Bone mineral is an ion-substituted carbonated apatite that is highly susceptible to lattice substitutions, and the commonly used Ca/P ratio does not adequately reflect this non-stoichiometric chemistry. We therefore propose cation-to-anion ratios (CARs) that account for major substitutions, including magnesium and sodium at calcium sites and carbonate at phosphate sites (B-type substitution). Bovine cortical bone slices were used as whole bone or after deproteinisation with 5% NaOCl for up to 168 h. B-type carbonate content determined by Raman spectroscopy and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used to correct phosphorus values measured by energy-dispersive X-ray spectroscopy (EDX). With progressive deproteinisation, Ca/P and Ca+Mg+Na/P increased significantly, revealing a substantial contribution of phosphorus from the organic matrix. Phosphorus correction by accounting for carbonate substitution generally lowered CAR values, showing that uncorrected CARs substantially overestimate bone mineral composition. These findings indicate that elemental ratios obtained directly from whole bone should be interpreted cautiously, while corrected CARs in deproteinised bone provide a more mineral-focussed basis for assessing bone mineral elemental composition.

Elective removal of bone-anchored hearing implants and epistheses is uncommon, limiting the understanding of the biological factors contributing to adverse reactions, implant success or failure. Therefore, the tissue-level response at the implant interface remains poorly understood. This study examined 21 explanted bone-anchored implants and interfacial tissues from 19 patients using a controlled retrieval protocol. The clinical data were correlated with histological, immunohistochemical, microbiological, and structural analyses. Pain (12/19) and infection (7/19) were the most common reasons for elective implant retrieval and in 5 cases occurred together. All explants, apart from one, showed significant osseointegration and maintained stability in situ, independent of their design characteristics. Staphylococcus aureus was isolated in five patients—four of whom reported pain even without overt infection. Staphylococcus epidermidis was detected in two patients: one with pain and infection and the other asymptomatic with a resolved infection. S. aureus colonization was associated with intense inflammation, marked by the presence of neutrophils, iNOS+ macrophages, CD3 + T-cells, and CD20 + B-cells. Pain and infection—often involving Staphylococcus species—are key drivers of implant retrieval. These findings emphasize the complex immune response at the implant site and the diagnostic value of analysing electively retrieved implants for improving patient outcomes.

Abstract Implants aim to restore skeletal dysfunction associated with ageing and trauma, yet infection and ineffective immune responses can lead to failure. This project characterized the microbiological and host cell responses to titanium alloy with or without electroplated metallic copper. Bacterial viability counting and scanning electron microscopy quantified and visualized the direct and indirect bactericidal effects of the Cu-electroplated titanium (Cu-Ep-Ti) against two different Staphylococcus aureus strains. Human THP-1 macrophage adhesion and viability was analyzed, along with phagocytosis. Results showed potent antimicrobial activity alongside promising host-immunomodulatory properties. Direct and indirect exposure to Cu-Ep-Ti produced potent bactericidal effects resulting in 94–100% reductions in bacterial viability at 24 h, with complete eradication in some cases. As expected, cytotoxicity was observed in THP-1 macrophages without media exchange, though when media was exchanged at 8, 24 and 48 h cell viability was equivalent to Control-Ti. Interestingly macrophages adhered to the copper material or grown in the presence of copper ions showed 7-fold increase in phagocytosis of S. aureus bioparticles compared to Control-Ti, suggesting a dual bactericidal and host immunomodulatory mechanism. In conclusion, this Cu-electroplated Ti biomaterial can limit bacterial contamination on the implant surface, whilst simultaneously promoting a beneficial antimicrobial immune response. Graphical Abstract

