Ultrastructure and Nanoporosity of Human Bone Shown with Correlative On-Axis Electron and Spectroscopic Tomographies.

Mineralized collagen fibrils are the building block units of bone at the nanoscale. While it is known that collagen fibrils are mineralized both inside their gap zones (intra-fibrillar mineralization) and on their outer surfaces (extra-fibrillar mineralization), a clear visualization of this architecture in three dimensions (3D), combining structural and compositional information over large volumes, but without compromising the resolution, remains challenging. In this study, we demonstrate the use of on-axis Z-contrast electron tomography (ET) with correlative energy-dispersive X-ray spectroscopy (EDX) tomography to examine rod-shaped samples with diameters up to 700 nm prepared from individual osteonal lamellae in the human femur. Our work mainly focuses on two aspects: (i) low-contrast nanosized circular spaces ("holes") observed in sections of bone oriented perpendicular to the long axis of a long bone, and (ii) extra-fibrillar mineral, especially in terms of morphology and spatial relationship with respect to intra-fibrillar mineral and collagen fibrils. From our analyses, it emerges quite clearly that most "holes" are cross-sectional views of collagen fibrils. While this had been postulated before, our 3D reconstructions and reslicing along meaningful two-dimensional (2D) cross-sections provide a direct visual confirmation. Extra-fibrillar mineral appears to be composed of thin plates that are interconnected and span over several collagen fibrils, confirming that mineralization is cross-fibrillar, at least for the extra-fibrillar phase. EDX tomography shows mineral signatures (Ca and P) within the gap zones, but the signal appears weaker than that associated with the extra-fibrillar mineral, pointing toward the existence of dissimilarities between the two types of mineralization.

Leptin receptor gene deficiency minimally affects osseointegration in rats.

Metabolic syndrome represents a cluster of conditions such as obesity, hyperglycaemia, dyslipidaemia, and hypertension that can lead to type 2 diabetes mellitus and/or cardiovascular disease. Here, we investigated the influence of obesity and hyperglycaemia on osseointegration using a novel, leptin receptor-deficient animal model, the Lund MetS rat. Machined titanium implants were installed in the tibias of animals with normal leptin receptor (LepR+/+) and those harbouring congenic leptin receptor deficiency (LepR-/-) and were left to heal for 28 days. Extensive evaluation of osseointegration was performed using removal torque measurements, X-ray micro-computed tomography, quantitative backscattered electron imaging, Raman spectroscopy, gene expression analysis, qualitative histology, and histomorphometry. Here, we found comparable osseointegration potential at 28 days following implant placement in LepR-/- and LepR+/+ rats. However, the low bone volume within the implant threads, higher bone-to-implant contact, and comparable biomechanical stability of the implants point towards changed bone formation and/or remodelling in LepR-/- rats. These findings are corroborated by differences in the carbonate-to-phosphate ratio of native bone measured using Raman spectroscopy. Observations of hypermineralised cartilage islands and increased mineralisation heterogeneity in native bone confirm the delayed skeletal development of LepR-/- rats. Gene expression analyses reveal comparable patterns between LepR-/- and LepR+/+ animals, suggesting that peri-implant bone has reached equilibrium in healing and/or remodelling between the animal groups.

High-resolution Raman spectroscopy reveals compositional differences between pigmented incisor enamel and unpigmented molar enamel in Rattus norvegicus.

Dental enamel is a peculiar biological tissue devoid of any self-renewal capacity as opposed to bone. Thus, a thorough understanding of enamel composition is essential to develop novel strategies for dental enamel repair. While the mineral found in bone and dental enamel is generally viewed as the biologically-produced equivalent of hydroxy(l)apatite, the formation of these bioapatites is controlled by different organic matrix frameworks-mainly type-I collagen in bone and amelogenin in enamel. In lower vertebrates, such as rodents, two distinct types of enamel are produced. Iron-containing pigmented enamel protects the continuously growing incisor teeth while magnesium-rich unpigmented enamel covers the molar teeth. Using high-resolution Raman spectroscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy, this work explores the differences in acid phosphate (HPO42-), carbonate (CO32-), hydroxyl (OH-), iron, and magnesium content of pigmented incisor enamel and unpigmented molar enamel of Sprague Dawley rats. Bundles of hydroxy(l)apatite nanowires comprise the enamel prisms, where prisms in pigmented enamel are wider and longer than those in unpigmented molars. In contrast to magnesium-rich unpigmented enamel, higher mineral crystallinity, and higher HPO42- and OH- levels are hallmark features of iron-rich pigmented enamel. Furthermore, the apparent absence of iron oxides or oxy(hydroxides) indicates that iron is introduced into the apatite lattice at the expense of calcium, albeit in amounts that do not alter the Raman signatures of the PO43- internal modes. Compositional idiosyncrasies of iron-rich pigmented and nominally iron-free unpigmented enamel offer new insights into enamel biomineralisation supporting the notion that, in rodents, ameloblast function differs significantly between the incisors and the molars.

