous collagen scaffolds (1:1 and 5:1 weight ratios of nHA/collagen). Mechanical assessment demonstrated that increasing nHA incorporation correlated with increasing Young's moduli, which could be further amplified using cross-linking treatments. Typically, the porosity of a scaffold is sacrificed to produce a stiffer material; however, through the use of nanosized particles the inclusion of up to 5:1 nHA/collagen content still preserved the high 99% porosity of the composite scaffold, allowing for maximum cell infiltration. Moreover, increasing nHA presence induced significant bioactive responses, achieving superior cellular attachment and enhanced osteogenesis, promoting earlier expression of bone markers and cell-mediated mineralization versus nHA-free collagen controls. Interestingly, these content-dependent results observed in vitro did not directly translate in vivo. Instead, similar levels of bone formation were achieved within critical-sized rat calvarial defects, independent of nHA content, following acellular implantation. The addition of nHA, both 1:1 and 5:1, induced significantly higher levels of mineralization and de novo bone ingrowth versus collagen controls as demonstrated by microcomputed tomography, histological, and histomorphometric analyses. Ultimately, these results demonstrate the immense osteoinductivity of nonaggregated nanoparticles of HA incorporated into collagen-composite scaffolds and emphasize the importance of in vivo-based evaluation of therapies intended for clinical use.