Static 3D Osteoblast Cell Culture on 3D Printed Titanium Scaffolds.

In vitro cell cultures are a very useful tool for the validation of biomaterial cytocompatibility, especially for bone tissue engineering scaffolds and bone implants. In this chapter, a protocol for a static three-dimensional osteoblast cell culture on titanium scaffolds and subsequent analysis of osteogenic capacity is presented. The protocol is explained for additively manufactured titanium scaffolds, but it can be extrapolated to other scaffolds with similar size and structure, while differing in composition or manufactured technology. Additionally, the protocol can be used for culture of other adherent cell types beyond osteoblast cells such as mesenchymal stem cells.

High-aspect-ratio nanostructured hydroxyapatite: towards new functionalities for a classical material.

Hydroxyapatite-based materials have been widely used in countless applications, such as bone regeneration, catalysis, air and water purification or protein separation. Recently, much interest has been given to controlling the aspect ratio of hydroxyapatite crystals from bulk samples. The ability to exert control over the aspect ratio may revolutionize the applications of these materials towards new functional materials. Controlling the shape, size and orientation of HA crystals allows obtaining high aspect ratio structures, improving several key properties of HA materials such as molecule adsorption, ion exchange, catalytic reactions, and even overcoming the well-known brittleness of ceramic materials. Regulating the morphogenesis of HA crystals to form elongated oriented fibres has led to flexible inorganic synthetic sponges, aerogels, membranes, papers, among others, with applications in sustainability, energy and catalysis, and especially in the biomedical field.

Trabecular Titanium for Orthopedic Applications: Balancing Antimicrobial with Osteoconductive Properties by Varying Silver Contents.

Periprosthetic joint infection (PJI) and implant loosening are the most common complications after joint replacement surgery. Due to their increased surface area, additively manufactured porous metallic implants provide optimal osseointegration but they are also highly susceptible to bacterial colonization. Antibacterial surface coatings of porous metals that do not inhibit osseointegration are therefore highly desirable. The potential of silver coatings on arthroplasty implants to inhibit PJI has been demonstrated, but the optimal silver content and release kinetics have not yet been defined. A tight control over the silver deposition coatings can help overcome bacterial infections while reducing cytotoxicity to human cells. In this regard, porous titanium sputtered with silver and titanium nitride with increasing silver contents enabled controlling the antibacterial effect against common PJI pathogens while maintaining the metabolic activity of human primary cells. Electron beam melting additively manufactured titanium alloys, coated with increasing silver contents, were physico-chemically characterized and investigated for effects against common PJI pathogens. Silver contents from 7 at % to 18 at % of silver were effective in reducing bacterial growth and biofilm formation. Staphylococcus epidermidis was more susceptible to silver ions than Staphylococcus aureus. Importantly, all silver-coated titanium scaffolds supported primary human osteoblasts proliferation, differentiation, and mineralization up to 28 days. A slight reduction of cell metabolic activity was observed at earlier time points, but no detrimental effects were found at the end of the culture period. Silver release from the silver-coated scaffolds also had no measurable effects on primary osteoblast gene expression since similar expression of genes related to osteogenesis was observed regardless the presence of silver. The investigated silver-coated porous titanium scaffolds may thus enhance osseointegration while reducing the risk of biofilm formation by the most common clinically encountered pathogens.

The Effect of PCL Addition on 3D-Printable PLA/HA Composite Filaments for the Treatment of Bone Defects.

The still-growing field of additive manufacturing (AM), which includes 3D printing, has enabled manufacturing of patient-specific medical devices with high geometrical accuracy in a relatively quick manner. However, the development of materials with specific properties is still ongoing, including those for enhanced bone-repair applications. Such applications seek materials with tailored mechanical properties close to bone tissue and, importantly, that can serve as temporary supports, allowing for new bone ingrowth while the material is resorbed. Thus, controlling the resorption rate of materials for bone applications can support bone healing by balancing new tissue formation and implant resorption. In this regard, this work aimed to study the combination of polylactic acid (PLA), polycaprolactone (PCL) and hydroxyapatite (HA) to develop customized biocompatible and bioresorbable polymer-based composite filaments, through extrusion, for fused filament fabrication (FFF) printing. PLA and PCL were used as supporting polymer matrices while HA was added to enhance the biological activity. The materials were characterized in terms of mechanical properties, thermal stability, chemical composition and morphology. An accelerated degradation study was executed to investigate the impact of degradation on the above-mentioned properties. The results showed that the materials' chemical compositions were not affected by the extrusion nor the printing process. All materials exhibited higher mechanical properties than human trabecular bone, even after degradation with a mass loss of around 30% for the polymer blends and 60% for the composites. It was also apparent that the mineral accelerated the polymer degradation significantly, which can be advantageous for a faster healing time, where support is required only for a shorter time period.