Bone structure and composition in a hyperglycemic, obese, and leptin receptor-deficient rat: Microscale characterization of femur and calvarium.

Metabolic abnormalities, such as diabetes mellitus and obesity, can impact bone quantity and/or bone quality. In this work, we characterize bone material properties, in terms of structure and composition, in a novel rat model with congenic leptin receptor (LepR) deficiency, severe obesity, and hyperglycemia (type 2 diabetes-like condition). Femurs and calvaria (parietal region) from 20-week-old male rats are examined to probe bones formed both by endochondral and intramembranous ossification. Compared to the healthy controls, the LepR-deficient animals display significant alterations in femur microarchitecture and in calvarium morphology when analyzed by micro-computed X-ray tomography (micro-CT). In particular, shorter femurs with reduced bone volume, combined with thinner parietal bones and shorter sagittal suture, point towards a delay in the skeletal development of the LepR-deficient rodents. On the other hand, LepR-deficient animals and healthy controls display analogous bone matrix composition, which is assessed in terms of tissue mineral density by micro-CT, degree of mineralization by quantitative backscattered electron imaging, and various metrics extrapolated from Raman hyperspectral images. Some specific microstructural features, i.e., mineralized cartilage islands in the femurs and hyper-mineralized areas in the parietal bones, also show comparable distribution and characteristics in both groups. Overall, the altered bone microarchitecture in the LepR-deficient animals indicates compromised bone quality, despite the normal bone matrix composition. The delayed development is also consistent with observations in humans with congenic Lep/LepR deficiency, making this animal model a suitable candidate for translational research.

The many facets of micropetrosis - Magnesium whitlockite deposition in bisphosphonate-exposed human alveolar bone with osteolytic metastasis.

The lacuno-canalicular space of apoptotic osteocytes eventually becomes mineralised in vivo. This condition is known as micropetrosis and is a fundamental characteristic of ageing bone. Increased prevalence of such hypermineralised osteocyte lacunae is viewed as a structural marker of impaired bone function - both mechanical and biological. Within the lacuno-canalicular space, mineralised apoptotic debris typically occurs as micrometre-sized, spherical nodules of magnesium-rich, carbonated apatite. Moreover, characteristically facetted, rhomboidal nodules of magnesium whitlockite [Mg-whitlockite; Ca18Mg2(HPO4)2(PO4)12] have been reported in human alveolar bone exposed to the bisphosphonate alendronate. This work provides supporting evidence for Mg-whitlockite formation in the alveolar bone of a 70-year-old male exposed to the bisphosphonate zoledronic acid to suppress osteolytic changes in skeletal metastasis. Backscattered electron scanning electron microscopy (BSE-SEM) revealed spherical and rhomboidal nodules within the lacuno-canalicular space. A variant of spherical nodules exhibited a fuzzy surface layer comprising radially extending acicular crystallites. The rhomboidal nodules ranged between ∼200 nm to ∼2.4 µm across the widest dimension (652 ± 331 nm). Micro-Raman spectroscopy and energy dispersive X-ray spectroscopy confirmed that rhomboidal nodules are compositionally distinct from spherical nodules, exhibiting higher Mg content and lower Ca/P ratio. Formation of Mg-whitlockite within osteocyte lacunae is multifactorial in nature and suggests altered bone biomineralisation. Nevertheless, the underlying mechanism(s) and sequence of events remain poorly understood and warrant further investigation. The possibility to discriminate between carbonated apatite and Mg-whitlockite nodules within osteocyte lacunae, based on particle morphology, attests to the diagnostic potential of BSE-SEM with or without additional analyses of material composition.

Magnesium implant degradation provides immunomodulatory and proangiogenic effects and attenuates peri-implant fibrosis in soft tissues.

