Aims. This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation. Methods. In this study, 60 rats were included in a titanium rod implantation model and underwent subsequent guanylate cyclase treatment. Imaging, histology, and biomechanics were used to evaluate the osseointegration of rats in each group. First, the impact of VIT on bone integration in aged rats with iron overload was investigated. Subsequently, VIT was employed to modulate the differentiation of MC3T3-E1 cells and RAW264.7 cells under conditions of iron overload. Results. Utilizing an OVX rat model, we observed significant alterations in bone mass and osseointegration due to VIT administration in aged rats with iron overload. The observed effects were concomitant with reductions in bone metabolism, oxidative stress, and inflammation. To elucidate whether these effects are associated with osteoclast and osteoblast activity, we conducted in vitro experiments using MC3T3-E1 cells and RAW264.7 cells. Our findings indicate that iron accumulation suppressed the activity of MC3T3-E1 while enhancing RAW264.7 function. Furthermore, iron overload significantly decreased oxidative stress levels; however, these detrimental effects can be mitigated by VIT treatment. Conclusion. Collectively, our data provide compelling evidence that VIT has the potential to reverse the deleterious consequences of iron overload on osseointegration and bone mass during ageing. Cite this article: Bone Joint Res 2024;13(9):427–440

Objectives. Ultraviolet (UV) light-mediated photofunctionalisation is known to improve osseointegration of pure titanium (Ti). However, histological examination of titanium alloy (Ti6Al4V), which is frequently applied in orthopaedic and dental surgery, has not yet been performed. This study examined the osseointegration of photofunctionalised Ti6Al4V implants. Methods. Ti and Ti6Al4V implants were treated with UV light, and the chemical composition and contact angle on the surfaces were evaluated to confirm photofunctionalisation. The implants were inserted into femurs in rats, and the rats were killed two or four weeks after the surgery. For histomorphometric analysis, both the bone–implant contact (BIC) ratio and the bone volume (BV) ratio were calculated from histological analysis and microcomputed tomography data. Results. The amount of carbon and the contact angle on both implants were significantly reduced after UV irradiation. The BIC ratios for both UV light-treated implants significantly increased at two weeks, but there was no significant difference at four weeks. There was no significant difference in the BV ratios between the UV light-treated and control implants at two or four weeks. Conclusions. This study suggests that photofunctionalisation of Ti6Al4V implants, similar to that of Ti implants, may promotes osseointegration in early but not in the late phase of osseointegration. Cite this article: R. Yamauchi, T. Itabashi, K. Wada, T. Tanaka, G. Kumagai, Y. Ishibashi. Photofunctionalised Ti6Al4V implants enhance early phase osseointegration. Bone Joint Res 2017;6:331–336. DOI: 10.1302/2046-3758.65.BJR-2016-0221.R1

Failure of osseointegration and periprosthetic joint infection (PJI) are the two main reasons of implant failure after total joint replacement (TJR). Nanofiber (NF) implant surface coating represents an alternative local drug eluting device that improves osseointegration and decreases the risk of PJI. The purpose of this study was to investigate the therapeutic efficacies of erythromycin (EM)-loaded coaxial PLGA/PCL-PVA NF coating in a rat S. aureus-infected tibia model. NF coatings with 100mg and 1000mg EM were prepared. NF without EM was included as positive control. 56 Sprague Dawley rats were divided into 4 groups. A titanium pin (1.0-mm x 8 mm) was placed into the tibia through the intercondylar notch. S. aureus (SA) was introduced by both direct injection of 10 μl broth (1 × 10. 4. CFU) into the medullary cavity and single dip of Ti pins into a similar solution prior to insertion. Rats were sacrificed at 8 and 16 weeks after surgery. The outcome measurements include μCT based quantitative osteolysis evaluation and hard tissue histology. Results: EM-NF coating (EM100 and EM1000) reduced osteolysis at 8 and 16 weeks, compared to EM0 and negative control. The effective infection control by EM-NFs was further confirmed by hard tissue section analysis. The Bone implant contact (BIC) and bone area fraction Occupancy (BAFO) within 200 µm of the surface of the pins were used to evaluate the osseointegration and new bone formation around the implants. At 16 weeks, the bone implant contact (BIC) of EM 100 (35.08%) was higher than that of negative control (3.43%) and EM0 (0%). The bone area fraction occupancy within 200 µm (BAFO) of EM100 (0.63 mm2) was higher than that of negative control (0.390 mm2) and EM0 (0.0 mm. 2. ). The BAFO of EM100 was also higher than that of EM1000 (0.3mm. 2. ). There was much less osteolysis observed with EM100 and EM1000 NF coatings at 16 weeks, as compared to EM0 positive control, p=0.08 and p=0.1, respectively. Osseointegration and periprosthetic bone formation was enhanced by EM-NFs, especially EM100. Data from this pilot study is promising for improving implant surface fabrication strategies