Orthopedic implants made of biodegradable magnesium (Mg) provide an alternative to nondegradable implants for fracture repair. Widely reported to be pro-osteogenic, Mg implants are also believed to be anti-inflammatory and anti-osteoclastic, but this is difficult to reconcile with the early clinical inflammation observed around these implants. Here, by surveying implant healing in a rat bone model, we determined the cellular responses and structural assembly of bone correlated with the surface changes of Mg implants inherent in degradation. We show that, compared to titanium, both high-purity (99.998%) and clinical-grade, rare earth-alloyed (MgYREZr) Mg implants create an initial, transient proinflammatory environment that facilitates inducible nitric oxide synthase-mediated macrophage polarization, osteoclastogenesis, and neoangiogenesis programs. While this immunomodulation subsequently reinforces reparative osteogenesis at the surface of both Mg implants, the faster degradation of high-purity Mg implants, but not MgYREZr implants, elicits a compositional alteration in the interfacial bone and a previously unknown proadipogenic response with persistent low-grade inflammation in the surrounding bone marrow. Beyond the need for rigorous tailoring of Mg implants, these data highlight the need to closely monitor osseointegration not only at the immediate implant surface but also in the peri-implant bone and adjacent bone marrow.

This study addresses the critical clinical challenge of implant failures due to mechanical overload by developing a novel rat model to investigate re-osseointegration. Metal implants, essential in dental, maxillofacial, and orthopaedic treatments, rely on osseointegration for stability. However, the fate of mechanically overloaded implants remains poorly understood. We introduced intentional traumatic loosening of submicron-modified titanium implants (treated with NaOH) through snap rotational overload in rat tibiae. After four weeks of initial healing, implants were disrupted and then allowed to re-heal for another four weeks. Evaluations using removal torque, histology, histochemistry, and Raman spectroscopy demonstrated successful re-healing with regained mechanical stability, bone-implant contact, and bone volume. Dynamic histology revealed bone tissue remodelling near the implant interface, indicating fractures due to mechanical disruption. These findings confirm that osseointegrated implants can re-heal under normal conditions. The validated rat model offers a controlled platform for future studies on re-osseointegration following traumatic mechanical overload. The potential applications of this experimental model may extend to investigating compromised healing conditions, early/direct loading conditions, and the cellular and molecular mechanisms involved in peri-implant bone repair.

Gristina et al. (1987) suggested that the fate of a biomaterial is decided in a "race for the surface" between pathogens and the host. To gain deeper insight into the mechanisms behind this concept, we investigated the "race for the surface" across three co-culture scenarios with THP-1 macrophages and Staphylococcus aureus (1:1 ratio), varying the order of addition: (i) simultaneous, (ii) macrophages first, and (iii) S. aureus first, on six Ti6Al4V-ELI surfaces modified with specific topographies and wettability. The outcome of the race for the surface was not influenced by these biomaterials but by the chronological introduction of macrophages and S. aureus. When macrophages and S. aureus arrived simultaneously, macrophages won the race, leading to the lowest number of viable S. aureus through rapid phagocytosis and killing. When macrophages arrived and established first, macrophages still prevailed but under greater challenge resulting from the lower bacterial killing efficiency of adherent macrophages and numerous viable intracellular bacteria, supporting the concept of the so-called immunocompromised zone around implants (upregulation of TLR-2 receptor and pro-inflammatory IL-1 beta). When S. aureus arrived first establishing a biofilm, bacteria won the race, leading to macrophage dysfunction and cell death (upregulation of Fc gamma R and TLR-2 receptors, NF-kappa B signaling, NOX2 mediated reactive oxygen species), contributing to a persistent biofilm phenotype (upregulation of clfA, icaA, sarA, downregulation of agrA, hld, lukAB) and intracellular survival of S. aureus (lipA upregulation). The clinical implications are bacterial colonization of the implant and persistence of intracellular bacteria in periprosthetic tissues, which can lead to infection chronicity. Statement of Significance: Gristina et al. (1987) suggested that the fate of a biomaterial is decided in a "race for the surface" between bacterial pathogens and host cells. There is a lack of in vitro co-culture models and knowledge on macrophage-S. aureus interactions on biomaterial surfaces, and no studies have evaluated the expression of virulence factors in S. aureus biofilms. We have successfully developed co-culture models and molecular panels, and elucidated important cellular and molecular interactions between macrophages and S. aureus on a broad range of titanium biomaterials with welldefined surface topography and wettability. Our findings highlight the critical role of biofilm formation and the chronological order of bacteria or macrophage arrival in determining the fate of the race for the surface.

