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Osseointegrated implants inserted in the temporal bone are a vital component of bone-anchored hearing systems (BAHS). Despite low implant failure levels, early loading protocols and simplified procedures necessitate the application of implants which promote bone formation, bone bonding and biomechanical stability. Here, screw-shaped, commercially pure titanium implants were selectively laser ablated within the thread valley using an Nd:YAG laser to produce a microtopography with a superimposed nanotexture and a thickened surface oxide layer. State-of-the-art machined implants served as controls. After eight weeks' implantation in rabbit tibiae, resonance frequency analysis (RFA) values increased from insertion to retrieval for both implant types, while removal torque (RTQ) measurements showed 153% higher biomechanical anchorage of the laser-modified implants. Comparably high bone area (BA) and bone-implant contact (BIC) were recorded for both implant types but with distinctly different failure patterns following biomechanical testing. Fracture lines appeared within the bone ~30-50 μm from the laser-modified surface, while separation occurred at the bone-implant interface for the machined surface. Strong correlations were found between RTQ and BIC and between RFA at retrieval and BA. In the endosteal threads, where all the bone had formed de novo, the extracellular matrix composition, the mineralised bone area and osteocyte densities were comparable for the two types of implant. Using resin cast etching, osteocyte canaliculi were observed directly approaching the laser-modified implant surface. Transmission electron microscopy showed canaliculi in close proximity to the laser-modified surface, in addition to a highly ordered arrangement of collagen fibrils aligned parallel to the implant surface contour. It is concluded that the physico-chemical surface properties of laser-modified surfaces (thicker oxide, micro- and nanoscale texture) promote bone bonding which may be of benefit in situations where large demands are imposed on biomechanically stable interfaces, such as in early loading and in compromised conditions.

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). 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. 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. 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.

Osseointegrated implants are frequently used in reconstructive surgery, both in the dental and orthopedic field, restoring physical function and improving the quality of life for the patients. The bone anchorage is typically evaluated at micrometer resolution, while bone tissue is a dynamic composite material composed of nanoscale collagen fibrils and apatite crystals, with defined hierarchical levels at different length scales. In order to understand the bone formation and the ultrastructure of the interfacial tissue, analytical strategies needs to be implemented enabling multiscale and multimodal analyses of the intact interface. This paper describes a sample preparation route for successive analyses allowing assessment of the different hierarchical levels of interest, going from macro to nano scale and could be implemented on single samples. Examples of resulting analyses of different techniques on one type of implant surface is given, with emphasis on correlating the length scale between the different techniques. The bone-implant interface shows an intimate contact between mineralized collagen bundles and the outermost surface of the oxide layer, while bone mineral is found in the nanoscale surface features creating a functionally graded interface. Osteocytes exhibit a direct contact with the implant surface via canaliculi that house their dendritic processes. Blood vessels are frequently found in close proximity to the implant surface either within the mineralized bone matrix or at regions of remodeling.

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.

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

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.

The orientation of nanoscale mineral platelets was quantitatively evaluated in relation to the shape of lacunae associated with partially embedded osteocytes (osteoblastic–osteocytes) on the surface of deproteinised trabecular bone of adult sheep. By scanning electron microscopy and image analysis, the mean orientation of mineral platelets at the osteoblastic–osteocyte lacuna (Ot.Lc) floor was found to be 19° ± 14° in the tibia and 20° ± 14° in the femur. Further, the mineral platelets showed a high degree of directional coherency: 37 ± 7 % in the tibia and 38 ± 9 % in the femur. The majority of Ot.Lc in the tibia (69.37 %) and the femur (74.77 %) exhibited a mean orientation of mineral platelets between 0° and 25°, with the largest fraction within a 15°–20° range, 17.12 and 19.8 % in the tibia and femur, respectively. Energy dispersive X-ray spectroscopy and Raman spectroscopy were used to characterise the features observed on the anorganic bone surface. The Ca/P (atomic %) ratio was 1.69 ± 0.1 within the Ot.Lc and 1.68 ± 0.1 externally. Raman spectra of NaOCl-treated bone showed peaks associated with carbonated apatite: ν 1 , ν 2 and ν 4 PO 4 3− , and ν 1 CO 3 2− , while the collagen amide bands were greatly reduced in intensity compared to untreated bone. The apatite-to-collagen ratio increased considerably after deproteinisation; however, the mineral crystallinity and the carbonate-to-phosphate ratios were unaffected. The ~19°–20° orientation of mineral platelets in at the Ot.Lc floor may be attributable to a gradual rotation of osteoblasts in successive layers relative to the underlying surface, giving rise to the twisted plywood-like pattern of lamellar bone.