The use of implant biomaterials for prosthetic reconstructive surgery and osteosynthesis is consolidated in the orthopaedic field, improving the quality of life of patients and allowing for healthy and better ageing. However, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials, particularly for the study of local effects of implant and osteointegration. Despite the complex process of osseointegration is difficult to recreate in vitro, the growing challenges in developing alternative models require to set-up and validate new approaches. Aim of the present study is to evaluate an advanced in vitro tissue culture model of osteointegration of titanium implants in human trabecular bone. Cubic samples (1.5×1.5 cm) of trabecular bone were harvested as waste material from hip arthroplasty surgery (CE AVEC 829/2019/Sper/IOR); cylindrical defects (2 mm Ø, 6 mm length) were created, and tissue specimens assigned to the following groups: 1) empty defects- CTR-; 2) defects implanted with a cytotoxic copper pin (Merck cod. 326429)- CTR+; 3) defects implanted with standard titanium pins of 6 µm-rough (ZARE S.r.l) -Ti6. Tissue specimens were cultured in mini rotating bioreactors in standard conditions, weekly assessing viability. At the 8-week-timepoint, immunoenzymatic, microtomographic, histological and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, differently from Ti6 which appears to have a trophic effect on the bone. MicroCT and histological analysis supported the results, with lower BV/TV and Tb.Th values observed in CTR- compared to CTR+ and Ti6 and signs of matrix and bone deposition at the implant site. The collected data suggest the reliability of the tested model which can recreate the osseointegration process in vitro and can therefore be used for preliminary evaluations to reduce and refine in vivo preclinical models. Acknowledgment: This work was supported by Emilia-Romagna Region for the project “Sviluppo di modelli biologici in vitro ed in silico per la valutazione e predizione dell'osteointegrazione di dispositivi medici da impianto nel tessuto osseo”

3D Printed polyether-ether-ketone (PEEK) has gained widespread use in clinical practice due to its excellent biocompatibility, biomechanical compatibility, and personalization. However, pre-printed PEEK implants are not without their flaws, including bioinert, optimization distortion of 3D printing digital model and prosthetic mismatching. Recent advancements in mechanical processing technology have made it possible to print bone implants with PEEK fused deposition, allowing for the construction of mechanically adaptable implants. In this study, we aimed to synthesize silanized polycitrate (PCS) via thermal polymerization and in situ graft it to PEEK surface to construct an elastomer coating for 3D printed PEEK implants (PEEK-PCS). This incorporation of PCS allows the implant to exhibit adaptive space filling ability and stress dispersal. In vivo and in vitro results, PEEK-PCS exhibited exceptional osseointegration and osteogenesis properties along with macrophage M2 phenotypic polarization, inflammatory factors reducing, promotion of osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Additionally, PEEK-PCS displays good autofluorescence properties in vitro and in vivo, with stable fluorescence for 14 days, suggesting potential bioimaging applications. The study confirms that PEEK in situ grafting with thermo-polymerized PCS elastomers is a viable approach for creating multifunctional (bone defect adaptation, bioimaging, immune regulation, and osseointegration) implants for bone tissue engineering

