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Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. 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 remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation.

Inspired by nature, poly(acrylic acid) (PAA) and other polyelectrolytes have been used as noncollagenous proteins (NCPs) surrogates for biomimetic intrafibrillar mineralization of collagen fibrils and, thus, to model the ultrastructure of bone, to study the mechanism of bone mineralization, and more scarcely to fabricate scaffolds for hard tissue engineering. The objective of this study was to systematically investigate the effect of the molecular weight (MW) and the concentration of PAA on the rate and pattern of biomineralization of collagen matrices. Densified type I collagen films were mineralized in supersaturated PAA-stabilized amorphous calcium-phosphate (PAA-ACP) solutions containing increasing MW (2 kDa, 50 kDa, 450 kDa) and concentrations (10, 25, 50 mg/L) of PAA up to 7 days. The stability and physical properties of collagen-free PAA-ACP solutions were also investigated. In our system, lowering PAA MW and increasing PAA concentration resulted in solutions with increasing stability. Overstable PAA-ACP solutions that fully inhibited mineralization of the collagen matrices were achieved using PAA 2kDa-50mg/L. Conversely, unstable solutions were obtained using high PAA MW at low concentrations. Nucleation and growth of a significant amount of extrafibrillar minerals on the collagen fibrils was obtained using these solutions. In a wide range of combined MW and concentration of PAA, we obtained intrafibrillar mineralization of collagen with hydroxyapatite crystals aligned parallel to the collagen fibril as in natural tissues. Intrafibrillar mineralization was correlated with PAA-ACP stability and growth of the PAA-ACP particles in solution. Our results support using PAA to act as a surrogate to NCPs function as selective inhibitors or promoters of biological mineralization and provide parameters to manufacture new biomimetic scaffolds and constructs for bone and dentin tissue engineering.

Calcium phosphates have long been used as synthetic bone grafts. Recent studies have shown that the modulation of composition and textural properties, such as nano-, micro- and macro-porosity, is a powerful strategy to control and synchronize material resorption and bone formation.Biomimetic calcium phosphates, which closely mimic the composition and structure of bone mineral, can be produced using low-temperature processing routes, and offer the possibility to modulate the material properties to a larger extent than conventional high temperature sintering processes.Advanced technologies open up new possibilities in the design of bioceramics for bone regeneration; 3D-printing technologies, in combination with the development of hybrid materials with enhanced mechanical properties, supported by finite element modelling tools, are expected to enable the design and fabrication of mechanically competent patient-specific bone grafts.The association of ions, drugs and cells allows leveraging of the osteogenic potential of bioceramic scaffolds in compromised clinical situations, where the intrinsic bone regeneration potential is impaired.Cite this article: EFORTOpen Rev 2018;3 DOI: 10.1302/2058-5241.3.170056

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 (SSA) and micro- or nanometric crystal size. The aim of this article 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 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 SSA, 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, while 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 a higher alkaline phosphatase activity for both cell types compared to sintered CaPs, indicating a promotion of the osteoblastic phenotype.

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.

This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low- and high-temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize.

The success or failure of a material when implanted in the body is greatly determined by the surface properties of the material and the host tissue reactions. The very first event that takes place after implantation is the interaction of soluble ions, molecules and proteins from the biological environment with the material surface leading to the formation of an adsorbed protein layer that will later influence cell attachment. In this context, the particular topography and surface charge of a material become critical as they influence the nature of the proteins that will adsorb. However, very limited information is available on the surface charge of porous substrates. Only until very recently was the determination of the zeta potential on porous membranes accurately determined. The goal of this work was to implement the previous findings for the determination of the zeta potential of a series of porous hydroxyapatite (HA) substrates and to assess how porosity affects the measurements. In addition, studies using various electrolytes were also performed to prove how the specific affinity of certain ions for HA can further impact surface charge. The results showed that all materials exhibited very similar external surface charge (approximately −23 mV), consistent with their almost identical topographies. However, the presence of interconnected pores underneath the sample surface resulted in an additional internal zeta potential that varied with the porosity content. Measurements with different electrolytes confirmed the selectivity of divalent ions for HA underlying the importance of testing biomaterials using relevant electrolytes.

Since the discovery that nanostructured surfaces were able to kill bacteria, many works have been published focusing on the design of nanopatterned surfaces with antimicrobial properties. Synthetic bone grafts, based on calcium phosphate (CaP) formulations, can greatly benefit from this discovery if adequate nanotopographies can be developed. However, CaP are reactive materials and experience ionic exchanges when placed into aqueous solutions which may in turn affect cell behaviour and complicate the interpretation of the bactericidal results. The present study explores the bactericidal potential of two nanopillared CaP prepared by hydrolysis of two different sizes of α-tricalcium phosphate (α-TCP) powders under biomimetic or hydrothermal conditions. A more lethal bactericidal response toward Pseudomonas aeruginosa(~75% killing efficiency of adhered bacteria) was obtained from the hydrothermally treated CaP which consisted in a more irregular topography in terms of pillar size (radius: 20–60 nm), interpillar distances (100–1500 nm) and pillar distribution (pillar groups forming bouquets) than the biomimetically treated one (radius: 20–40 nm and interpillar distances: 50–200 nm with a homogeneous pillar distribution). The material reactivity was greatly influenced by the type of medium (nutrient-rich versus nutrient-free) and the presence or not of bacteria. A lower reactivity and superior bacterial attachment were observed in the nutrient-free medium while a lower attachment was observed for the nutrient rich medium which was explained by a superior reactivity of the material paired with the lower tendency of planktonic bacteria to adhere on surfaces in the presence of nutrients. Importantly, the ionic exchanges produced by the presence of materials were not toxic to planktonic cells. Thus, we can conclude that topography was the main contributor to mortality in the bacterial adhesion tests.

Immune cells play a vital role in regulating bone dynamics. This has boosted the interest in developing biomaterials that can modulate both the immune and skeletal systems. In this study, calcium phosphates discs (i.e., beta‐tricalcium phosphate, β‐TCP) are functionalized with heparin to investigate the effects on immune and stem cell responses. The results show that the functionalized surfaces downregulate the release of hydrogen peroxide and proinflammatory cytokines (tumor necrosis factor alpha and interleukin 1 beta) from human monocytes and neutrophils, compared to nonfunctionalized discs. The macrophages show both elongated and round shapes on the two ceramic substrates, but the morphology of cells on heparinized β‐TCP tends toward a higher elongation after 72 h. The heparinized substrates support rat mesenchymal stem cell (MSC) adhesion and proliferation, and anticipate the differentiation toward the osteoblastic lineage as compared to β‐TCP and control. The coupling between the inflammatory response and osteogenesis is assessed by culturing MSCs with the macrophage supernatants. The downregulation of inflammation in contact with the heparinized substrates induces higher expression of bone‐related markers by MSCs. Heparinized calcium phosphatesrepresent an excellent platform to modulate osteoimmune response. Heparinization offers a wide range of benefits observed by the downregulation of oxidative stress, inflammation and the improvement of mesenchymal stem cell differentiation. Moreover, a coupling of inflammation and osteogenesis, investigated through the cytokine containing supernatants from macrophage cell cultures, is evidenced by a pro‐osteogenic effect on rat mesenchymal stem cell.