Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and β-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.

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The regulatory role of the immune system in maintaining bone homeostasis and restoring its functionality, when disturbed due to trauma or injury, has become evident in recent years. The polarization...

Immune cells are sensitive to the microstructural and textural properties of materials. Tuning the structural features of synthetic bone grafts could be a valuable strategy to regulate the specific response of the immune system, which in turn modulates the activity of bone cells. The aim of this study was to analyse the effect of the structural characteristics of biomimetic calcium deficient hydroxyapatite (CDHA) on the innate immune response of macrophages and the subsequent impact on osteogenesis and osteoclastogenesis. Murine RAW 264.7 cells were cultured, under standard and inflammatory conditions, on chemically identical CDHA substrates that varied in microstructure and porosity. The impact on osteogenesis was evaluated by incubating osteoblastic cells (SaOS-2) with RAW-CDHA conditioned extracts. The results showed that macrophages were sensitive to different textural and structural properties of CDHA. Under standard conditions, the impact of inflammatory cytokine production by RAW cells cultured on CDHA played a significant role in the degradation of substrates, suggesting the impact of resorptive behaviour of RAW cells on biomimetic surfaces. Osteoblast differentiation was stimulated by the conditioned media collected from RAW cells cultured on needle-like nanostructured CDHA. The results demonstrated that needle-like nanostructured CDHA was able to generate a favourable osteoimmune environment to regulate osteoblast differentiation and osteogenesis. Under inflammatory conditions, the incubation of RAW cells with less porous CDHA resulted in a decreased gene expression and release of pro-inflammatory cytokines.

The fabrication of calcium phosphates using biomimetic routes, namely, precipitation processes at body temperature, results in distinct features compared to conventional sintered calcium phosphate ceramics, such as a high specific surface area and micro or nanometric crystal size. The aim of this paper is to analyze the effects of these parameters on cell response, focusing on two bone cell types: rat mesenchymal stem cells (rMSCs) and human osteoblastic cells (SaOS-2). Biomimetic calcium deficient hydroxyapatite (CDHA) was obtained by a low temperature setting reaction, and α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP) were subsequently obtained by sintering CDHA either at 1400 °C or 1100 °C. Sintered stoichiometric hydroxyapatite (HA) was also prepared using ceramic routes. The materials were characterized in terms of specific surface area, skeletal density, porosity and pore size distribution. SaOS-2 cells and rMSCs were seeded either directly on the surfaces of the materials or on glass coverslips subsequently placed on top of the materials to expose the cells to the CaP-induced ionic changes in the culture medium while avoiding any topography-related effects. CDHA produced higher ionic fluctuations in both cell culture media than sintered ceramics, with a strong decrease of calcium and a release of phosphate. Indirect contact cell cultures revealed that both cell types were sensitive to these ionic modifications, resulting in a decrease in proliferation rate, more marked for CDHA, this effect being more pronounced for rMSCs. In direct contact cultures, good cell adhesion was found on all materials, but whereas cells were able to proliferate on the sintered calcium phosphates, cell number was significantly reduced with time on biomimetic CDHA, which was associated to a higher percentage of apoptotic cells. Direct contact of the cells with biomimetic CDHA resulted also in higher ALP activity for both cell types as compared to sintered CaPs, indicating a promotion of the osteoblastic phenotype.

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Fluid mediated interactions play a significant role in the bioactivity of calcium phosphates. Ionic exchange is amplified in the case of biomimetic hydroxyapatite, which makes the in vitro characterisation of cell-material interactions especially challenging. The present work proposes a novel and simple strategy to explore the mechanisms of interaction of biomimetic and sintered calcium phosphates with mesenchymal stem cells. The effects of topography and ion exchange are analysed separately by modifying the volume ratio between cell culture medium and biomaterial. High ionic fluctuations interfered in the maturation of focal adhesions, hampering cell adhesion and leading to increased apoptosis and reduced proliferation rate.

Bone apatite consists in carbonated calcium deficient hydroxyapatite nanocrystals (CDHA). Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely nanocrystal morphology (plate vs. needle-like crystals) and carbonate content on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical Coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone healing capacity showed by the Fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodelling cycle, promoting a tight synchronization between scaffold degradation and bone formation.