Abstract. Introduction. The long-term biological success of cementless orthopaedic prostheses is highly dependent on osteointegration. Pre-clinical testing of new cementless implant technology however, requires live animal testing, which has anatomical, loading, ethical and cost challenges. This proof-of-concept study aimed to develop an in vitro model to examine implant osteointegration under known loading/micromotion conditions. Methods. Fresh cancellous bone cylinders (n=8) were harvested from porcine femur and implanted with additive manufactured porous titanium implants (Ø4 × 15 mm). To simulate physiological conditions, n=3 bone cylinders were tested in a bioreactor system with a cyclic 30 µm displacement at 1Hz for 300 cycles every day for 15 days in a total of 21 days culture. The chamber was also perfused with culture medium using a peristaltic pump. Control bone cylinders were cultured under static conditions (n=5). Samples were calcein stained at day 7. Post-testing, bone cylinders were formalin fixed and bony ingrowth was measured via microscopy. Results. Viability of the freshly harvested ex vivo bone cylinders was maintained for up to 28 days. Two samples remain unanalysed due to COVID lockdown, one in each group. Similar to osteointegration seen in live animal models, evidence of bony ingrowth was seen more markedly at the bone-implant interface under dynamic conditions. This was evident by a greater intensity of calcein staining, confirming the deposition of new bone, at the bone-implant interface. In comparison, under static conditions, calcein staining was observed randomly all over the cylinder. Conclusion. This proof-of-concept study demonstrates that implant bony adaptation and ingrowth can be measured in vitro under known cyclic micromotion/loading conditions. This comparatively low cost, low ethical impact, controlled loading laboratory method has potential to accelerate the rate of implant development whilst conforming with the principles of NC3Rs. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Hypoxic Inducible Factor and Hypoxic mimicking agents (HMA) trigger the initiation and promotion of angiogenic-osteogenic cascade events. However, there has been paucity of studies investigating how HIF could be over expressed under chronic hypoxic conditions akin to that seen in sickle cell disease patients to help form a template for tackling the matter of macrocellular avascular necrosis. Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration, and the hypoxia-inducible factor-1 alpha (HIF-1) pathway has been identified as a key component in this process studies have shown. There are still no established experimental models showing how this knowledge can be used for the evaluation of bone implant integration and suggest ways of improving osseointegration in sickle cell disease patients with hip arthroplasty and thereby prevent increased implant loosening. The aim of this study is to help develop an in vitro experimental model which would mimic the in vivo pathologic state in the bone marrow of sickle cell disease patients. It also seeks to establish if the hypoxic inducible factor (HIF) could be over expressed in vitro and thus enhancing osseointegration. MG63 osteoblastic cells were cultured under normoxia and hypoxic conditions (20%; and 1% oxygen saturation) for 48 and 72 hours. Cobalt chloride was introduced to the samples in order to mimic true hypoxia. Cells cultured under normoxic conditions and without cobalt chloride was used as the control in this study. The expression of the hypoxic inducible factor was assessed using the reverse transcriptase qualitative polymerase chain reaction (RT-qPCR). There was increased expression of HIF1-alpha at 72hours as compared to 48hours under the various conditions. The level of expression of HIF increased from 48hrs (mean rank= 4.60) to 72hrs (mean rank =5.60) but this difference was not statistically significant, X2(1) = 0.24, p =0.625. The mean rank fold change of HIF in hypoxic samples decreased compared to the normoxic samples but this difference was not statistically significant, X2(1) = 0.54, p= 0.462. Therefore, the expression of HIF is only increased with prolonged hypoxia as seen in the 72hours samples. The expression of HIF increased in samples with CoCl2 (mean rank=5.17), compared with samples without CoCl2 (mean rank 4.67), however this was not statistically significant, X2(1) = 0.067, p=0.796, p value > 0.05. The over expression of HIF was achieved within a few days (72hours) with the introduction of Cobalt Chloride, which is a mimetic for hypoxia similar to the in vivo environment in sickle cell disease patients. This is an in vitro model which could help investigate osseointergation in such pathologic bone conditions

Focal knee resurfacing implants (FKRIs) are typically intended to treat focal cartilage defects in middle-aged patients. All currently available FKRIs are (partly) composed of metal, which potentially leads to degeneration of the opposing articulating cartilage and hampers follow-up using magnetic resonance imaging (MRI). The purpose of this study was to investigate the in vivo osseointegration process of a novel non-degradable thermoplastic polycarbonate-urethane (TPU) osteochondral implant. Bi-layered implants measuring 6 mm in diameter, with a double-curvature to match the approximate curvature of the goat medial femoral condyle were fabricated. TPU implants were composed of an articulating Bionate® II 80A top layer, and a Bionate® 75D bottom layer (DSM Biomedical, Geleen, the Netherlands) which is intended to osseointegrate. A biphasic calcium phosphate coating formulation, optimized during a prior in vitro study, was applied to half of the TPU implants, while the other half was left uncoated. Bi-layered metal implants (articulating cobalt-chromium top layer and titanium bottom layer) were used as positive control implants. Eight implants per group were implanted bilaterally in the medial femoral condyle of the stifle joints in 12 Dutch milk goats. 18F-sodium fluoride (18F-NaF) positron emission tomography-computed tomography (PET-CT) scanning was performed at 3 and 12 weeks postoperatively, and the corrected maximum standard uptake values (cSUVmax) was calculated to assess the peri-implant bone metabolism. After sacrifice 12 weeks postoperatively, bone histomorphometric analysis was performed to assess the bone-to-implant contact area (BIC). Student's T-test was used in case of normal distribution and the Mann-Whitney-U-test was used in case of abnormal distribution for comparison of BIC and cSUVmax. The BIC value of 10.27 ± 4.50% (mean ± SD) for the BCP-coated TPU implants was significantly (P=0.03) higher than the 4.50 ± 2.61% for the uncoated TPU implants. The uncoated TPU implants scored significantly (P=0.04) lower than the BIC of 12.81 ± 7.55% for the metal implants, whereas there was no significant difference between BCP-coated TPU implants and the metal implants (P=0.68). There was a strong correlation between the cSUVmax values and the BIC values at 12 weeks (Pearson's R=0.74, P=0.001). The cSUVmax values significantly decreased between 3 and 12 weeks for the metal implants (p=0.04). BCP-coated TPU implants followed a similar trend but did not reach statistical significance (p=0.07). cSUVmax in the uncoated TPU implants did not show a significant difference between the time-points (p=0.31). Osseointegration of BCP-coated TPU implants did not significantly differ from metal implants. 18F-NaF PET-CT is a feasible modality to assess osseointegration patterns and showed a similar trend between the BCP-coated and metal implants. Hence, an implant fully composed of TPU may avoid the typical metal-related drawbacks of currently available FKRIs. Long-term follow-up studies are advocated to address the effects of the implant to the opposing cartilage, and are therefore warranted

