Recent studies suggested that both the soluble protein of the mesenchymal stromal cell (MSC) secretome, as well as the secreted extracellular vesicles (EVs) promote bone regeneration. However, there is limited knowledge of the changes in MSC secretome vesicular fraction during aging. We therefore aimed to characterize the release profiles and cargo of EVs from MSCs of different chronological ages. Conditioned medium (CM) was collected from 13 bone marrow MSC strains (20-89 years) and from one MSC strain derived from human induced pluripotent stem cells (iPSCs). The EV-containing fraction was enriched with ultracentrifugation. The number of particles in the CM was evaluated by nanoparticle tracking analysis (NTA), and the number of EVs was evaluated by flow cytometry (FC) after staining with cell-mask-green and anti-CD81 antibody. EV cargo analysis was conducted using next-generation sequencing (NGS). Our data confirmed the release of EVs from all MSC strains used in the study. There were no correlations between the number of particles and the number of EVs released in the CM, and between the number of EVs released and the strain age. Nevertheless, some of the lowest concentrations of EVs were found in the CM of strains over 70 years of age, which exhibited a low/absent chondrogenic and osteogenic differentiation potential. In contrast, iPSC-MSCs, which exhibited a high growth and three-lineage differentiation potential, released a similar amount of EVs as the best performing bone marrow MSC strain. NGS analysis identified several microRNAs that were significantly enriched in EVs of young MSC strains exhibiting low senescence, and those that were enriched in EVs of strains exhibiting high differentiation potentials. Gender had no influence on microRNA profiles in EVs or releasing MSCs. Taken together, our data provides new insights into the properties of MSC vesicular secretome and its therapeutic potential during aging

Introduction. Herein, a tri-layered core-shell microfibrous scaffold with layer-specific growth factors (GFs) release is developed using coaxial electrohydrodynamic (EHD) printing for in situ cell recruitment and differentiation to facilitate gradient enthesis tissue repair. Our findings suggest that the microfibrous scaffolds with layer-specific GFs release may offer a promising clinical solution for enthesis regeneration. Method. Utilizing coaxial electrohydrodynamic (EHD) printing, we engineered tri-layered core-shell microfibrous scaffolds, each layer tailored with specific growth factors (GFs) for targeted enthesis tissue repair. This configuration aims to sequentially guide cell migration and differentiation, mirroring the natural enthesis’ gradient structure. SDF-1 was strategically loaded into the shell, while bFGF, TGF-β, and BMP-2 were encapsulated in the core, each selected for their roles in stimulating the regeneration of corresponding enthesis tissue layers. Result. The coaxial EHD-printed microfibrous scaffolds demonstrated a core-shell fiber width of 24.3 ± 6.3 μm, supporting distinct tenogenic, chondrogenic, and osteogenic layers with pore sizes of 81.5 ± 4.6 μm, 173.3 ± 6.9 μm, and 388.9 ± 6.9 μm, respectively. This structure facilitated a targeted and effective release of growth factors, optimizing stem cell recruitment and differentiation. In vivo assessments demonstrated that the scaffolds significantly enhanced biomechanical properties and facilitated the formation of gradient enthesis structures, with improved biomechanical strength approximately 2-3 times that of control groups. These results highlight the scaffold's capability to mimic the native enthesis structure, encouraging a conducive environment for cell-mediated repair and regeneration. Conclusion. The integration of layer-specific growth factors not only fostered a conducive environment for tissue regeneration but also exemplified a leap in the design of scaffolds that closely mimic the native tendon-to-bone interface. The findings illuminate the scaffold's capacity to direct cellular behavior and tissue formation, heralding a new era in regenerative strategies and offering a promising avenue for clinical translation in the treatment of rotator cuff injuries