A microfluidic-based approach to investigate the inflammatory response of macrophages to pristine and drug-loaded nanostructured hydroxyapatite.

The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 â€‹cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.

3D-printed porous Ti6Al4V alloys with silver coating combine osteocompatibility and antimicrobial properties.

Additive manufacturing allows for the production of porous metallic implants for use in orthopaedics, providing excellent mechanical stability and osseointegration. However, the increased surface area of such porous implants also renders them susceptible to bacterial colonization. In this work, two trabecular porous Ti6Al4V alloys produced by electron beam melting were investigated for their osteocompatibility and antimicrobial effects, comparing samples with a silver-coated surface to uncoated samples. Dense grit-blasted Ti samples were used for comparison. The porous samples had pore sizes of 500-600 µm and 5 to 10 µm surface roughness, the silver-coated samples contained 7 at.% Ag, resulting in a cumulative Ag release of 3.5 ppm up to 28 days. Silver reduced the adhesion of Staphylococcus aureus to porous samples and inhibited 72 h biofilm formation by Staphylococcus epidermidis but not that of S. aureus. Primary human osteoblast adhesion, proliferation and differentiation were not impaired in the presence of silver, and expression of osteogenic genes as well as production of mineralized matrix were similar on silver-coated and uncoated samples. Our findings indicate that silver coating of porous titanium implants can achieve antimicrobial effects without compromising osteocompatibility, but higher silver contents may be needed to yield a sustained protection against fast-growing bacteria.

A practical guide for evaluating the osteoimmunomodulatory properties of biomaterials.

Biomaterials offer a promising approach to repair bone defects. Whereas traditional studies predominantly focused on optimizing the osteogenic capacity of biomaterials, less focus has been on the immune response elicited by them. However, the immune and skeletal systems extensively interact, a concept which is referred to as 'osteoimmunology'. This realization has fuelled the development of biomaterials with favourable osteoimmunomodulatory (OIM) properties, aiming to modulate the immune response and to support bone regeneration, thereby affecting the success of an implant. Given the plethora of in vitro assays used to evaluate the OIM properties of biomaterials, it may be challenging to select the right methods to produce conclusive results. In this review, we aim to provide a comprehensive and practical guide for researchers interested in studying the OIM properties of biomaterials in vitro. After a concise overview of the concept of osteoimmunology, emphasis is put on the methodologies that are regularly used to evaluate the OIM properties of biomaterials. First, a description of the most commonly used cell types and cell culture media is provided. Second, typical experimental set-ups and their relevant characteristics are discussed. Third, a detailed overview of the generally used methodologies and readouts, including cell type-specific markers and time points of analysis, is given. Finally, we highlight the promise of advanced approaches, namely microarrays, bioreactors and microfluidic-based systems, and the potential that these may offer to the osteoimmunology field. STATEMENT OF SIGNIFICANCE: Osteoimmunology focuses on the connection and communication between the skeletal and immune systems. This interaction has been recognized to play an important role in the clinical success of biomaterials, which has resulted in an increasing amount of research on the osteoimmunomodulatory (OIM) properties of biomaterials. However, the amount of literature makes it challenging to extract the information needed to design experiments from beginning to end, and to compare obtained results to existing work. This article intends to serve as a guide for those aiming to learn more about the commonly used experimental approaches in the field. We cover early-stage choices, such as cell types and experimental set-ups, but also discuss specific assays, including cell markers and time points of analysis.

Hexagonal pore geometry and the presence of hydroxyapatite enhance deposition of mineralized bone matrix on additively manufactured polylactic acid scaffolds.

Additive manufacturing (AM) has revolutionized the design of regenerative scaffolds for orthopaedic applications, enabling customizable geometric designs and material compositions that mimic bone. However, the available evidence is contradictory with respect to which geometric designs and material compositions are optimal. There is a lack of studies that systematically compare different pore sizes and geometries in conjunction with the presence or absence of calcium phosphates. We therefore evaluated the physicochemical and biological properties of additively manufactured scaffolds based on polylactic acid (PLA) in combination with hydroxyapatite (HA). HA was either incorporated in the polymeric matrix or introduced as a coating, yielding 15 and 2% wt., respectively. Pore sizes of the scaffolds varied between 200 and 450 µm and were shaped either triangularly or hexagonally. All scaffolds supported the adhesion, proliferation and differentiation of both primary mouse osteoblasts and osteosarcoma cells up to four weeks, with only small differences in the production of alkaline phosphatase (ALP) between cells grown on different pore geometries and material compositions. However, mineralization of the PLA scaffolds was substantially enhanced in the presence of HA, either embedded in the PLA matrix or as a coating at the surface level, and by larger hexagonal pores. In conclusion, customized HA/PLA composite porous scaffolds intended for the repair of critical size bone defects were obtained by a cost-effective AM method. Our findings indicate that the analysis of osteoblast adhesion and differentiation on experimental scaffolds alone is inconclusive without the assessment of mineralization, and the effects of geometry and composition on bone matrix deposition must be carefully considered in order to understand the regenerative potential of experimental scaffolds.