Abstract. Objectives. Currently, total hip replacement surgery is an effective treatment for osteoarthritis, where the damaged hip joint is replaced with an artificial joint. Stress shielding is a mechanical phenomenon that refers to the reduction of bone density as a result of altered stresses acting on the host bone. Due to solid metallic nature and high stiffness of the current orthopaedic prostheses, surrounding bones undergo too much bone resorption secondary to stress shielding. With the use of 3D printing technology such as selective laser melting (SLM), it is now possible to produce porous graded microstructure hip stems to mimics the surrounding bone tissue properties. Method. In this study we have compared the physical and mechanical properties of two triply periodic minimal surface (TPMS) lattice structure namely gyroid and diamond TPMS. Based on initial investigations, it was decided to design, and 3D print the gyroid and diamond scaffolds having pore size of 800 and 1100 um respectively. Scaffold of each type of structure were manufactured and were tested mechanically in compression (n=8), tension (n=5) and bending (n=1). Results. Upon FEA validation of the scaffold in Abaqus, the desired scaffold for hip implant application was evaluated to have a young's modules of 12.15 GPa, yield strength of 242 MPa and porosity of 55%. Topology and lattice optimization were performed using nTopology to design an optimised graded porous hip implant based on stress shielding reduction. It was understood that the designed optimised hip implant can reduce the stress shielding effect by more than 65% when compared to the conventional generic implant. Conclusions. The designed hip implant presented in this work shows clinical promise in reducing bone loss while having enhanced osseointegration with the surrounding cortical bones. Hence, this will help reduce the risk of periprosthetic fracture and the probability of revision surgery

Background. Following endosteal uncemented orthopaedic device implantation, the initial implant/bone interface retains spaces and deficiencies further exacerbated by pressure necrosis and resultant bone resorption. This implant-bone space requires native bone infill through the process of de novo osteogenesis. New appositional bone formation on the implant surface is known as contact osteogenesis and is generated by osteogenic cells, including skeletal stem cells (SSCs), colonising the implant surface and depositing the extracellular bone matrix. Surface nanotopographies provide physical cues capable of triggering SSC differentiation into osteoblasts, thus inducing contact osteogenesis, translated clinically into enhanced osseointegration and attainment of secondary stability. The current study has investigated the in vitro and in vivo effects of unique nanotopographical pillar substrates on SSC phenotype and function. Methods. Adult human SSCs were immunoselected, enriched using STRO-1 antibody and cultured on control and test surfaces for 21 days in vitro. The test groups comprised Ti-coated substrates with planar or modified surfaces with nanopillar. Osteoinductive potential was analysed using qPCR and immunostaining to examine gene expression and protein synthesis. Results. Following in vitro (n=5) culture on nanopillars, the expression of osteogenic genes (ALP, Collagen 1, OPN and OCN) and of Osteopontin protein (a bone matrix protein), on Ti pillars were both significantly enhanced when compared to control or Ti planar surfaces. Conclusions. Discrete raised surface nanopillars modulate adult SSC populations in the absence of any chemical cues and enhance their osteogenic properties, an effect not observed on planar Ti constructs. Hence, these findings herald exciting opportunities to improve the implant surface design, implant osseointegration, and, ultimately, implant survival. Level of evidence. Original experimental study

Degree of early integration of titanium alloy implants into bone is an important predictor of long term implant success in arthroplasty. The correlation between observations on early cell adhesion and the ability of modified surfaces to affect osseointegration of implants in in vivo models is unclear. We hypothesised that observation of increased focal adhesion complexes in early cultures of osteoblasts would correlate with increased osseointegration of treated implants in an animal model. Longer term culture of rat osteoblasts for alkaline phosphatase activity indicated that cells cultured on the 9V treated surfaces were displaying greater alkaline phosphatase activity at 14 days. Bone nodule formation at 28 days demonstrated a trend towards smaller area of bone nodules on the surfaces treated at 9V then those treated at 3V and 5V. A rat model was employed for testing mechanical push-out strength of experimental implants and demonstrated a trend towards increased yield strength of the bone-implant interface for implants treated at 3V180s and 5V180s. Histomorphometry was performed and no statistically significant differences in percentage area of contact with mineralised bone matrix were seen, although there was a trend for greater mineralised matrix contact on the polished and 9V180s treated implants. Previous experiments demonstrated cells on the 9V treated surfaces were well spread and had significantly increased size and number of focal adhesions. This was regarded as indicating more successful cell adhesion. The above results demonstrate that this early trend disappeared in longer term culture did not persist in experiments in an animal model