Introduction and Objective. The continued effectiveness of antibiotic loaded bone cements is threatened by antibiotic resistance. The common antiseptic, chlorhexidine (CHX), is a potential alternative to antibiotics in bone cements, but conventional salts are highly soluble, causing burst release and rapid decline to subinhibitory local CHX concentrations. Here, chlorhexidine triphosphate (CHX-TP), a low solubility CHX salt, is investigated as an alternative antimicrobial in PMMA bone cements. The aim was to assess duration of antimicrobial release and antimicrobial efficacy, along with handling, setting and mechanical properties of CHX-TP loaded cements, compared with an existing cement formulation containing gentamicin. Materials and Methods. Palacos R (Heraeus Medical, Newbury, UK) with 0, 1, 4, 7 and 12% CHX-TP (w/w) cements were prepared by combining solid CHX-TP with Palacos R components, and compared with Palacos R+G. All cements were prepared without vacuum and under ISO 5833:2002 conditions. Cements were tested under ISO 5833:2002 for compressive and bending properties, setting time, maximum temperature and doughing time. Antimicrobial release from the cements into deionised water was studied and antimicrobial efficacy of unaged and aged cements against Staphylococcus aureus (ATCC 29213) was assessed using a disc diffusion assay. Results. Compressive strength of CHX-TP loaded cements was not significantly different to Palacos R or Palacos R+G (p > 0.05, all exceeding ISO 5833:2002 minimum of 70 MPa). Mean bending strength was significantly lower with CHX-TP loading (p < 0.05) than bending strength of Palacos R and Palacos R+G, though all bending moduli exceeded the ISO 5833:2002 minimum (1800 MPa). All cements studied were within the ISO 5833:2002 limits for setting time (3 to 15 min), doughing time (≤ 5 min) and maximum temperature (90 . o. C). Mean doughing time for Palacos R, Palacos R+G and Palacos R + 12 % CHX-TP respectively: 52.5 s, 45 s and 45 s. Mean setting time and mean maximum temperature for Palacos R, Palacos R+G and Palacos R + 1, 4, 7 and 12% CHX-TP respectively: 11.00 min (73 . o. C), 11.25 min (72 . o. C), 12.25 min (66 . o. C), 10.50 min (70 . o. C), 10.00 min (70 . o. C), 10.75 min (62 . o. C). Sustained CHX release into deionised water was observed from all Palacos R + CHX-TP cements. Duration varied according to CHX-TP dosing and diminished over time, although to an extent that itself varied with dosing. 1 % CHX-TP ceased releasing CHX at 6.9 weeks; 4 % CHX-TP ceased at 67.7 weeks; 7 % and 12 % CHX-TP were ongoing at 75.5 weeks. Palacos R+G cements ceased releasing detectable levels of gentamicin after 14.4 weeks. Palacos R+G and Palacos R + CHX-TP cement discs showed efficacy against S. aureus (ATCC 29213) when applied as prepared (unaged) to S. aureus bacterial lawns in disc diffusion assays, with CHX-TP cements showing dose dependency. Zone of inhibition (ZOI) size was significantly reduced for Palacos R+G cements and Palacos R + 1% CHX-TP cements after 1 week and 6 weeks aging, compared to ZOI from unaged cements (p < 0.05). ZOI size produced by Palacos R + 4, 7, and 12 % CHX-TP cements did not decline significantly after 6 weeks aging (p > 0.05). Conclusions. CHX-TP can be incorporated into the Palacos R cement matrix up to 12% w/w without deterioration of compressive strength, bending modulus, doughing time, setting time or maximum temperature. Bending strength was significantly reduced at all CHX-TP loadings studied. Palacos R + 4, 7 and 12% CHX-TP cements provided sustained CHX release, exceeding the duration of gentamicin release from Palacos R+G, and showed sustained efficacy against S. Aureus after 6 weeks aging, which was not achieved by Palacos R+G cements