Bacterial infections are one major cause of metallic implant failure. A metastable beta-type 96(Ti-45Nb)-4Cu alloy was developed for potential load-bearing implant applications with tailored antibacterial properties. The cast alloy was initially subjected to homogenization (1000 degrees C, 24 h, water-quenched), and then aged at two temperatures (425 degrees C, 640 degrees C, 8 h). The influence of aged microstructures on structural (XRD, SEM, APT), mechanical (microhardness, tensile tests), and corrosion (potentiodynamic polarization, ion release) behaviour, as well as on macrophage cytocompatibility and antibacterial properties, was studied. The homogenized microstructure consists of a single-phase beta, which partially decomposes into beta+omega (aging at 425 degrees C), and into three phases, beta+Ti2Cu+alpha (aging at 640 degrees C). The omega phase leads to a drastic increase in yield strength (sigma(y) > 1 GPa), and concomitant decrease in elongation (epsilon(max) = 2 %). The three-phase alloy shows a moderate yield strength (sigma(y) = 578 MPa), acceptable elongation (epsilon(max) = 10 %), and low Young's modulus (E = 78 GPa). All three alloys exhibit, in simulated physiological solution, low free corrosion and direct transfer to stable anodic passivity, similar to Ti-6Al-4V. The alloy aged at 425 degrees C exhibits enhanced corrosion activity in the higher anodic polarization regime due to omega precipitates in the beta matrix. The three-phase alloy shows good cytocompatibility with THP-1 macrophages at 24 h. Moreover, it exhibits antibacterial effect against S. aureus at 4 h. In conclusion, the aged alloy containing Ti2Cu precipitates exhibits a good combination of properties: high strength and elongation, low Young's modulus, excellent corrosion resistance, good biocompatibility, and antibacterial properties.

In bone, critical size defects pose substantial challenge in maxillofacial and orthopedic reconstructions as they are incapable of spontaneous regeneration. In such cases, autografts, allografts, and bone graft substitutes are used. Calcium phosphates (CaP) are widely used bone graft substitutes due to their biocompatibility, osteoconductive properties, and potential for osteoinductivity. CaP materials containing monetite, beta‐tricalcium phosphate (β‐TCP), and a small amount of calcium pyrophosphate (Ca‐PP) possess both osteoconductive and osteoinductive properties. However, the role of Ca‐PP in osteoinduction and material degradation remains unexplored. This study investigates heterotopic bone formation in response to five CaP compositions, maintaining a constant monetite to β‐TCP ratio, with varying amounts of Ca‐PP (0–12.5%). Twelve adult female sheep ( Ovis aries ) are subcutaneously implanted with constructs made of six CaP tiles interconnected by a Ti6Al4V frame and a control implant. Histological analysis, backscattered electron scanning electron microscopy, and Raman spectroscopy of samples retrieved at 12‐ and 52 weeks reveal that Ca‐PP does not hinder heterotopic bone formation and minimally impacts CaP degradation. While monetite and β‐TCP transform into apatite, the Ca‐PP phase remains unchanged. The addition of Ca‐PP to the CaP influences heterotopic bone quality and inflammatory response during tissue regeneration.