Implants made of magnesium (Mg) are increasingly employed in patients to achieve osteosynthesis while degrading in situ. Since Mg implants and Mg2+ have been suggested to possess anti-inflammatory properties, the clinically observed soft tissue inflammation around Mg implants is enigmatic. Here, using a rat soft tissue model and a 1-28 d observation period, we determined the temporo-spatial cell distribution and behavior in relation to sequential changes of pure Mg implant surface properties and Mg2+ release. Compared to nondegradable titanium (Ti) implants, Mg degradation exacerbated initial inflammation. Release of Mg degradation products at the tissue-implant interface, culminating at 3 d, actively initiated chemotaxis and upregulated mRNA and protein immunomodulatory markers, particularly inducible nitric oxide synthase and toll-like receptor-4 up to 6 d, yet without a cytotoxic effect. Increased vascularization was demonstrated morphologically, preceded by high expression of vascular endothelial growth factor. The transition to appropriate tissue repair coincided with implant surface enrichment of Ca and P and reduced peri-implant Mg2+ concentration. Mg implants revealed a thinner fibrous encapsulation compared with Ti. The detailed understanding of the relationship between Mg material properties and the spatial and time-resolved cellular processes provides a basis for the interpretation of clinical observations and future tailoring of Mg implants.

Bench-to-bedside: Feasibility of nano-engineered and drug-delivery biomaterials for bone-anchored implants and periodontal applications.

Nanotechnology and drug-release biomaterials have been thoroughly explored in the last few years aiming to develop specialized clinical treatments. However, it is rare to find biomaterials associated with drug delivery properties in the current dental market for application in oral bone- and periodontal-related procedures. The gap between basic scientific evidence and translation to a commercial product remains wide. Several challenges have been reported regarding the clinical translation of biomaterials with drug-delivery systems (BDDS) and nanofeatures. Therefore, processes for BDDS development, application in preclinical models, drug delivery doses, sterilization processes, storage protocols and approval requirements were explored in this review, associated with tentative solutions for these issues. The diversity of techniques and compounds/molecules applied to develop BDDS demands a case-by-case approach to manufacturing and validating a commercial biomaterial. Promising outcomes such as accelerated tissue healing and higher antibacterial response have been shown through basic and preclinical studies using BDDS and nano-engineered biomaterials; however, the adequate process for sterilization, storage, cost-effectiveness and possible cytotoxic effects remains unclear for multifunctional biomaterials incorporated with different chemical compounds; then BDDSs are rarely translated into products. The future benefits of BDDS and nano-engineered biomaterials have been reported suggesting personalized clinical treatment and a promising reduction in the use of systemic antibiotics. Finally, the launch of these specialized biomaterials with solid data and controlled traceability onto the market will generate strong specificity for healthcare treatments.

Contributions of Resin Cast Etching to Visualising the Osteocyte Lacuno-Canalicular Network Architecture in Bone Biology and Tissue Engineering.

Recent years have witnessed an evolution of imaging technologies towards sophisticated approaches for visualising cells within their natural environment(s) and for investigating their interactions with other cells, with adjacent anatomical structures, and with implanted biomaterials. Resin cast etching (RCE) is an uncomplicated technique involving sequential acid etching and alkali digestion of resin embedded bone to observe the osteocyte lacuno-canalicular network using scanning electron microscopy. This review summarises the applicability of RCE to bone and the bone-implant interface. Quantitative parameters such as osteocyte size, osteocyte density, and number of canaliculi per osteocyte, and qualitative metrics including osteocyte shape, disturbances in the arrangement of osteocytes and canaliculi, and physical communication between osteocytes and implant surfaces can be investigated. Ageing, osteoporosis, long-term immobilisation, spinal cord injury, osteoarthritis, irradiation, and chronic kidney disease have been shown to impact osteocyte lacuno-canalicular network morphology. In addition to titanium, calcium phosphates, and bioactive glass, observation of direct connectivity between osteocytes and cobalt chromium provides new insights into the osseointegration potential of materials conventionally viewed as non-osseointegrating. Other applications include in vivo and in vitro testing of polymer-based tissue engineering scaffolds and tissue-engineered ossicles, validation of ectopic osteochondral defect models, ex vivo organ culture of whole bones, and observing the effects of gene dysfunction/deletion on the osteocyte lacuno-canalicular network. Without additional contrast staining, any resin embedded specimen (including clinical biopsies) can be used for RCE. The multitude of applications described here attest to the versatility of RCE for routine use within correlative analytical workflows, particularly in biomaterials science.