Summary Statement. Preoperative bone-marrow-derived cell mobilization by G-CSF is a safe orthopaedic procedure and allows circulation in the blood of high numbers of CD34+ve cells, promoting osseointegration of a bone substitute. Introduction. Granulocyte-colony-stimulating-factor(G-CSF) has been used to improve repair processes in different clinical settings for its role in bone-marrow stem cell(CD34+ and CD34-) mobilization. Recent literature suggests that G-CSF may also play a role in skeletal-tissue repair processes. Aim of the study was to verify the feasibility and safety of preoperative bone-marrow cell (BMC) mobilization by G-CSF in orthopaedic patients and to evaluate G-CSF efficacy in accelerating bone regeneration following opening-wedge high tibial valgus osteotomy(HTVO) for genu varum. Patients/Methods. 24 patients were enrolled in a prospective phase II trial. The osteotomy gap was filled by a hydroxyapatite-tricalciumphosphate bone substitute(HATriC). Patients were randomised to receive (GROUP A) or not receive (GROUP B) preoperatively a daily dose of 10µg/kg of G-CSF for three consecutive days, with an additional dose 4 hours before surgery. BMC-mobilization was monitored by white blood cell (WBC)-count, flow-cytometry analysis of circulating CD34+cells and Colony-forming cell assays. Patients were evaluated by: Lysholm and SF-36 scores preoperatively and at 1, 2, 3, 6, and 12 months after surgery;. X-ray evaluation preoperatively and at 1, 2, 3, 6, and 12 months after surgery, in order to compare the percentage of osseointegration of the bone-graft junction using the semi-quantitative score of Dallari[1]. CT-scan of the host bone-substitute interface at 2 months, in order to estimate the quality of the newly formed bone at the bone-graft junction by a quantitative measure of bone density (by Hounsfield unit) at the proximal and distal bone-graft junctions. Results. All patients completed the treatment program without major side effects; G-CSF was well tolerated. BMC-mobilization occurred in all Group A patients, with median peak values of 110/µL (range 29–256) of circulating CD34+ve cells. Circulating clonogenic progenitors paralleled CD34+ve cell levels. A significant improvement in the SF-36-Role-Physical scale and in the Lysholm score was recorded at follow-up in Group A compared to Group B(p<0.05). At the X-ray-evaluation, there was a significant increase in osseointegration at the bone-graft junction in Group A at 1, 2, 3 and 6 months post-surgery compared to Group B(p<0.05). CT-scans of the grafted area at 2 months post-surgery showed no significant difference in the quality of the newly formed bone between the two Groups. Discussion/Conclusions. These results suggest that G-CSF can be safely administered preoperatively in subjects undergoing HTVO. In addition, the clinical, radiographic and CT monitoring indicate that preoperative G-CSF administration promotes bone graft substitute osseointegration. Enhanced osseointegration might be the result of the direct activity of G-CSF on the host bone or a cellular effect mediated by bone marrow-derived progenitors mobilised by G-CSF, or by a combination of all these factors. This study is a proof-of-principle that preoperative G-CSF might be an alternative treatment option to enhance bone regeneration in the field of bone marrow stem cell therapy and reconstructive orthopaedic surgery

