The success of biomaterials lies in the direct interaction with the host tissue. Calcium phosphates (CaP) stand as an alternative graft material for bone regeneration due to their similar composition to natural bone. Few studies have focused on the early stages of bone-like material remodeling by osteoclasts (OC), though the CaP fate is to be resorbed and then replaced by new bone. Instead, to understand how osteoclasts modify the CaP surface and initiate resorption, so as to influence subsequent osteoblast activities and bone formation, is mandatory.

Sintered hydroxyapatite (s-HA) and biomimetic hydroxyapatite with two different microstructures (b-HA-C, coarse and b-HA-F, fine) discs (1500×250 µm²) were produced from the same reagents [1]. Tissue culture polystyrene (TCPS) was used as control. Precursor human OC from buffy coats were seeded on ceramic substrates [6·10⁶cells/cm²] and supplemented with RANKL-containing osteoblast supernatant as differentiation medium over 21 days. Cell interaction with the biomaterials was investigated in terms of OC adhesion and differentiation, with gene expression, tartrate-resistant acid phosphatase (TRAP) and Hoechst staining for OC maturation. Cell culture supernatants were analyzed for ionic exchange, namely Ca and P, due to biomaterials or cells. Osteoclasts morphology was evaluated using SEM at 21 days. Innovatively, focused ion beam (FIB) was used to evaluate biomaterial structure beneath the OC to further investigate the resorption effects. To this aim, selected OC were cut cross-sectioned using a Gallium ion beam at an acceleration of 30KV, followed by a coarse milling at 10nA and a deposition of platinum to achieve a fine milling at 500pA.

Clear differences in cellular behavior were noted relative to the different substrate microstructures. Control TCPS and s-HA showed similar TRAP-positive staining and gene expression for mature OC. Several resorption pits with partial dissolution of the equiaxial grains of s-HA were noticed. b-HA substrates also showed attached and differentiated TRAP-positive OC, but gene expression resulted lower than control and s-HA. However, morphological evaluation with SEM-FIB interestingly showed early stages of osteoclast-mediated degradation on b-HA-F, i.e.an increased surface roughness in the substrate underlying cells. B-HA-C also showed attached and mature OC with a scarce degradation activity

FIB technique has been applied to cell-seeded CaP and shown as a viable method to investigate OC morphology and resorption. Though gene expression showed similarities for both biomimetic substrates, substrate morphology observed underneath OC was significantly different. b-HA-F showed early stages of OC mediated degradation underneath well spread cells similar to those seen on s-HA. No resorptive activity was found on b-HA-C even though gene expression values were similar to b-HA-F: both the acute ion exchange and the surface tortuosity on b-HA-C could explain the difficulty with the resorptive process by OC. In conclusion focused ion beam technique complements SEM imaging and may disclose changes in the inner structure of materials due to cell/material interactions.

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.