The role of silver coating for arthroplasty components.

Periprosthetic joint infection (PJI) is one of the most dreaded complications after arthroplasty surgery; thus numerous approaches have been undertaken to equip metal surfaces with antibacterial properties. Due to its antimicrobial effects, silver is a promising coating for metallic surfaces, and several types of silver-coated arthroplasty implants are in clinical use today. However, silver can also exert toxic effects on eukaryotic cells both in the immediate vicinity of the coated implants and systemically. In most clinically-used implants, silver coatings are applied on bulk components that are not in direct contact with bone, such as in partial or total long bone arthroplasties used in tumour or complex revision surgery. These implants differ considerably in the coating method, total silver content, and silver release rates. Safety issues, such as the occurrence of argyria, have been a cause for concern, and the efficacy of silver coatings in terms of preventing PJI is also controversial. The application of silver coatings is uncommon on parts of implants intended for cementless fixation in host bone, but this option might be highly desirable since the modification of implant surfaces in order to improve osteoconductivity can also increase bacterial adhesion. Therefore, an optimal silver content that inhibits bacterial colonization while maintaining osteoconductivity is crucial if silver were to be applied as a coating on parts intended for bone contact. This review summarizes the different methods used to apply silver coatings to arthroplasty components, with a focus on the amount and duration of silver release from the different coatings; the available experience with silver-coated implants that are in clinical use today; and future strategies to balance the effects of silver on bacteria and eukaryotic cells, and to develop silver-coated titanium components suitable for bone ingrowth. Cite this article: Bone Joint J 2021;103-B(3):423-429.

Porous polylactic acid scaffolds for bone regeneration: A study of additively manufactured triply periodic minimal surfaces and their osteogenic potential.

Three different triply periodic minimal surfaces (TPMS) with three levels of porosity within those of cancellous bone were investigated as potential bone scaffolds. TPMS have emerged as potential designs to resemble the complex mechanical and mass transport properties of bone. Diamond, Schwarz, and Gyroid structures were 3D printed in polylactic acid, a resorbable medical grade material. The 3D printed structures were investigated for printing feasibility, and assessed by morphometric studies. Mechanical properties and permeability investigations resulted in similar values to cancellous bone. The morphometric analyses showed three different patterns of pore distribution: mono-, bi-, and multimodal pores. Subsequently, biological activity investigated with pre-osteoblastic cell lines showed no signs of cytotoxicity, and the scaffolds supported cell proliferation up to 3 weeks. Cell differentiation investigated by alkaline phosphatase showed an improvement for higher porosities and multimodal pore distributions, suggesting a higher dependency on pore distribution and size than the level of interconnectivity.

3D-printed PLA/HA composite structures as synthetic trabecular bone: A feasibility study using fused deposition modeling.

Additive manufacturing has significant advantages, in the biomedical field, allowing for customized medical products where complex architectures can be achieved directly. While additive manufacturing can be used to fabricate synthetic bone models, this approach is limited by the printing resolution, at the level of the trabecular bone architecture. Therefore, the aim of this study was to evaluate the possibilities of using fused deposition modeling (FDM) to this end. To better mimic real bone, both in terms of mechanical properties and biodegradability, a composite of degradable polymer, poly(lactic acid) (PLA), and hydroxyapatite (HA) was used as the filament. Three PLA/HA composite formulations with 5-10-15 wt% HA were evaluated, and scaled up human trabecular bone models were printed using these materials. Morphometric and mechanical properties of the printed models were evaluated by micro-computed tomography, compression and screw pull out tests. It was shown that the trabecular architecture could be reproduced with FDM and PLA by applying a scaling factor of 2-4. The incorporation of HA particles reduced the printing accuracy, with respect to morphology, but showed potential for enhancement of the mechanical properties. The scaled-up models displayed comparable, or slightly enhanced, strength compared to the commonly used polymeric foam synthetic bone models (i.e. Sawbones). Reproducing the trabecular morphology by 3D printed PLA/HA composites appears to be a promising strategy for synthetic bone models, when high printed resolution can be achieved.