The recent description of progenitor/stem cells in degenerated intervertebral discs (IVDs) raised the possibility of harnessing their regenerative capacity for endogenous repair. The aim of this work is to develop an intradiscal polysaccharide microbead-based delivery system for the sequential release of chemokines and nucleopulpogenic factors. This delivery system would sequentially contribute to 1) the recruitment of resident progenitors (CXCL12 or CCL5), 2) the differentiation of the mobilized progenitors (TGF-β1 and GDF5), and 3) the subsequent regeneration of NP. To determine the effects of chemokines on in vitro cell recruitment, human mesenchymal stem cells (MSC) were cultured in Transwells for 4h, with or without CXCL12 or CCL5. In parallel, pullulan microbeads (PMBs) (100µm) were prepared by a simultaneous crosslinking protocol coupled to a water-in-oil emulsification process. Freeze-dried PMBs were loaded with biological factors then release assays were performed at 37°C for 21 days and supernatant concentrations were measured by ELISA. As compared to untreated MSC, MSC migration was improved with a 3.9 (CXCL12) and 7.5 (CCL5) fold increase, respectively. All factors were successfully adsorbed on PMBs and a burst release within the 1. st. day was observed. At day 7, 27.5% and 83% of CXCL12 and CCL5 were released, respectively and at day 21, 20% and 100% of TGF-β1 and GDF5 were released, respectively. Currently, released cytokine bioactivity is being analysed and an ex vivo ovine IVD model is developed to determine the repair potential of this controlled release approach

To prevent infections after orthopedic surgery, intravenous antibiotics are administered perioperatively. Cefazolin is widely used as the prophylactic antibiotic of choice. Systemic antibiotic therapy may however be less effective in longstanding surgery where bone allografts are used. Bone chips have been shown to be an effective carrier for certain types of antibiotics. Bone allografts impregnated with antibiotics may therefore provide the necessary local antibiotic levels for prophylaxis. To be efficient, a prolonged release from these bonechips is required. In contrast to vancomycin, for which prolonged release has clearly been proven effective from Osteomycin®, a commercially available impregnated bone allograft, no prolonged release bone chip preparations have been described so far for cefazolin. We developed a protocol to bind cefazolin in the porous structure of bone chips by means of a hydrogel composed of proteins naturally present in the human body. Three types of bone chips were evaluated: fresh frozen, decellularized frozen and decellularized lyophilized. Bone chips were incubated with 20 mg/ml cefazolin or treated with liquid hydrogel containing either 1 mg/ml fibrin or 1 mg/ml collagen and 20 mg/ml cefazolin. The cefazolin hydrogel was distributed in the porous structure by short vacuum treatment. Bone chips with cefazolin but without hydrogel were either incubated for 20 min- 4h or also treated with vacuum. Cefazolin elution of bone chips was carried out in fetal bovine serum and analyzed by Ultra Performance Liquid Chromatography – Diode Array Detection. Soaking of bone chips without hydrogel resulted in a quick release of cefazolin, which was limited to 4 hours. When vacuum was applied elution of >1 µg/ml cefazolin was measured for up to 36 hours. Combination with collagen hydrogel resulted in a higher cefazolin concentration released at 24 hours (3.9 vs 0.3 µg/ml), but not in a prolonged release. However, combination of decellularized frozen bone chips with fibrin hydrogel resulted in an initial release of 533 µg/ml followed by a gradual decline reaching the minimal inhibitory concentration for S. aureus at 72 hours (1.7 µg/ml), while not measurable anymore after 92 hours. Processed bone chips with hydrogel-cefazolin showed a markedly prolonged cefazolin release. When combined with a fibrin hydrogel, high initial peak levels of cefazolin were obtained, followed by a decreasing release over the following three days. This elution profile is desirable, since high initial levels are important to maximize anti-bacterial action whereas low levels of antibiotic for a limited time may stimulate osteogenesis. It is important that antibiotic release is ending after a few days as prolonged low levels of antibiotics are not clinically helpful and may lead to antibiotic resistance. Further preclinical studies are warranted to show effectiveness of hydrogel-cefazolin impregnated bone chips

