The regenerative potential of bone grafts is tightly linked to the interaction of the biomaterial with the host tissue environment. Hence, strategies to confer artificial extracellular matrix (aECM) cues on the material surface are becoming a powerful tool to trigger the healing cascade and to stimulate bone regeneration. The use of glycosaminoglycans (GAGs), such as heparin, as aECM components has gained interest in the last years as a strategy to improve biological response. Calcium phosphates (CaP) are extensively used as bone grafts, however no studies have investigated the effect of GAG functionalisation on their surface. Some authors have focused on the effects of GAGs on osteoblastic cells, however, little work has been performed on the interaction with osteoclasts (OC), and still the reported effects are controversial [1]. The aim of this study was to investigate the effect of heparin on osteoclastic fate in terms of adhesion and differentiation.

Sintered CaP (β-TCP) and biomimetic CaP (calcium-deficient hydroxyapatite, CDHA) discs were synthesized at 1100 ºC and at 37ºC, respectively. Heparinisation was achieved though silane coupling (APTES) followed by amidation in the presence of EDC/NHS to covalently link heparin. The osteoclast response of heparinised (H) vsnon-heparinised substrates was studied using human monocytes as OC precursors. Tissue culture plastic (TCPS) was used as a control sample. Cell densities were 6·10⁶and 3·10⁶cells/cm²for biomaterials and TCPS, respectively. Cell cultures were supplemented every 3 days with 25% supernatant of osteoblast-like cell line as a source of RANKL, as well as other stimulating factors [2]. Tartrate-resistant acid phosphatase and Hoechst staining were used to evaluate OC adhesion, differentiation and morphology at different time points from seeding on the surfaces (14–21–28 days).

OC precursors showed adhesion on all substrates. β-TCP and β-TCP-H hosted higher number of OC precursors which might be related to the smoother sintered surface of the materials. Oppositely, the high roughness of CDHA and CDHA-H hamper the adhesion of OC, hence a lower number of cells was observed on heparin-coated and uncoated biomimetic apatites. However, the maturation of OC precursors was found to take place at earlier times (14days) on biomimetic substrates compared to sintered ones. TCPS, CDHA, CDHA-H and β-TCP-H showed clearly differentiated OC at 14 days, as revealed by TRAP positivity and multinuclearity. Interestingly, CDHA-H and β-TCP-H induced the highest multinuclearity among all differentiated OC. Both heparinised substrates point at an enhancing effect of heparin on OC maturation.

OC precursors are able to differentiate on β-TCP and CDHA substrates, a process enhanced when heparin functionalisation is performed on the materials surface. In our hands heparinisation is promoting OC differentiation at early time points, similarly to TCPS control. Interestingly, heparin substrates induced larger TRAP positive-OC and higher multinuclearity in the mature OC than TCPS control. As pointed out by Irie et al., heparin might interact through the RANKL/OPG ratio [3], thus inhibiting OPG activity and enhancing RANKL which triggers OC maturation.