Introduction: Chronic infections are a major clinical challenge in hard-to-heal wounds and implanted devices. Pseudomonas aeruginosa is a common causative pathogen that produces numerous virulence factors. Due to the increasing problem of antibiotic resistance, new alternative treatment strategies are needed. Quorum sensing (QS) is a bacterial communication system that regulates virulence and dampens inflammation, promoting bacterial survival. QS inhibition is a potent strategy to reduce bacterial virulence and alleviate the negative impact on host immune response.Aim: This study investigates how secreted factors from P. aeruginosa PAO1, cultured in the presence or absence of the QS inhibitor sodium salicylate (NaSa), influence host immune response.Material and methods: In vitro, THP-1 macrophages and neutrophil-like HL-60 cells were used. In vivo, discs of titanium were implanted in a subcutaneous rat model with local administration of P. aeruginosa culture supernatants. The host immune response to virulence factors contained in culture supernatants (+/-NaSa) was characterized through cell viability, migration, phagocytosis, gene expression, cytokine secretion, and histology.Results: In vitro, P. aeruginosa supernatants from NaSa-containing cultures significantly increased THP-1 phagocytosis and HL-60 cell migration compared with untreated supernatants (-NaSa). Stimulation with NaSa-treated supernatants in vivo resulted in: (i) significantly increased immune cell infiltration and cell attachment to titanium discs; (ii) increased gene expression of IL-8, IL-10, ARG1, and iNOS, and (iii) increased GRO-α protein secretion and decreased IL-1β, IL-6, and IL-1α secretion, as compared with untreated supernatants.Conclusion: In conclusion, treating P. aeruginosa with NaSa reduces the production of virulence factors and modulates major immune events, such as promoting phagocytosis and cell migration, and decreasing the secretion of several pro-inflammatory cytokines.

In the present study, a model for simulations of removal torque experiments was developed using finite element method. The interfacial retention and fracturing of the surrounding material caused by the surface features during torque was analyzed. It was hypothesized that the progression of removal torque and the phases identified in the torque response plot represents sequential fractures at the interface. The 3-dimensional finite element model fairly accurately predicts the torque required to break the fixation of acid-etched implants, and also provides insight to how sequential fractures progress downwards along the implant side.

Osseointegration is a well-established concept used in applications including the percutaneous Bone-Anchored Hearing System (BAHS) and auricular rehabilitation. To date, few retrieved implants have been described. A systematic review including cases where percutaneous bone-anchored implants inserted in the temporal bone were retrieved and analyzed was performed. We also present the case of a patient who received a BAHS for mixed hearing loss. After the initial surgery, several episodes of soft tissue inflammation accompanied by pain were observed, leading to elective abutment removal 14 months post-surgery. Two years post-implantation, the implant was removed due to pain and subjected to a multiscale and multimodal analysis: microbial DNA using molecular fingerprinting, gene expression using quantitative real-time polymerase chain reaction (qPCR), X-ray microcomputed tomography (micro-CT), histology, histomorphometry, backscattered scanning electron microscopy (BSE-SEM), Raman spectroscopy, and fluorescence in situ hybridization (FISH). Evidence of osseointegration was provided via micro-CT, histology, BSE-SEM, and Raman spectroscopy. Polymicrobial colonization in the periabutment area and on the implant, including that with Staphylococcus aureus and Staphylococcus epidermidis , was determined using a molecular analysis via a 16S-23S rDNA interspace [IS]-region-based profiling method (IS-Pro). The histology suggested bacterial colonization in the skin and in the peri-implant bone. FISH confirmed the localization of S. aureus and coagulase-negative staphylococci in the skin. Ten articles (54 implants, 47 patients) met the inclusion criteria for the literature search. The analyzed samples were either BAHS (35 implants) or bone-anchored aural epitheses (19 implants) in situ between 2 weeks and 8 years. The main reasons for elective removal were nonuse/changes in treatment, pain, or skin reactions. Most samples were evaluated using histology, demonstrating osseointegration, but with the absence of bone under the implants’ proximal flange. Taken together, the literature and this case report show clear evidence of osseointegration, despite prominent complications. Nevertheless, despite implant osseointegration, chronic pain related to the BAHS may be associated with a chronic bacterial infection and raised inflammatory response in the absence of macroscopic signs of infection. It is suggested that a multimodal analysis of peri-implant health provides possibilities for device improvements and to guide diagnostic and therapeutic strategies to alleviate the impact of complications.