Effect of calcium phosphate heparinization on the in vitro inflammatory response and osteoclastogenesis of human blood precursor cells.

The immobilization of natural molecules on synthetic bone grafts stands as a strategy to enhance their biological interactions. During the early stages of healing, immune cells and osteoclasts (OC) modulate the inflammatory response and resorb the biomaterial, respectively. In this study, heparin, a naturally occurring molecule in the bone extracellular matrix, was covalently immobilized on biomimetic calcium-deficient hydroxyapatite (CDHA). The effect of heparin-functionalized CDHA on inflammation and osteoclastogenesis was investigated using primary human cells and compared with pristine CDHA and beta-tricalcium phosphate (ß-TCP). Biomimetic substrates led to lower oxidative stresses by neutrophils and monocytes than sintered ß-TCP, even though no further reduction was induced by the presence of heparin. In contrast, heparinized CDHA fostered osteoclastogenesis. Optical images of stained TRAP positive cells showed an earlier and higher presence of multinucleated cells, compatible with OC at 14 days, while pristine CDHA and ß-TCP present OC at 21-28 days. Although no statistically significant differences were found in the OC activity, microscopy images evidenced early stages of degradation on heparinized CDHA, compatible with osteoclastic resorption. Overall, the results suggest that the functionalization with heparin fostered the formation and activity of OC, thus offering a promising strategy to integrate biomaterials in the bone remodelling cycle by increasing their OC-mediated resorption.

Impact of Biomimicry in the Design of Osteoinductive Bone Substitutes: Nanoscale Matters.

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.

Osteogenesis by foamed and 3D-printed nanostructured calcium phosphate scaffolds: Effect of pore architecture.

There is an urgent need of synthetic bone grafts with enhanced osteogenic capacity. This can be achieved by combining biomaterials with exogenous growth factors, which however can have numerous undesired side effects, but also by tuning the intrinsic biomaterial properties. In a previous study, we showed the synergistic effect of nanostructure and pore architecture of biomimetic calcium deficient hydroxyapatite (CDHA) scaffolds in enhancing osteoinduction, i.e. fostering the differentiation of mesenchymal stem cells to bone forming cells. This was demonstrated by assessing bone formation after implanting the scaffolds intramuscularly. The present study goes one step forward, since it analyzes the effect of the geometrical features of the same CDHA scaffolds, obtained either by 3D-printing or by foaming, on the osteogenic potential and resorption behaviour in a bony environment. After 6 and 12 weeks of intraosseous implantation, both bone formation and material degradation had been drastically affected by the macropore architecture of the scaffolds. Whereas nanostructured CDHA was shown to be highly osteoconductive both in the robocast and foamed scaffolds, a superior osteogenic capacity was observed in the foamed scaffolds, which was associated with their higher intrinsic osteoinductive potential. Moreover, they showed a significantly higher cell-mediated degradation than the robocast constructs, with a simultaneous and progressive replacement of the scaffold by new bone. In conclusion, these results demonstrate that the control of macropore architecture is a crucial parameter in the design of synthetic bone grafts, which allows fostering both material degradation and new bone formation. Statement of Significance 3D-printing technologies open new perspectives for the design of patient-specific bone grafts, since they allow customizing the external shape together with the internal architecture of implants. In this respect, it is important to design the appropriate pore geometry to maximize the bone healing capacity of these implants. The present study analyses the effect of pore architecture of nanostructured hydroxyapatite scaffolds, obtained either by 3D-printing or foaming, on the osteogenic potential and scaffold resorption in an in vivo model. While nanostructured hydroxyapatite showed excellent osteoconductive properties irrespective of pore geometry, we demonstrated that the spherical, concave macropores of foamed scaffolds significantly promoted both material resorption and bone regeneration compared to the 3D-printed scaffolds with orthogonal-patterned struts and therefore prismatic, convex macropores.

Heparinization of Beta Tricalcium Phosphate: Osteo-immunomodulatory Effects.

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, ß-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 ß-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 ß-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.

Osteoinduction by Foamed and 3D-Printed Calcium Phosphate Scaffolds: Effect of Nanostructure and Pore Architecture.

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 ß-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.

Focus Ion Beam/Scanning Electron Microscopy Characterization of Osteoclastic Resorption of Calcium Phosphate Substrates.

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.

Osteoclast differentiation from human blood precursors on biomimetic calcium-phosphate substrates.

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium. STATEMENT OF SIGNIFICANCE: The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors' differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.