Complex geometry and integrated macro-porosity: Clinical applications of electron beam melting to fabricate bespoke bone-anchored implants.

The last decade has witnessed rapid advancements in manufacturing technologies for biomedical implants. Additive manufacturing (or 3D printing) has broken down major barriers in the way of producing complex 3D geometries. Electron beam melting (EBM) is one such 3D printing process applicable to metals and alloys. EBM offers build rates up to two orders of magnitude greater than comparable laser-based technologies and a high vacuum environment to prevent accumulation of trace elements. These features make EBM particularly advantageous for materials susceptible to spontaneous oxidation and nitrogen pick-up when exposed to air (e.g., titanium and titanium-based alloys). For skeletal reconstruction(s), anatomical mimickry and integrated macro-porous architecture to facilitate bone ingrowth are undoubtedly the key features of EBM manufactured implants. Using finite element modelling of physiological loading conditions, the design of a prosthesis may be further personalised. This review looks at the many unique clinical applications of EBM in skeletal repair and the ground-breaking innovations in prosthetic rehabilitation. From a simple acetabular cup to the fifth toe, from the hand-wrist complex to the shoulder, and from vertebral replacement to cranio-maxillofacial reconstruction, EBM has experienced it all. While sternocostal reconstructions might be rare, the repair of long bones using EBM manufactured implants is becoming exceedingly frequent. Despite the various merits, several challenges remain yet untackled. Nevertheless, with the capability to produce osseointegrating implants of any conceivable shape/size, and permissive of bone ingrowth and functional loading, EBM can pave the way for numerous fascinating and novel applications in skeletal repair, regeneration, and rehabilitation. STATEMENT OF SIGNIFICANCE: Electron beam melting (EBM) offers unparalleled possibilities in producing contaminant-free, complex and intricate geometries from alloys of biomedical interest, including Ti6Al4V and CoCr. We review the diverse range of clinical applications of EBM in skeletal repair, both as mass produced off-the-shelf implants and personalised, patient-specific prostheses. From replacing large volumes of disease-affected bone to complex, multi-material reconstructions, almost every part of the human skeleton has been replaced with an EBM manufactured analog to achieve macroscopic anatomical-mimickry. However, various questions regarding long-term performance of patient-specific implants remain unaddressed. Directions for further development include designing personalised implants and prostheses based on simulated loading conditions and accounting for trabecular bone microstructure with respect to physiological factors such as patient's age and disease status.

Bone without borders - Monetite-based calcium phosphate guides bone formation beyond the skeletal envelope.

Calcium phosphates (CaP) represent an important class of osteoconductive and osteoinductive biomaterials. As proof-of-concept, we show how a multi-component CaP formulation (monetite, beta-tricalcium phosphate, and calcium pyrophosphate) guides osteogenesis beyond the physiological envelope. In a sheep model, hollow dome-shaped constructs were placed directly over the occipital bone. At 12 months, large amounts of bone (∼75%) occupy the hollow space with strong evidence of ongoing remodelling. Features of both compact bone (osteonal/osteon-like arrangements) and spongy bone (trabeculae separated by marrow cavities) reveal insights into function/need-driven microstructural adaptation. Pores within the CaP also contain both woven bone and vascularised lamellar bone. Osteoclasts actively contribute to CaP degradation/removal. Of the constituent phases, only calcium pyrophosphate persists within osseous (cutting cones) and non-osseous (macrophages) sites. From a translational perspective, this multi-component CaP opens up exciting new avenues for osteotomy-free and minimally-invasive repair of large bone defects and augmentation of the dental alveolar ridge.

Multimodal and Multiscale Characterization of the Bone-Bacteria Interface in a Case of Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a known side effect of bisphosphonates (BPs). Although bacterial infection is usually present, the etiology of MRONJ remains unknown. Here we apply a multimodal and multiscale (micro-to-nano) characterization approach to investigate the interface between necrotic bone and bacteria in MRONJ. A non-necrotic bone sample was used as control. Both necrotic and non-necrotic bone samples were collected from the jaw of a female individual affected by MRONJ after using BPs for 23 years. For the first time, resin cast etching was used to expose bacteria at the necrotic site. The bone-bacteria interface was also resolved at the nanoscale by scanning transmission electron microscopy (STEM). Nanosized particulates, likely corresponding to degraded bone mineral, were often noted in close proximity to or enclosed by the bacteria. STEM also revealed that the bone-bacteria interface is composed of a hypermineralized front fading into a highly disordered region, with decreasing content of calcium and phosphorus, as assessed by electron energy loss spectroscopy (EELS). This, combined with the variation in calcium, phosphorus, and carbon across the necrotic bone-bacteria interface evaluated by scanning electron microscopy (SEM)-energy dispersive X-ray spectroscopy (EDX) and the lower mineral-to-matrix ratio measured by micro-Raman spectroscopy in necrotic bone, indicates the absence of a mineralization front in MRONJ. It appears that the bone-bacteria interface originates not only from uncontrolled mineralization but also from the direct action of bacteria degrading the bone matrix. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Modelling the resonant frequency associated with the spatio-temporal evolution of the bone-dental implant interface.