PURPOSE:This study aimed to evaluate the mechanical performance, heat generation, bone distortion, and characteristics of bone chips generated during drilling using a novel one-step guided drill system (MONO) for installing the bone-anchored hearing system (BAHS). A comparison was made with an existing three-stage drill system (MIPS). MATERIALS AND METHODS:Drill force and torque were measured during drilling in cow tibia at different feed rates. Compact artificial bone was utilized to determine temperature increases using thermocouples placed at specific positions around the osteotomy site during drilling with the two systems at different feed rates and levels of irrigation. The effects of drilling on osteotomy characteristics and the formation of bone fragments were evaluated through micro-CT, Raman spectroscopy, and histology. RESULTS:Force and torque increased with the feed rate in both systems, whereas the total work required to perform the osteotomy significantly decreased as the feed rate increased. Compared to the three-stage MIPS system, the MONO system required less work for one-step osteotomy creation, generated equal or less heat, and exhibited greater tolerance for procedural deviations in irrigation and drilling sequence. Additionally, heat generation for both systems decreased when drilling at higher feed rates. Compositional changes within the osteotomy were primarily observed under reduced irrigation protocols, while no differences were identified in bone chips across drilling protocols. CONCLUSION:Compared with a multistep conventional drilling procedure, MONO drilling is less affected by variations in the drilling protocol, particularly in flapless and blind procedures, resulting in safer and more efficient osteotomy creation. The MONO system demonstrated superior performance in terms of energy efficiency and temperature control.

Persistent inflammation and infection, often linked to staphylococcal colonization, affect bone-anchored hearing system (BAHS) outcomes. Although antibiotics are often used to treat skin complications, the roles of biofilms and antimicrobial resistance (AMR) in clinical success remain unclear. This clinical prospective study characterized biofilm formation and antibiotic resistance in Staphylococcus spp. from BAHS patients, and examined associations with inflammation, pain, and hygiene. Adults eligible for BAHS were prospectively enrolled at a tertiary university hospital in Sweden during 2014-2015. Fifteen patients were followed clinically and microbiologically at surgery, 3- and 12- months. Abutment, peri-abutment exudate and soft-tissue samples were cultured. Fifty-seven Staphylococcus spp. isolates underwent biofilm phenotyping (Crystal Violet, Congo Red), antimicrobial susceptibility testing (minimum inhibitory concentration [MIC], minimum biofilm eradication concentration [MBEC]) and whole-genome sequencing (lineage, AMR and virulence genes). Clinical status was scored (Holgers, pain, debris). Individual patients harbored the same staphylococcal clone on abutment, exudate, and tissue for 12 months. S. aureus was more prevalent in patients with inflammation (Holgers score >0), S. epidermidis correlated with pain, and slime production was associated with debris accumulation. Overall, 56 % of isolates showed resistance to fusidic acid, and 11-34 % carried tetracycline resistance genes. S. epidermidis carried multidrug resistance genes (beta-lactams, tetracycline, sulfamethoxazole, fosfomycin), and resistance increased under biofilm conditions (MBEC > MIC). The ica operon was detected in all S. aureus and S. epidermidis ST7, ST297, ST749 and ST278. These findings indicate that staphylococci from BAHS exhibit persistent colonization, diverse clonal lineages, and high biofilm-associated AMR. Early microbial diagnostics and biofilm-targeted strategies, alongside cautious use of topical antibiotics, may improve outcomes.

Ti-based bulk metallic glasses (Ti-BMGs) are promising candidates for mini-invasive dental implant devices due to their unique properties. Among them, Ti40Zr10Cu36Pd14 has received particular attention in the literature for its potential biocompatibility. However, its high copper content limits corrosion resistance by promoting localised corrosion (pitting or crevice). This study presents a rapid and straightforward surface modification technique known as chemical pseudo-dealloying to reduce copper content in the top surface layer, enriching palladium and enhancing corrosion resistance limiting pitting occurrence. Additionally, the selective etching of Cu creates nanoscale surface features that could be interesting for cell adhesion and differentiation. The Pd-rich nanoporous layer was thoroughly characterized by its topography, chemical composition, electrochemical behaviour, and biocompatibility. These findings pave the way for tailoring the topography and surface properties of metallic amorphous alloys for biomedical applications.