Summary Statement. A porcine model using Yucatan minipigs was found to be very promising for the investigation of healing around transcutaneous osseointegrated implants. Pigs demonstrated surprising agility and adaptability including the ability to ambulate on three legs during the immediate postoperative period. Introduction. Previous non weight-bearing and weight-bearing caprine, canine and ovine models have evaluated design, material, and biological coating variations in an attempt to improve the wound healing and skin-implant seal around transcutaneous osseointegrated implants. Although these models have primarily been used as a window into the application of transcutaneous osseointegrated implants in humans, some important model characteristics affecting wound healing and infection have been missing including: 1) replication of the physiological tissue response, and 2) availability of a transcutaneous site with sufficient soft tissue coverage. Pig skin, like human, is relatively hairless, tightly attached to the subcutaneous tissue, vascularised by a cutaneous blood supply, and healed by means of epithelialization. Swine have been extensively utilised for superficial and deep wound healing studies and can offer ample soft tissue coverage following a lower limb amputation. Development of a porcine model is important for continued understanding and improvement of weight-bearing transcutaneous osseointegration. Methods. Two male Yucatan mini-pigs (9 months, 36kg) were fit with transcutaneous osseointegrated prostheses using a single-stage transtibial amputation and prosthesis implantation procedure. The endo-prosthesis consisted of a cylindrical intraosseous threaded section and a smooth transcutaneous section. The transcutaneous sections were smooth to promote epithelialization and deter direct skin-implant adhesion. The implants were custom manufactured from medical grade Ti-6Al-4V alloy. The exo-prosthesis, consisting of an adjustable length leg and foot, was attached by clamp to the supercutaneous portion of the implant following either one or two days of sling constraint to limit initial weight-bearing. Various exo-prosthesis designs and configurations were trialed. The animals’ behavior and gait were closely observed. Weight-bearing was monitored using a force plate. At 5 and 8 weeks, clinical, microbiological, and histological data were examined to assess wound healing and infection at the skin-bone-implant interface. Results. The pigs demonstrated surprising agility and adaptability. They were able to successfully ambulate on three legs during the post-op period before weight-bearing was permitted. They adapted quickly to changes in exo-prosthesis design, position, and length. Although bacterial colonization was verified, neither of the animals exhibited clinical signs of infection over the respective eight and five week studies. Histological results indicated that there was no skin to implant adhesion but that epithelial growth was progressing towards the implant in one animal. Healing of the transcutaneous wound site showed substantial progress but a definitive skin seal was non-existent at the eight week time point. Discussion/Conclusion. This is likely the first animal model developed, having soft tissue characteristics similar to those found in humans, in which an axially-loaded, weight-bearing implant was successfully used. Results indicated that this porcine model offers many advantages over previous models for the development, evaluation, and comparison of the various techniques being advocated to achieve successful transcutaneous osseointegration in humans. The Yucatan miniature pig's ability to ambulate on three legs during the immediate post-operative period and quickly adjust to changes in the exo-prosthesis design, coupled with their physiological similarity to humans, makes them a valuable model for future studies

We implanted nails made of titanium (Ti6Al4V) and of two types of glass ceramic material (RKKP and AP40) into healthy and osteopenic rats. After two months, a histomorphometric analysis was performed and the affinity index calculated. In addition, osteoblasts from normal and osteopenic bone were cultured and the biomaterials were evaluated in vitro. In normal bone the rate of osseointegration was similar for all materials tested (p > 0.5) while in osteopenic bone AP40 did not osseointegrate (p > 0.0005). In vitro, no differences were observed for all biomaterials when cultured in normal bone-derived cells whereas in osteopenic-bone-derived cells there was a significant difference in some of the tested parameters when using AP40. Our findings suggest that osteopenic models may be used in vivo in the preclinical evaluation of orthopaedic biomaterials. We suggest that primary cell cultures from pathological models could be used as an experimental model in vitro

Introduction. A modified anodisation technique where a titanium surface releases bactericidal concentrations of silver was developed and called Agluna. Our hypothesis was that silver incorporation was bactericidal and had no effects on the viability of fibroblasts and osteoblasts, would have no negative effect on interfacial shear strength and bone contact in an in vivo trans-cortical implant ovine model. Methods. In vitro: Titanium alloy discs were either polished (Ti), anodised (Ano), anodised or Agluna treated (Ag) or anodised and Agluna treated followed by a conditioning step (Ag C). Conditioning was achieved by incubating discs in culture fluid for 48 hrs. The bactericidal effect of these discs was tested by measuring the zone of inhibition of different bacteria grown on agar. Live/dead staining was carried out and silver levels measured using atomic emission spectroscopy. 8 implants were inserted into each sheep (60 in total (n=5)). Grit blasted Titanium alloy (Gb) and Agluna treated grit blasted titanium alloy (Ag) at a silver concentration of 4-6 micrograms/cm2 were compared at 6 weeks. Gb implants, Ag (at 4-6micrograms/cm2), high dose Agluna implants with silver concentrations at 15-20micrograms/cm2 (HdAg) and a grit blasted anodised titanium alloy (Ano) were compared at 12 weeks. Pullout strength and bone-implant contact was quantified. Results. On Ti, Ano and Ag C surfaces the number of live fibroblasts was significantly greater than on Ag (non-conditioned) surfaces. Data from pull out tests at 6 weeks showed a lower but significant interfacial shear strength in the Ag group (310.4N) when compared with the Gb group (561.2N) (p=0.01). At 12 weeks, there were no significant differences between each of the 4 treatment groups. Histological analysis showed no significant differences in bone-implant contact between groups at 6 and 12 weeks. Discussion. The initial non-conditioned Agluna surface is bactericidal and cytotoxic but on conditioning, osteoblasts and fibroblasts attached and remained viable. The condition Agluna surface remains bactericidal. Silver incorporation at a concentration up to 20 micrograms/cm2 has no adverse toxic effect on osteointegration and the interfacial shear strength of implants. This coating has been used clinically in situations where the infection rate is high