Dental implant stability is greatly affected by the mechanical properties of the bone-implant interface (BII), and it is key to long-term successful osseointegration. Implant stability is often evaluated using the Resonant Frequency Analysis (RFA) method, and also by the quality of this interface, namely the bone-implant contact (BIC). True to this day, there is a scarcity of models tying BIC, RFA and a spatially and mechanically evolving BII. In this paper, based on the contact/distance osteogenesis concept, a novel numerical spatio-temporal model of the implant, surrounding bone and evolving interface, was developed to assess the evolution of the interfacial stresses on the one hand and the corresponding resonant frequencies on the other. We postulate that, since the BIC percentage reaches saturation over a very short time, long before densification of the interface, it becomes irrelevant as to load transmission between the implant and the bone due to the existence of an open gap. Gap closure is the factor that provides continuity between the implant and the surrounding bone. The results of the calculated RFA evolution match and provide an explanation for the multiple clinical observations of a sharp initial decline in RFA, followed by a gradual increase and plateau formation. STATEMENT OF SIGNIFICANCE: A novel three-dimensional numerical model of an evolving bone-dental implant interface (BII) is presented. The spatio-temporal evolution of the bone-implant contact (BIC) and the BII, based on contact/distance (CO/DO) osteogenesis, is modeled. A central outcome is that, until BII maturation into a solid continuous bone (no open gap between CO-DO fronts), the bone-implant load transfer is hampered, irrespective of the BIC. The resonant frequencies' evolution of the jawbone-BII-implant is calculated to reproduce the well-established implant stability analysis based on the Resonant Frequency Analysis. The results resemble those reported clinically, and here too, the determinant transition occurs only after interfacial gap closure. Those results should motivate clinicians to re-consider structural continuity of the BII rather than the BIC only.

The impact of medication on osseointegration and implant anchorage in bone determined using removal torque-A review.

Permanently anchored metal implants are frequently used in dental, craniomaxillofacial, and orthopaedic rehabilitation. The success of such therapies is owed to the phenomenon of osseointegration-the direct connection between the living bone and the implant. The extent of biomechanical anchorage (i.e., physical interlocking between the implant and bone) can be assessed with removal torque (RTQ) measurement. Implant anchorage is strongly influenced by underlying bone quality, involving physicochemical and biological properties such as composition and structural organisation of extracellular matrix, extent of micro-damage, and bone turnover. In this review, we evaluated the impact of various pharmacological agents on osseointegration, from animal experiments conducting RTQ measurements. In addition to substances whose antiresorptive and/or anti-catabolic effects on bone are well-documented (e.g., alendronate, zoledronate, ibandronate, raloxifene, human parathyroid hormone, odanacatib, and the sclerostin monoclonal antibody), positive effects on RTQ have been reported for substances that do not primarily target bone (e.g., aminoguanidine, insulin, losartan, simvastatin, bone morphogenetic protein, alpha-tocopherol, and the combination of silk fibroin powder and platelet-rich fibrin). On the contrary, several substances (e.g., prednisolone, cyclosporin A, cisplatin, and enamel matrix derivative) tend to adversely impact RTQ. While morphometric parameters such as bone-implant contact appear to influence the biomechanical anchorage, increased or decreased RTQ is not always accompanied by corresponding fluctuations in bone-implant contact. This further confirms that factors such as bone quality underpin biomechanical anchorage of metal implants. Several fundamental questions on drug metabolism and bioavailability, drug dosage, animal-to-human translation, and the consequences of treatment interruption remain yet unanswered.

Long-term osseointegration of laser-ablated hearing implants in sheep cranial bone.