The use of bone-repair biomaterials is rapidly expanding to meet the needs of an ageing and increasingly active population, often with compromised bone quality. However, inconsistencies in how materials are assessed preclinically, across animal models, sampling strategies, and analytical techniques, have led to flawed comparisons and misleading claims. Fundamental differences in material properties and the biological responses they elicit are frequently ignored, conflating distinct mechanisms of bone formation. This "apples vs. oranges" problem is magnified by the growing diversity of biomaterials. Here, we call for a more systematic, context-aware approach to biomaterial evaluation that emphasises standardisation and biological relevance.

To compare the osseointegration properties of two different types of nitrogen-containing bisphosphonate (N-BP)-coated dental implants in an experimental in vivo sheep model. In total, eight sheep were divided into two groups receiving implants at two time points. Each animal received four types of implants, AddBIO STL implants coated with Zoledronate (Zol) and AddBIO STL implants coated with Ibandronate and Pamidronate (IbaPam) as test groups and uncoated AddBIO STL implants (UC) and Straumann original SLA implants (SSLA) as controls. Implants were placed in the metatarsus bilaterally, and healing times were either 10 or 28 days. Implant stability quotient (ISQ) was measured at baseline and at euthanasia. Removal torque (RT) was assessed post-mortem, followed by histomorphometric analysis to evaluate bone-to-implant contact (BIC) and bone area (BA). The differences between implants were evaluated using paired t-tests. The significance level was set at p value < 0.05 After 10 days, Zol implants presented significantly higher RT (Ncm mean ± SD) values 26.0 (± 18.0) than IbaPam implants 8.3 (± 14.9) (p = 0.011). SSLA implants demonstrated significantly higher RT values 34.6 (± 22.2) than Zol and IbaPam-coated implants (p = 0.048) and Zol-coated implants showed significantly higher RT values than UC implants 12.0 (± 7.6) than UC implants (p = 0.015). No significant differences in RT were detected at 28 days. No differences were observed among the groups regarding ISQ, BIC, or BA at either time point. Zoledronate-coated implants exhibited enhanced early mechanical stability compared to ibandronate/pamidronate-coated implants. However, this advantage was eradicated after 28 days, suggesting that the early anabolic effect of zoledronate may be time-limited.

Tartrate-resistant acid phosphatase (TRAP) serum levels reflect osteoclast number, bone remodeling activity, and fracture risk. Deletion or loss of function of TRAP results in short stature in mice and man. Yet, the impact and mechanisms of TRAP for the site- and sex-specific development of bone and cartilage is not well understood. Here, we use a global TRAP knockout (TRAPKO) and wildtype littermate control (WT) mice of both sexes to investigate TRAP as a possible sex- and site-specific regulator of bone and growth plate development. TRAPKO mice of both sexes weighed less and had shorter tibial length than their WT, features that were more accentuated in male than female TRAPKO mice. These changes were not associated with a general reduction in growth as not all organs displayed a proportionally lower mass, and serum IGF-1 was unchanged. Using μCT and site-specificity analysis of the cortical bone revealed wider proximal tibia, a higher trabecular thickness, and lower trabecular separation in male TRAPKO compared to WT mice, an effect not seen in female mice. Histomorphometric analysis revealed that the growth plate height as well as height of terminal hypertrophic chondrocytes were markedly increased, and the number of columns was decreased in TRAPKO mice of both sexes. These effects were more accentuated in female mice. Proliferation and differentiation of bone marrow derived macrophages into osteoclasts, as well as C-terminal cross links were normal in TRAPKO mice of both sexes. Collectively, our results show that TRAP regulates bone and cartilage development in a sex-and site-specific manner in mice.