It is well documented that implant loosening rate in sickle cell disease patients is higher than that seen in patients with hip arthroplasty from other indications. The Hypoxic inducible factor(HIF) - is activated in the microcellular hypoxic environment and this through a cascade of other enzymatic reactions promotes the activity of other factors and further help enhance angiogenesis and osteogenesis. The aim of this study was to investigate and propose a potential model for investigating osseointegration in a hypoxic microcellular environment using osteoblasts(MG63). Human MG63 osteoblastic cells were cultured under normoxia and hypoxic conditions (20%; and 1% oxygen saturation) for 72 hours under two different condition- with and without cobalt chloride. The samples cultured under normoxic condtions without cobalt chloride acted as control. Using qualitative polymerase chain reaction-(qPCR) - HIF expression was assessed under the above conditions in relation to the control. The results showed there was significant expression of the HIF 1 alpha protein under hypoxic condition with cobalt chloride in comparison with the control samples- all at 72hours incubation. Mann-Whitney U test was used to deduce level of significance of fold change.(p=0.002; <0.05). This was deemed as being a significant difference in the level of expression of HIF compared to the control. The results show that the hypoxic inducible factor can be expressed using the above tested. experimental invitro-model with significant results which can be a foundation for further research into improving hip implant prosthesis design to help enhance osseo-integration in sickle cell disease patient with AVN

Titanium is a popular orthopaedic implant material, but it requires surface modification techniques to improve osseointegration and long term functionality. This project compares a new method of modifying surface topography (nano-patterning) with an existing clinical technology (grit-blasting and acid-etching (GAE)). Titanium discs were blasted with aluminium oxide and etched in sulphuric and acetic acid. Injection moulded discs (with two different nano-patterns) were coated in titanium by evaporation. The topography and chemistry of the discs was assessed using atomic force microscopy (AFM), scanning electron microscopy (SEM), water contact angle measurements, and X-ray photo-electron spectroscopy (XPS). Two discs were plated bilaterally onto a flattened area of the tibiae of 12 rabbits. Tibiae were removed after 4 and 8 weeks for histological assessment of the bone-implant contact (BIC) ratio. AFM and SEM demonstrated a difference in pattern between the square array of nano-pits (SQ) and the randomly positioned nano-pits (RAND). The GAE implants exhibited increased surface roughness (Ra = 570nm) compared to the titanium coated SQ and RAND implants (Ra = 12nm). Water contact angle measurements showed the surface had comparable wettability and XPS demonstrated similar chemical compositions, except GAE surfaces contained 6.8% aluminium. Histological samples analysed at 4 weeks showed a BIC ratio of 36% for GAE, 56% for SQ, and 48% for RAND. At 8 weeks, the BIC ratio was 52% for GAE, 80% for SQ, and 72% for RAND implants. This increase in BIC at 8 weeks for both SQ and RAND implants compared to GAE was statistically significant (P < 0.05). This project demonstrated there was an increase in interfacial bone to implant contact when using a nano-scale topography incorporating nano-pits compared to conventional grit-blasted acid-etched micro-scale topographies. This enhancement of BIC may reduce long term loosening of orthopaedic implants due to mechanical and biological attrition at the interface

