Introduction: The next-generation of bone graft substitutes are aimed at better handling properties and improved clinical effectiveness. Currently, injectable calcium phosphates bioceramics are combined with mesenchymal stromal cells (MSC) for effective bone regeneration[1]. The aim of this study was to assess the in vitro biocompatibility of a novel injectable multiphasic bone substitute (MBS) based on polysaccharide gel-coated OsproLife® hydroxyapatite (HA)/tetracalcium phosphate (TTCP) granules combined with bone marrow concentrate (BMC).
Materials and Methods: Gel-coated granules consisted by biphasic blend HA/TTCP (60/40 Wt %, 300–600 µm, Eurocoating, Italy) and an external layer of dried polysaccharide (around 4 Wt %) able to crosslink in presence of divalent cations. BMC was obtained using the IOR-G1 device (Novagenit, Italy). The concentration and viability of nucleated cells (NC) were analysed before and after their isolation from the bone marrow, then the NC recovery was calculated. BMC was characterized for MSC progenitors content performing the CFU assay, and cells were expanded in vitro until passage 10 in order to evaluate cell kinetics. The in vitro experiments were performed combining 200µL of BMC diluted in 200µL of gel cross-linking solution with 0.5cc of gel-coated granules loaded in syringe. The MBS was maintained in culture (Figure.1) for assessing cell viability at time 0, 24, and 96 hours from the preparation using the LIVE/DEAD staining kit. The distribution of viable cells in the product was also studied through confocal microscopy, whereas the osteogenic potential of cells was assessed by Alizarin Red-S (AR-S) staining after 28 days of stimuli-induced differentiation.
Results: NC and MSC progenitors were enriched in the BMC compared to BM by a factor of 7.4 and 3.4, respectively. Biocompatibility analysis highlighted that cells remained viable hereafter the combination with the gel-coated HA/TTCP granules and viability was maintained as long as observed (Figure.2). Confocal imaging showed the cells immersed in the gel immediately after the preparation and the absence of cell adhesion to the granules even after 6 days in culture. The MSC progenitors contained in the MBS were also able to differentiate toward osteoblasts producing an osteogenic matrix as evidenced by AR-S staining.
Discussion: The manipulation for injectable MBS preparation did not cause cell death and MSC progenitors remained viable over time maintaining their differentiation potential. It is noteworthy that mobility of MSC is important when the scaffold is used in clinical surgery for bone reconstruction, and this is possible in gel-based graft substitutes. In fact, MSC progenitors can be potentially released during polysaccharide gel degradation and freely proliferate/differentiate or simply migrate to the inflammatory sites.
Conclusion: Injectable MBS based on gel-coated OsproLife® HA/TTCP granules was proved to be biocompatible and the easy combination with BMC might lead to a more clinically successful “single-step procedure” for bone regeneration.
This work is part of “CaP Project” co-sponsored by Provincia Autonoma di Trento and Eurocoating SpA, Trento, Italy. The authors wish to thank Novagenit S.r.l. for providing the IOR-G1 device for the BM concentration.
References:
[1] Jager et al., Bone Marrow Concentrate: A Novel Strategy for Bone Defect Treatment, Current Stem Cell Research & Therapy, Volume 4, Number 1, 1 January 2009, pp. 34-43(10)