Osteoarthritis (OA) poses significant therapeutic challenges, particularly OA that affects the hand. Currently available treatment strategies are often limited in terms of their efficacy in managing pain, regulating invasiveness, and restoring joint function. The APRICOT((R)) implant system developed by Aurora Medical Ltd (Chichester, UK) introduces a minimally invasive, bone-conserving approach for treating hand OA ( https://apricot-project.eu/ ). By utilizing polycarbonate urethane (PCU), this implant incorporates a caterpillar track-inspired design to promote the restoration of natural movement to the joint. Surface modifications of PCU have been proposed for the biological fixation of the implant. This study investigated the biocompatibility of PCU alone or in combination with two surface modifications, namely dopaminecarboxymethylcellulose (dCMC) and calcium-phosphate (CaP) coatings. In a rat soft tissue model, native and CaP-coated PCU foils did not increase cellular migration or cytotoxicity at the implant-soft tissue interface after 3 d, showing gene expression of proinflammatory cytokines similar to that in non-implanted sham sites. However, dCMC induced an amplified initial inflammatory response that was characterized by increased chemotaxis and cytotoxicity, as well as pronounced gene activation of proinflammatory macrophages and neoangiogenesis. By 21 d, inflammation subsided in all the groups, allowing for implant encapsulation. In a rat bone model, 6 d and 28 d after release of the periosteum, all implant types were adapted to the bone surface with a surrounding fibrous capsule and no protracted inflammatory response was observed. These findings demonstrated the biocompatibility of native and CaP-coated PCU foils as components of APRICOT((R)) implants.

Salmonid fish include some of the most valued cultured fish species worldwide. Unlike most other fish, the hearts of salmonids, including Atlantic salmon and rainbow trout, have a well-developed coronary circulation. Consequently, their hearts' reliance on oxygenation through coronary arteries leaves them prone to coronary lesions, believed to precipitate myocardial ischemia. Here, we mimicked such coronary lesions by subjecting groups of juvenile rainbow trout to coronary ligation, assessing histomorphological myocardial changes associated with ischemia and scarring in the context of cardiac arrhythmias using electrocardiography (ECG). Notable ECG changes resembling myocardial ischemia-like ECG in humans, such as atrioventricular blocks and abnormal ventricular depolarization (prolonged and fragmented QRS complex), as well as repolarization (long QT interval) patterns, were observed during the acute phase of myocardial ischemia. A remarkable 100% survival rate was observed among juvenile trout subjected to coronary ligation after 24 wk. Recovery from coronary ligation occurred through adaptive ventricular remodeling, coupled with a fast cardiac revascularization response. These findings carry significant implications for understanding the mechanisms governing cardiac health in salmonid fish, a family particularly susceptible to cardiac diseases. Furthermore, our results provide valuable insights into comparative studies on the evolution, pathophysiology, and ontogeny of vertebrate cardiac repair and restoration. NEW & NOTEWORTHY Juvenile rainbow trout exhibit a remarkable capacity to recover from cardiac injury caused by myocardial ischemia. Recovery from cardiac damage occurs through adaptive ventricular remodeling, coupled with a rapid cardiac revascularization response. These findings carry significant implications for understanding the mechanisms governing cardiac health within salmonid fishes, which are particularly susceptible to cardiac diseases.

The ability of additive manufacturing to generate intricate structures has led to its popularity and widespread use in a variety of applications, ranging from the production of biomedical implants to aircraft components. Additive manufacturing techniques can overcome the limitations of the traditional manufacturing methods to create complex near-net-form structures. A vast array of clinical applications effectively employ Ti-6Al-4V as a biomaterial. The evolution of additive manufacturing has accelerated the development of patient-specific implants. The surface characteristics play a critical role in tissue healing and adaptation to implants. The present research set out to examine the effects of powder recycling with respect to the powder itself and the surface properties resulting from the electron beam melting (EBM) of the implant material. The printed implants, as well as the powder samples, underwent morphological, surface chemistry, and microstructure analyses. The in vitro cytotoxicity was evaluated with THP-1 macrophages. The overall microstructure of the implant samples showed little variation in terms of powder recycling based on the results. Higher oxygen levels were found in the solid and lattice sections of those implants manufactured with batches of recycled powder, along with marginally better cell viability. This emphasizes how crucial powder quality is to the process of additive manufacturing.