Summary Statement. The problem facing this research is to promote rapid osteointegration of titanium implants and to minimise the risks of infections by the functionalization with different agents, each designed for a specific action. A patented process gives a multifunctional titanium surface. Introduction. A patented process of surface modification is described. It gives a multifunctional surface with a multiscale roughness (micro and nano topography), that is excellent for osteoblast adhesion and differentiation. It has a high degree of hydroxylation, that is relevant for inorganic bioactivity (apatite-HA precipitation) and it is ready for a functionalization with biological factors. A direct grafting of ALP has been obtained. Moreover, the growth of an antibacterial agent within the surface oxide layer can be useful in order to combine the osteoinduction ability to antimicrobial effects. The selection of an inorganic agent (metal nanoparticles) has the advantage to avoid an eventual development of antibiotic resistance by bacteria. Experimental Methods. Ti-cp and Ti6Al4V samples were polished or blasted, etched in diluted hydrofluoric acid (step 1a), oxidised in hydrogen peroxide (step 1b), incubated in Tresyl chloride (step 2a) and Alkaline phosphatase (ALP) enzyme (step 2b) [1, 2]. A water solution, containing a salt of the metal to be added to the surface as an inorganic antibacterial agent, can be introduced during the oxidation in hydrogen peroxide. Surface morphology and chemical composition were investigated by Scanning Electron Microscopy (SEM) and Field Emission Scanning Electron Microscopy (FESEM) equipped with Energy Dispersive Spectroscopy (EDS). The composition of the outermost surface layer and the chemical state of elements were analyzed by X-Ray Photoelectron Spectroscopy (XPS). The activity of grafted enzyme was studied by an enzymatic activity test. In vitro bioactivity was evaluated by soaking the samples in simulated body fluid and SEM observation to verify hydroxyapatite (HA) precipitation. Antibacterial activity has been determined by inhibition halo test against S aureus. Results and Discussion. A peculiar multi-scale topography, with spongy-like nanometric features, was obtained after the inorganic treatment (step 1a-1b). This morphology can be superimposed on the micro-or macro roughness deriving from acid etching or blasting, by properly optimizing the process parameters. Moreover, the treated surfaces present a high density of hydroxyl groups (XPS data) and they are bioactive (HA precipitation after soaking in SBF for 15 days). Metal (Ag, Cu, Zn) nanoparticles can be grown within the surface oxide layer and they are effective as antimicrobial inorganic agents. The amount of the metal nanoparticles can be tailored in order to have an antibacterial or a bacteriostatic surface. The effective grafting of ALP (step 2a-2b) has been shown by XPS because of the appearance of characteristic peaks in the carbon region. Moreover, it has been observed that ALP maintains its activity after grafting by an enzymatic activity test. ALP grafting improves HA precipitation kinetics. Conclusions. An innovative process was applied to titanium surfaces in order to obtain a better bone integration ability and antibacterial activity. A multi scale surface topography (micro and nano features) was successfully obtained together with an high hydroxylation degree. Modified surfaces are able to induce hydroxyapatite precipitation in vitro and to graft ALP, maintaining its activity and improving bioactivity. Metal nanoparticles embedded in the surface oxide layer have an antibacterial effect

Coating titanium alloy implants with titanium nitride (TiN) by the method of Powder Immersion Reaction Assisted Coating (PIRAC) produces a stable layer on their surface. We have examined the ability of the new TiN coating to undergo osseointegration. We implanted TiN-coated and uncoated Ti6Al4V alloy pins into the femora of six-month-old female Wistar rats. SEM after two months showed a bone collar around both TiN-coated and uncoated implants. Morphometrical analysis revealed no significant differences between the percentage of the implant-bone contact and the area and volume of the bone around TiN-coated compared with uncoated implants. Electron-probe microanalysis indicated the presence of calcium and phosphorus at the implant-bone interface. Mineralisation around the implants was also confirmed by labelling with oxytetracycline. Strong activity of alkaline phosphatase and weak activity of tartrate-resistant acid phosphatase were shown histochemically. Very few macrophages were detected by the non-specific esterase reaction at the site of implantation. Our findings indicate good biocompatibility and bone-bonding properties of the new PIRAC TiN coatings which are comparable to those of uncoated Ti6Al4V alloy implants

Periprosthetic joint infections (PJI) are one of the most common reasons for orthopedic revision surgeries. In previous studies, it has been shown that silver modification of titanium (Ti-6Al-4V) surfaces by PMEDM (powder mixed electrical discharge machining) has an antibacterial effect on Staphylococcus aureus adhesion. Whether this method also influences the proliferation of bacteria has not been investigated so far. Furthermore, the effect is only limitedly investigated on the ossification processes. Therefore, the aim of this work is to investigate the antibacterial effect as well as the in vitro ossification process of PMEDM machined surfaces modified by integration of silver.

In this study, we analyzed adhesion and proliferation of S. aureus in comparison to of surface roughness, silver content and layer thickness of the silver-integrated-PMEDM surfaces (N = 5). To test the in vitro ossification, human osteoblasts (SaOs-2) and osteoclasts (differentiated from murine-bone-marrow-macrophages) were cultured on the silver surfaces (N = 3).

We showed that the attachment of S. aureus on the surfaces was significantly lower than on the comparative control surfaces of pure Ti-6Al-4V without incorporated silver, independently of the measured surface properties. Bacterial proliferation, however, was not affected by the silver content. No influence on the in vitro ossification was observed, whereas osteoclast formation was drastically reduced on the silver-modified surfaces.

We showed that 1 to 3% of silver in the surface layer significantly reduced the adhesion of S. aureus, but not the proliferation of already attached bacteria. At the same time, no influence on the in vitro ossification was observed, while no osteoclasts were formed on the surface. Therefore, we state that PMEDM with simultaneous silver modification of the machined surfaces represents a promising technology for endoprostheses manufacturing to reduce infections while at the same time optimizing bone ingrowth.