Introduction and Objective. Carpal tunnel syndrome (CTS) is a very common compressive neuropathy involving the median nerve. The typical symptoms are paraesthesia, dysesthesia and loss of strength; in severe case, this compression deteriorates the sensorimotor control of the hand and interferes with the adjustment of the forces at the level of the fingers, thus affecting the components that are the basis of dexterity and control of fine movements. For these reasons, the CTS has repercussions on various activities of daily life, including writing skills. Word processing via PC and mobile device (touch-typing) require a fine control of the hand-wrist movement and of the opposition of the thumb, while in handwriting, gripping and gripping movements are carried out in a protracted manner. In modern society, present skills play a role of fundamental importance from an educational, professional and social point of view. The aim of the study is to describe the effects of carpal tunnel release (CTR) on handwriting and digital writing performance. Materials and Methods. We recruited patients suffering from carpal tunnel syndrome (CTS) who were candidates for CTR surgery and collected clinical and demographic data, including age, occupation, duration of symptoms and electromyography outcomes. The first trial session was carried out before surgery and the subsequent ones at 1, 2, 3, 4, 8 and 12 weeks after the CTR. These trials involved copying a 500-character paragraph by handwriting, personal computer (PC) and mobile device, for which a dedicate Google Colab web page was computed. We used as parameters the speed, expressed in words per minute (wpm), and the accuracy of copying, which was measured in number of errors (en). Moreover in each session the patient filled in the QuickDASH (Disabilities of the Arm, Shoulder, and Hand) questionnaire. We used the one-way anova to evaluate the change in the three performances and in the QuickDASH score in follow-up sessions. We used the two-way anova to detect a possible interactions between speed improvement and groups of variables, namely gender, writing frequency, schooling, diabetes, dysthyroidism and metabolic syndrome. Results. We recruited 20 patients of whom 7 dropped out for personal reasons and 13 had completed all trial sessions. The PC writing performance had an average speed and accuracy of 15.1 ± 6.8 wpm and 13.1 ± 8.2 en, respectively, while post-operatively it returned values of 17.6 ± 5.0 wpm and 9.9 ± 5.6 en. Regarding touch-typing, a pre-operative average of 16.9 ± 5.8 wpm and 14.3 ± 14.4 en was recorded, while post-operatively an average of 21.7 ± 6.5 wpm and 11.5 ± 14.7 en was reported. Handwriting performance initially had a mean of 20.5 ± 7.1 wpm and 0.1 ± 0.6 en and after three months returned a mean of 22.4 ± 4.0 wpm and 0 ± 0 n. The QuickDASH score had a pre-operative mean of 39.1 ± 9.1 and post-operative mean of 17 ± 6 points. The only statistically significant improvements were those related to touch-typing (P = 0.022) and QuickDASH score (P < 0.001). There was no significant interaction between gender, comorbidity, writing frequency, level of schooling and recovery of writing ability. Conclusions. The data collected showed, in agreement with previous studies, that CTS has a significant impact on the patient's writing ability, who benefits from the surgical treatment, especially in terms of touch-typing and general manual dexterity. In addition, the recovery of writing ability did not show significant correlation with other variables

Aseptic loosening is a major cause of revision surgeries and occurs when osteolysis is stimulated around the implant by pro-inflammatory cytokines including IL-1β. Production of active IL-1β in response to orthopedic wear particles depends on processing by the NLRP3 inflammasome which requires priming followed by activation. We found that pathogen associated molecular patterns (PAMPs) adherent to wear particles are necessary to prime the NLRP3 inflammasome. In contrast, in pre-primed macrophages, particles themselves are sufficient to activate the NLRP3 inflammasome and induce secretion of active IL-1β. Particles themselves also induce cell death, kinetically preceding the release of active IL-1β. Phagocytosis of particles is required to initiate both responses as the phagocytosis inhibitor cytochalasin blocks cell death and IL-1β release. Lysosome membrane destabilization is also critical as inhibition of lysosomal function with bafilomycin or chloroquine significantly abrogated the release of active IL-1β and cell death in response to wear particles. The pan-cathepsin inhibitors Ca-074-Me or K777 also inhibit cell death and IL-1β release indicating that cathepsin release from lysosomes is also a necessary step in the particle-induced response. Our results open the possibility of clinical intervention with lysosomal or cathepsin inhibitors to treat aseptic loosening as these drugs have better specificity and less in vivo toxicity than the phagocytosis inhibitors. Testing of these inhibitors in vivo in models of particle induced osteolysis is a key future direction