Osseointegration, the ability for an implant to be anchored in bone tissue with direct bone-implant contact and allowing for continuous adaptive remodelling, is clinically used in different reconstructive fields, such as dentistry, orthopedics and otology. The latter uses a bone conducting sound processor connected to a skin-penetrating abutment that is mounted on a titanium implant placed in the temporal bone, thereby acting as a path for transmission of the vibrations generated by the sound processor. The success of the treatment relies on bone healing and osseointegration, which could be improved by surface modifications. The aim of this study was to evaluate the long-term osseointegration in a sheep skull model and compare a laser-ablated implant surface with a machined implant. Commercially available 4 mm titanium implants, either with a machined (Wide Ponto) or a laser-ablated surface (Ponto BHX, Oticon Medical, Sweden), were used in the current study. The surfaces were evaluated by scanning electron microscopy. The implantation was performed with a full soft tissue flap and the osteotomy was prepared using the MIPS drill kit (Oticon Medical, Sweden) prior to installation of the implants in the frontal bone of eight female sheep. After five months, biopsies including the implant and surrounding bone tissue obtained, processed and analysed using histology, histomorphometry, scanning electron microscopy and Raman spectroscopy. The animals healed well, without signs of adverse events. Histomorphometry showed a large amount of bone tissue around both implant types, with 75% of the threaded area occupied by bone for both implant types. A large amount of bone-implant contact was observed for both implant types, with 67%-71% of the surface covered by bone. Both implant types were surrounded by mature remodelled lamellar bone with high mineral content, corroborating the histological observations. The current results show that the laser-ablated surface induces healing similar to the well-known clinically used machined surface in ovine cranial bone. In conclusion, the present long-term experimental results indicate that a laser-ablated implant performs equally well as a clinically used implant with a machined surface. This, together with previously reported, improved early biomechanical anchorage, suggests future, safe and efficient clinical potential.

The long-term impact of alveolar ridge preservation with xenograft bone mineral on peri-implant health after 5 years in function: A retrospective cohort study of 108 patients assessed clinically and radiologically.

OBJECTIVES: When teeth are lost, dental implants contribute to improved oral function and quality of life. Limitations in dental implant placement arising from poor bone anatomy may be circumvented via alveolar ridge preservation (ARP). The aim is to evaluate the long-term impact of ARP on peri-implant health and the relationship with common risk indicators such as smoking and history of periodontitis. MATERIALS AND METHODS: One hundred and eight patients were enrolled in this retrospective cohort study with 308 implants. Of these, ∼41% were placed in bone sites that had previously received ARP with deproteinized bovine bone mineral xenograft. Association between baseline variables: ARP, age, gender, number of implants per patient, anatomical site, smoking, and previous history of grade III/IV periodontitis, and outcome variables: mucositis, peri-implantitis, implant loss, full-mouth plaque score (FMPS), full-mouth bleeding score, and marginal bone loss (MBL) was evaluated using both univariate and multivariate models. RESULTS: After 5 years, the overall survival rate was 93.7%. The occurrence of peri-implantitis was 21.3% and the extent of MBL was ~2.2 mm. Both peri-implantitis occurrence and MBL were comparable between ARP+  and ARP- . Smoking is associated with higher FMPS and MBL. CONCLUSIONS: The findings indicate that peri-implant health can be maintained around dental implants for up to 5 years in ARP+  sites using Bio-Oss®. Smoking is a major risk indicator for peri-implantitis, whereas the association between history of periodontitis and the risk of peri-implantitis, based on this specific, well-maintained cohort and the specific implants used, remains inconclusive.

Bone mineral organization at the mesoscale: A review of mineral ellipsoids in bone and at bone interfaces.