Macrophages and osteocytes are important regulators of inflammation, osteogenesis and osteoclastogenesis. However, their interactions under adverse conditions, such as biomaterial-associated infection (BAI) are not fully understood. We aimed to elucidate how factors released from macrophages modulate osteocyte responses in an in vitro indirect 3D co-culture model. Human monocyte-derived macrophages were cultured on etched titanium disks and activated with either IL-4 cytokine (anti-inflammatory M2 phenotype) or Staphylococcus aureus secreted virulence factors to simulate BAI (pro-inflammatory M1 phenotype). Primary osteocytes in collagen gels were then stimulated with conditioned media (CM) from these macrophages. The osteocyte response was analyzed by gene expression, protein secretion, and immunostaining. M1 phenotype macrophages were confirmed by IL-1β and TNF-α secretion, and M2 macrophages by ARG-1 and MRC-1.Osteocytes receiving M1 CM revealed bone inhibitory effects, denoted by reduced secretion of bone formation osteocalcin (BGLAP) and increased secretion of the bone inhibitory sclerostin (SOST). These osteocytes also downregulated the pro-mineralization gene PHEX and upregulated the anti-mineralization gene MEPE. Additionally, exhibited pro-osteoclastic potential by upregulating pro-osteoclastic gene RANKL expression. Nonetheless, M1-stimulated osteocytes expressed a higher level of the potent pro-osteogenic factor BMP-2 in parallel with the downregulation of the bone inhibitor genes DKK1 and SOST, suggesting a compensatory feedback mechanisms. Conversely, M2-stimulated osteocytes mainly upregulated anti-osteoclastic gene OPG expression, suggesting an anti-catabolic effect. Altogether, our findings demonstrate a strong communication between M1 macrophages and osteocytes under M1 (BAI)-simulated conditions, suggesting that the BAI adverse effects on osteoblastic and osteoclastic processes in vitro are partly mediated via this communication. Statement of Significance Biomaterial-associated infections are major challenges and the underlying mechanisms in the cellular interactions are missing, especially among the major cells from the inflammatory side (macrophages as the key cell in bacterial clearance) and the regenerative side (osteocyte as main regulator of bone). We evaluated the effect of macrophage polarization driven by the stimulation with bacterial virulence factors on the osteocyte function using an indirect co-culture model, hence mimicking the scenario of a biomaterial-associated infection. The results suggest that at least part of the adverse effects of biomaterial associated infection on osteoblastic and osteoclastic processes in vitro are mediated via macrophage-to-osteocyte communication.

Given the hierarchical nature of bone and bone interfaces, osseointegration, namely the formation of a direct bone-implant contact, is best evaluated using a multiscale approach. However, a trade-off exists between field of view and spatial resolution, making it challenging to image large volumes with high resolution. In this study, we combine established electron microscopy techniques to probe bone-implant interfaces at the microscale and nanoscale with plasma focused ion beam-scanning electron microscopy (PFIB-SEM) tomography to evaluate osseointegration at the mesoscale. This characterization workflow is demonstrated for bone response to an additively manufactured Ti-6Al-4V implant which combines engineered porosity to facilitate bone ingrowth and surface functionalization via genistein, a phytoestrogen, to counteract bone loss in osteoporosis. SEM demonstrated new bone formation at the implant site, including in the internal implant pores. At the nanoscale, scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy confirmed the gradual nature of the bone-implant interface. By leveraging mesoscale analysis with PFIB-SEM tomography that captures large volumes of bone-implant interface with nearly nanoscale resolution, the presence of mineral ellipsoids varying in size and orientation was revealed. In addition, a well-developed lacuno-canalicular network and mineralization fronts directed both towards the implant and away from it were highlighted.