Background. The doses of local rhBMP-2 in commercially available materials are high with known drawbacks such as inflammation and premature bone resorption. The latter can be prevented by adding bisphosphonates like zoledronic acid (ZA) but systemic ZA has side effects and patient adherence to treatment is low. In a recent study, we have shown that local co-delivery of rhBMP-2 and ZA via a calcium sulphate/hydroxyapatite (CS-HA) biomaterial can be used to regenerate both cortical and trabecular bone in a rat model of metaphyseal bone defect. Even low doses of local ZA in the biomaterial showed promising results and increased bone formation within the defect compared to the controls. A step before clinical translation of the local treatment regimen is to evaluate the in-vivo release kinetics of these additives and thus in this study, we aimed to investigate the in-vivo pharmacokinetics of rhBMP-2 and ZA from the CS-HA biomaterial in a rat abdominal muscle pouch model over a period of 4-weeks. Methods. In-vivo release kinetics of 125I labeled rhBMP-2 and 14C labeled ZA was performed using an abdominal muscle pouch model in rats (n=6). Both rhBMP-2 and ZA were labeled commercially with a radiochemical purity of >95%. The detection of 125I -rhBMP-2 release was performed by implanting pellets of the CS-HA biomaterial containing 125I -rhBMP-2 and ZA and the same animals followed over a period of 4-weeks (day 1, 3, 7, 14, 21& 28) using SPECT imaging. Similarly, the 14C-ZA was detected by implanting CS-HA pellets containing rhBMP-2 and 14C-ZA. Release was detected via scintillation counting and at each time point (Day 1, 7, 14& 28) 6-animals were sacrificed. Results. BMP Release. The CS-HA biomaterial retained 95±11% after 3-days, 88±12% after a week, 66±9% after 2 weeks, 51±5% after 3 weeks and 43±7% of 125I labeled rhBMP-2 after 4-weeks in-vivo (SPECT-CT). ZA Release. The CS-HA biomaterial retained 89±14% after a week, 84±8% after 2 weeks, 83±9% after 3 weeks and 77±3% of 14C labeled ZA after 4 weeks of in-vivo implantation. Discussion. Improved carriers and better knowledge of the release might improve the effect of bone active drugs in orthopedics. Our previous study shows that an off-the-shelf ceramic biomaterial combined with ZA alone or with both rhBMP-2 and ZA can be used to regenerate bone with potential for clinical translational. This study demonstrates long-term co-delivery of both rhBMP-2 and ZA in-vivo via the biomaterial. Constant availability of rhBMP-2 over a long period of time can give osteoinductive properties to the material while presence of local ZA prevents premature bone loss. The pharmacokinetic release pattern differs from what we have reported in vitro with less BMP and more ZA being released in vivo during the first 4 weeks. We speculate that rapid protein passivation of the ceramic material slows the release of BMP and partly preventing the ZA binding to apatite

Articular cartilage is often damaged, and its treatment is usually performed by surgical operation. Today, tissue engineering offers an alternative treatment option for injuries or diseases with increasing importance. Infrapatellar fat pad (IPFP) is a densely vascularized and innervated extra synovial tissue that fills the anterior knee compartment. Adipose-derived stem cells from infrapatellar fat pad (IPFP-ASCs) have multipotency means that they can differentiate into connective tissue cells and have age-independent differentiation capacity as compared to other stem cells. In this study, the osteochondral tissue construct was designed with different inner pattern due to original osteochondral tissue structure and fabrication of it was carried out by 3D printing. For this purpose, alginate (3% w/v) and carboxymethylcellulose (CMC) (9%w /v) were used as bioink. Also, IPFP-ASCs were isolated with enzymatic degradation. Osteogenic and chondrogenic differentiation of IPFP-ASCs were investigated with Alizarin Red and Alcian Blue staining, respectively. IPFP-ASCs-laden osteochondral graft differentiation will be induced by controlled release of growth factor BMP-2 and TGF-β. Before this step, nanocapsules formation with double emission technique with model protein BSA was carried out with different concentration of PCL (5%,10% and 20%). The morphology and structure of the nanocapsules were determined with scanning electron microscopy (SEM). Also, we successfully designed and printed alginate and CMC based scaffold with 20 layers. Chondrogenic and osteogenic differentiation of IPFP-ASCs with suitable culture conditions was obtained. The isolation of IPFP-ASCs, formation of the nanocapsules, and 3D printing of osteochondral graft were carried out successfully