Much debate still revolves around bone architecture, especially at the nano- and microscale. Bone is a remarkable material where high strength and toughness coexist thanks to an optimized composition of mineral and protein and their hierarchical organization across several distinct length scales. At the nanoscale, mineralized collagen fibrils act as building block units. Despite their key role in biological and mechanical functions, the mechanisms of collagen mineralization and the precise arrangement of the organic and inorganic constituents in the fibrils remains not fully elucidated. Advances in three-dimensional (3D) characterization of mineralized bone tissue by focused ion beam-scanning electron microscopy (FIB-SEM) revealed mineral-rich regions geometrically approximated as prolate ellipsoids, much larger than single collagen fibrils. These structures have yet to become prominently recognized, studied, or adopted into biomechanical models of bone. However, they closely resemble the circular to elliptical features previously identified by scanning transmission electron microscopy (STEM) in two-dimensions (2D). Herein, we review the presence of mineral ellipsoids in bone as observed with electron-based imaging techniques in both 2D and 3D with particular focus on different species, anatomical locations, and in proximity to natural and synthetic biomaterial interfaces. This review reveals that mineral ellipsoids are a ubiquitous structure in all the bones and bone-implant interfaces analyzed. This largely overlooked hierarchical level is expected to bring different perspectives to our understanding of bone mineralization and mechanical properties, in turn shedding light on structure-function relationships in bone. STATEMENT OF SIGNIFICANCE: In bone, the hierarchical organization of organic (mainly collagen type I) and inorganic (calcium-phosphate mineral) components across several length scales contributes to a unique combination of strength and toughness. However, aspects related to the collagen-mineral organization and to mineralization mechanisms remain unclear. Here, we review the presence of mineral prolate ellipsoids across a variety of species, anatomical locations, and interfaces, both natural and with synthetic biomaterials. These mineral ellipsoids represent a largely unstudied feature in the organization of bone at the mesoscale, i.e., at a level connecting nano- and microscale. Thorough understanding of their origin, development, and structure can provide valuable insights into bone architecture and mineralization, assisting the treatment of bone diseases and the design of bio-inspired materials.

Enhancing osteoblast survival through pulsed electrical stimulation and implications for osseointegration.

Electrical stimulation has been suggested as a means for promoting the direct structural and functional bonding of bone tissue to an artificial implant, known as osseointegration. Previous work has investigated the impact of electrical stimulation in different models, both in vitro and in vivo, using various electrode configurations for inducing an electric field with a wide range of stimulation parameters. However, there is no consensus on optimal electrode configuration nor stimulation parameters. Here, we investigated a novel approach of delivering electrical stimulation to a titanium implant using parameters clinically tested in a different application, namely peripheral nerve stimulation. We propose an in vitro model comprising of Ti6Al4V implants precultured with MC3T3-E1 preosteoblasts, stimulated for 72 h at two different pulse amplitudes (10 µA and 20 µA) and at two different frequencies (50 Hz and 100 Hz). We found that asymmetric charge-balanced pulsed electrical stimulation improved cell survival and collagen production in a dose-dependent manner. Our findings suggest that pulsed electrical stimulation with characteristics similar to peripheral nerve stimulation has the potential to improve cell survival and may provide a promising approach to improve peri-implant bone healing, particularly to neuromusculoskeletal interfaces in which implanted electrodes are readily available.

Immunomodulatory effects exerted by extracellular vesicles from Staphylococcus epidermidis and Staphylococcus aureus isolated from bone-anchored prostheses.

Staphylococcus aureus and Staphylococcus epidermidis are the bacteria that most frequently cause osteomyelitis. This study aimed to determine whether staphylococci isolated from osteomyelitis associated with septic loosening of orthopedic prostheses release extracellular vesicles (EVs) and, if so, to determine tentative immunomodulatory effects on the human monocytic cell line THP-1. EVs were isolated from bacterial cultures using filtration and ultracentrifugation and characterized by scanning electron microscopy, nanoparticle tracking analysis and Western Blot. The cytotoxic effect of EVs was analyzed by NucleoCounter and lactate dehydrogenase (LDH) analyses. Confocal laser scanning microscopy was employed to visualize the uptake of EVs by THP-1 cells. Activation of the transcription factor nuclear factor-κB (NF-κB) was determined in THP1-Blue™ NF-κB cells, and the gene expression and secretion of cytokines were determined by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. All investigated strains, irrespective of their biofilm formation ability, were able to secrete EVs in vitro. The S. aureus strains produced significantly more EVs than the S. epidermidis strains. Both S. aureus-derived EVs and S. epidermidis-derived EVs were internalized by THP-1 cells, upregulated Toll-like receptor 3 (TLR3) gene expression, activated NF-κB, and promoted the gene expression and secretion of interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1, matrix metallopeptidase (MMP)-9 and IL-10. Whereas EVs from both staphylococcal species upregulated the proapoptotic DNA damage-inducible transcript 4 (DDIT4) gene and downregulated the antiapoptotic B-cell lymphoma 2 (Bcl-2) gene, cytolysis was preferentially induced in S. aureus EV-stimulated cells, possibly related to the expression of cytolytic proteins predominantly in S. aureus EVs. In conclusion, staphylococcal EVs possess potent cytolytic and immunomodulatory properties.

The effects of controlled nanotopography, machined topography and their combination on molecular activities, bone formation and biomechanical stability during osseointegration.

