Analysing the cell response to biomaterials for spinal fusion surgery

Abstract

PART1: To date, the gold standard treatment to achieve bone regeneration and new-bone deposition is the Autologous Bone Graft (ABG), a technique that suffers from limited availability and the need for an additional surgical operation. To overcome these limitations, strategies to augment the overall osteoinductivity of synthetic bone grafts have been developed. Among them, strategies aiming to promote bone regeneration through the immune system have reached the forefront of bone regeneration. In fact, immune cells can promote the local release of soluble factors that recruit bone precursor cells and promote their differentiation towards osteoblasts. Recently, a new biphasic calcium phosphate with sub-micron needle-shaped surface topography (BCP<µm) has been developed at Kuros Biosciences capable of promoting macrophage polarization to the M2 phenotype, which has been associated with augmented bone regeneration. A new formulation has been developed consisting of BCP<µm granules embedded in a hydratable collagen matrix (BCP<µm-Collagen). Since the exposure of the needles is key to BCP<µm granules bioactivity, in this study we investigated whether the collage matrix hinders granules’ function. The Alamar blue assay on hMSCs cultured on the surface of the BCP<µm discs or BCP<µm-Collagen for 24 hours showed up to 20-fold increase in cell metabolic activity. Moreover, ALP and DNA quantification after 10 days of stimulation with OM showed up to 2-fold increase in ALP production per cell. These results suggest that collagen substantially favours cell attachment and metabolic activity within the first 24 hours of hMSC culture. Moreover, an increase in osteogenic differentiation towards osteoblast was reported in BCP<µm-Collagen. Hence, the addition of Collagen to BCP<µm granules does not hinder Granules’ function and BCP<µm-Collagen represents a promising bone filler with osteostimulating properties. PART 2: Blood vessels serve as a structure around which bone regeneration occurs, and newly formed blood vessels promote the local accumulation of soluble factors that promote the recruitment and differentiation of bone precursor cells towards osteoblasts. Elastin is a key component of elastic fibers, and in vitro tests showed that elastin promotes the migration and differentiation of HUVECs, which are the most employed cell types to study angiogenesis. To discover whether the addition of elastin to collagen matrices augments their pro-angiogenic properties resulting in more predictable bone regeneration, we cultured HUVECs for 16 hours on the surface of reconstituted collagen or collagen-elastin sponges to assess whether elastin positively regulated network formation. Then, an Alamar blue assay was performed on HUVECs cultured directly onto the surface of the lyophilized sponges to assess whether elastin promotes a higher metabolic activity in HUVECs. The network analysis revealed an increase in the number of junctions, total length, and total branching length when HUVECs were cultured in the presence of elastin compared to pure collagen gels. In particular, a 100% increase in the number of junctions, a 40% increase in total length, and an 82% increase in total branching length were observed in the presence of elastin. Finally, the metabolic assays revealed a 2-fold increase in reduction capability when HUVECs were cultured on the surface of collagen-elastin sponges compared to pure collagen sponges. In conclusion, elastin positively regulates network formation and metabolic activity in HUVECs, hence it represents a promising candidate to confer angiogenic properties to bone implants aiming to augment their overall osteoinductivity.