Aims. Animal models have been developed that allow simulation of post-traumatic joint contracture. One such model involves contracture-forming surgery followed by surgical capsular release. This model allows testing of antifibrotic agents, such as rosiglitazone. Methods. A total of 20 rabbits underwent contracture-forming surgery. Eight weeks later, the animals underwent a surgical capsular release. Ten animals received rosiglitazone (intramuscular initially, then orally). The animals were sacrificed following 16 weeks of free cage mobilisation. The joints were tested biomechanically, and the posterior capsule was assessed histologically and via genetic microarray analysis. Results. There was no significant difference in post-traumatic contracture between the rosiglitazone and control groups (33° (standard deviation (. sd. ) 11) vs 37° (. sd. 14), respectively; p = 0.4). There was no difference in number or percentage of myofibroblasts. Importantly, there were ten genes and 17 pathways that were significantly modulated by rosiglitazone in the posterior capsule. Discussion. Rosiglitazone significantly altered the genetic expression of the posterior capsular tissue in a rabbit model, with ten genes and 17 pathways demonstrating significant modulation. However, there was no significant effect on biomechanical or histological properties. Cite this article: M. P. Abdel. Effectiveness of rosiglitazone in reducing flexion contracture in a rabbit model of arthrofibrosis with surgical capsular release: A biomechanical, histological, and genetic analysis. Bone Joint Res 2016;5:11–17. doi: 10.1302/2046-3758.51.2000593

The aim of this study is to compare the release of titanium (Ti) and zirconium (Zr) into the tissue surrounding Ti- and ZrO. 2. -implants. Methyl methacrylate embedded mini pig maxillae with 6 Ti-implants and 4 ZrO2-implants were analysed after 12-weeks of implantation. The spatial distribution of elemental Ti and Zr in maxillae near implants was assessed with laser ablation (LA)-inductively coupled plasma (ICP)-mass spectrometry (MS). From each maxilla two bone slices adjacent to the implants were measured. The contents of Ti and Zr in these bone slices were determined by ICP-MS and ICP-optical emission spectrometry. Increased intensity of Ti and Zr could be detected in bone tissues at a distance of 891±398 µm (mean ± SD) from Ti-implants and 927±404 µm from ZrO2-implants. The increased intensity was mainly detected near implant screw threads. The average Ti content detected in 11 bone slices from samples with Ti-implants was 1.67 mg/kg, which is significantly higher than the Ti content detected in 8 slices from samples with ZrO. 2. -implants. The highest Ti content detected was 2.17 mg/kg. The average Zr content in 4 bone slices from samples with ZrO. 2. -implants is 0.59 mg/Kg, the other 4 bone slices showed Zr contents below the detection limit (. After 12-weeks of implantation, increased intensity of Ti and Zr can be detected in bone tissues near Ti- and ZrO. 2. -implants. The results show that Ti content released from Ti-implants is higher than the Zr content released from ZrO. 2. -implants