The initial cellular and molecular activities at the bone interface of implants with controlled nanoscale topography and microscale roughness have previously been reported. However, the effects of such surface modifications on the development of osseointegration have not yet been determined. This study investigated the molecular events and the histological and biomechanical development of the bone interface in implants with nanoscale topography, microscale roughness or a combination of both. Polished and machined titanium implants with and without controlled nanopatterning (75 nm protrusions) were produced using colloidal lithography and coated with a thin titanium layer to unify the chemistry. The implants were inserted in rat tibiae and subjected to removal torque (RTQ) measurements, molecular analyses and histological analyses after 6, 21 and 28 days. The results showed that nanotopography superimposed on microrough, machined, surfaces promoted an early increase in RTQ and hence produced greater implant stability at 6 and 21 days. Two-way MANOVA revealed that the increased RTQ was influenced by microscale roughness and the combination of nanoscale and microscale topographies. Furthermore, increased bone-implant contact (BIC) was observed with the combined nanopatterned machined surface, although MANOVA results implied that the increased BIC was mainly dependent on microscale roughness. At the molecular level, the nanotopography, per se, and in synergy with microscale roughness, downregulated the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In conclusion, controlled nanotopography superimposed on microrough machined implants promoted implant stability during osseointegration. Nanoscale-driven mechanisms may involve attenuation of the inflammatory response at the titanium implant site. STATEMENT OF SIGNIFICANCE: The role of combined implant microscale and nanotopography features for osseointegration is incompletely understood. Using colloidal lithography technique, we created an ordered nanotopography pattern superimposed on screwshaped implants with microscale topography. The midterm and late molecular, bone-implant contact and removal torque responses were analysed in vivo. Nanotopography superimposed on microrough, machined, surfaces promoted the implant stability, influenced by microscale topography and the combination of nanoscale and microscale topographies. Increased bone-implant contact was mainly dependent on microscale roughness whereas the nanotopography, per se, and in synergy with microscale roughness, attenuated the proinflammatory tumor necrosis factor alpha (TNF-α) expression. It is concluded that microscale and nanopatterns provide individual as well as synergistic effects on molecular, morphological and biomechanical implant-tissue processes in vivo.

A stochastic micro to macro mechanical model for the evolution of bone-implant interface stiffness.

Upon placement of an implant into living bone, an interface is formed through which various biochemical, biological, physical, and mechanical interactions take place. This interface evolves over time as the mechanical properties of peri-implant bone increase. Owing to the multifactorial nature of interfacial processes, it is challenging to devise a comprehensive model for predicting the mechanical behavior of the bone-implant interface. We propose a simple spatio-temporally evolving mechanical model - from an elementary unit cell comprising randomly oriented mineralized collagen fibrils having randomly assigned stiffness all the way up to a macroscopic bone-implant interface in a gap healing scenario. Each unit cell has an assigned Young's modulus value between 1.62 GPa and 25.73 GPa corresponding to minimum (i.e., 0) and maximum (i.e., 0.4) limits of mineral volume fraction, respectively, in the overlap region of the mineralized collagen fibril. Gap closure and subsequent stiffening are modeled to reflect the two main directions of peri-implant bone formation, i.e., contact osteogenesis and distance osteogenesis. The linear elastic stochastic finite element model reveals highly nonlinear temporal evolution of bone-implant interface stiffness, strongly dictated by the specific kinetics of contact osteogenesis and distance osteogenesis. The bone-implant interface possesses a small stiffness until gap closure, which subsequently evolves into a much higher stiffness, and this transition is reminiscent of a percolation transition whose threshold corresponds to gap closure. The model presented here, albeit preliminary, can be incorporated into future calculations of the bone-implant system where the interface is well-defined mechanically. STATEMENT OF SIGNIFICANCE: A simple, physically informed model for the mechanical characteristics of the bone-implant interface is still missing. Here, we start by extending the reported mechanical characteristics of a one cubic micrometre unit cell to a 250 µm long interface made of 1 µm thick layers. The stiffness of each cell (based on mineral content) is assigned randomly to mimic bone micro-heterogeneity. The numerical study of this interface representative structure allows for the simultaneous determination of the spatio-temporal evolution of the mechanical response at local (discrete element) and global (overall model) scales. The proposed model is the first of this kind that can easily be incorporated into realistic future models of bone-implant interaction with emphasis on implant stability and different loading conditions.