Total knee arthroplasty is a well-established treatment for degenerative joint disease, on the other hand metal ion release of cobalt or chromium and particle formation can trigger intolerance reactions. Biotribological examinations can help to assess the metal ion release in different settings. The purpose of this study was the evaluation of inter-laboratory differences in the metal ion concentration analysis. Samples were generated in a 3+1 station knee wear simulator (EndoLab GmbH, Thansau, Germany) with a medium size Columbus Knee System with or without AS multilayer coating. The wear simulation was performed under highly demanding activity (HDA) profile and samples were taken after 0.5, 5.0, 5.5. and 8.0 million cycles. The samples were blinded and sent to three different laboratories and the content of chromium, cobalt, molybdenum, nickel, and zirconium was assessed by inductively coupled plasma mass spectrometry (ICP-MS). The AS multilayer coating clearly reduced the release of chromium, cobalt and molybdenum. Mean levels were: Chromium 9329.78µg/l ± 985.44 vs 503.75µg/l ± 54.19, cobalt 10419.00µg/l ± 15.517.53 vs 2.60µg/l ± 1.35, molybdenum 2496.33µg/l ± 102.62 vs 2.46µg/l ± 2.31. Interestingly we found especially for nickel and zirconium big inter-laboratory differences in the metal assessment. There were up to 10-fold higher values in comparison of one laboratory to another. The data demonstrate that results of metal ion assessment should be evaluated by interlaboratory comparison and should be critically interpreted

Introduction. Prosthetic joint infections (PJI) occur infrequently, but due to its increased clinical use represent the most devastating complication with high morbidity and substantial cost. Staphylococcus aureus and coagulase-negative staphylococci are the most common infecting agents associated with PJI. A possible therapeutic approach could be the local antibiotic by fluoride-TiO2 nanostructured anodic layers in order to prevent surface colonisation during the early moments after surgery. Here we describe the first results of this model using two common antibiotics. Methods. Fluoride-TiO2 nanostructured anodic layers on Ti6Al4V alloy were produced as described previously by Arenas et al (2013). Discs shaped pieces of Ti6Al4V alloy were loaded with a solution of 150 mg antibiotic (vancomycin or gentamicin)/20 ml sterile distilled water. Samples were immersed in this solution during 24 hours at room temperature with agitation, and then were dried during 48 hours at 20°C. Antibiotic release was studied by introducing both discs in sterile PBS and samples were taken at different times. Samples were then frozen at −80°C until HPLC measurements and biological activity tests using Bacillus subtilis ATCC 6051 (vancomycin) and Escherichia coli ATCC 25922 (gentamicin) were performed. Results. Release kinetic constant was not be obtained by HPLC-UV due to chemical degradation, therefore it was needed to prove biological activity. According to biological activity test, vancomycin and gentamicin estimated concentration mean and standard deviation were 2.9±0.61 and 2.71±0.64 mg/L over time respectively. Conclusions. Fluoride-TiO2 nanostructured anodic layers on Ti6Al4V alloy could be considered as promising vector of antibiotics for the prevention of PJI during early moments after surgery

Although the response of macrophages to polyethylene debris has been widely studied, it has never been compared with the cellular response to ceramic debris. Our aim was to investigate the cytotoxicity of ceramic particles (Al. 2. O. 3. and ZrO. 2. ) and to analyse their ability to stimulate the release of inflammatory mediators compared with that of high-density polyethylene particles (HDP). We analysed the effects of particle size, concentration and composition using an in vitro model. The J774 mouse macrophage cell line was exposed to commercial particles in the phagocytosable range (up to 4.5 μm). Al. 2. O. 3. was compared with ZrO. 2. at 0.6 μm and with HDP at 4.5 μm. Cytotoxicity tests were performed using flow cytometry and macrophage cytokine release was measured by ELISA. Cell mortality increased with the size and concentration of Al. 2. O. 3. particles. When comparing Al. 2. O. 3. and ZrO. 2. at 0.6 μm, we did not detect any significant difference at the concentrations analysed (up to 2500 particles per macrophage), and mortality remained very low (less than 10%). Release of TNF-α also increased with the size and concentration of Al. 2. O. 3. particles, reaching 195% of control (165 pg/ml v 84 pg/ml) at 2.4 μm and 350 particles per cell (p < 0.05). Release of TNF-α was higher with HDP than with Al. 2. O. 3. particles at 4.5 μm. However, we did not detect any significant difference in the release of TNF-α between Al. 2. O. 3. and ZrO. 2. at 0.6 μm (p > 0.05). We saw no evidence of release of interleukin-1α or interleukin-1ß after exposure to ceramic or HDP particles

Total knee arthroplasty is a well established treatment for degenerative joint disease with good clinical results. However, complications may occur due to a biological response to polyethylene wear particles, leading to osteolysis and aseptic loosening, as well as local and systemic hypersensitivity reactions triggered by metal ions and particles such as chromium, cobalt and molybdenum. Moreover, there is an increasing demand on the performance of these implants, as this treatment is also performed in heavier, younger and middle-aged adults who have a significant physical activity and higher life expectancy. The purpose of the following study was to compare the wear characteristics and performance of a zirconium nitride (ZrN) coated knee implant, designed for patients with metal ion hypersensitivity, against the clinically established cobalt-chromium (CoCr) version under a high demanding activities wear simulation. Medium size AS Columbus® DD (Aesculap AG, Tuttlingen, Germany) femoral and tibial components with a ZrN surface were tested in comparison with the cobalt-chromium version Columbus® DD. For both groups, ultra-high-molecular weight polyethylene (UHMWPE) gliding surfaces (size T3, high 10 mm) were used. Wear simulation was performed on a load controlled 4 station knee wear simulator (EndoLab GmbH, Thansau, Germany) capable of reproducing loads and movement of daily activities measured in vivo (Bergmann et al, 2014) on 8 patients and normalized to a patient weight of 100 kg (Schwiesau et al, 2014). The load profiles were applied for 5 million cycles in a combination of 40% stairs up, 40% stairs down, 10% level walking, 8% chair raising and 2% deep squatting. Test serum was changed every 0.5 million cycles and all the components were cleaned and analyzed according to ISO 14243-2:2009(E). The gliding surfaces were evaluated for gravimetric wear and wear patterns, femur components analyzed for scratches and the test medium analyzed for metal ion concentration (cobalt, chromium, molybdenum and zirconium) using ICP-MS according to ISO 17294-2. The present study showed a wear rate reduction for the ZrN group (1.01 ± 0.29 mg/million) in comparison with the CoCr group (2.40 ± 1.18 mg/million cycles). The articulation surface of the ZrN coated femurs remained polished after the testing period, whereas the uncoated femurs showed wear scratches. Furthermore, the metal ion release from the ZrN coated implants was reduced orders of magnitude in comparison with the CoCr implants through the entire test. These results demonstrate the efficiency of ZrN coated knee implants to reduce wear as well as to prevent metal ion release in the knee joint

Pulsed Electromagnetic Fields (PEMFs) promote joint tissue anabolic activities, particularly in cartilage and bone. Here we investigated the effect of selected PEMFs (75Hz, 1.5mT, 1.3msec) in a differentiating model of murine myoblasts (C2C12) in vitro. C2C12 were seeded at 5×10. 3. cells/cm. 2. in 4 well plates and left to adhere for 24h. Subsequently, cells were either maintained in growth medium (GM) or induced towards myogenic differentiation in low-serum conditions, with and without PEMF exposure, for 4 days. Morphological analysis, myotube formation and fusion index (FI) were assessed with fluorescence microscopy techniques. Metabolic activity was determined by MTT; moreover, a multiplex cytokine array (RayBiotech) allowed cell supernatant molecule quantification. Cells exposed to PEMFs in GM acquired a distinctive elongated morphology, with increased bi-nuclear figures (3.2-fold FI increase over PEMF-unexposed cells) and displayed a significantly higher metabolic activity (+31%, p<0.05 over PEMF-unexposed cells). PEMF exposure increased metabolic activity also under myogenic differentiation (+15% over PEMF-unexposed differentiating cells, p<0.05), with the formation of long, thick polynuclear myotubes, suggesting a role of PEMFs in enhancing myogenesis (7.7-fold FI increase over PEMF-unexposed cells). 4-day culture supernatants revealed the presence of several myokines (KC/CXCL1, LIX, MCP-1, TIMP-1). Preliminary analysis showed a 1.16-fold increase (n=2) of LIX and, notably, a 1.91-fold increase (n=2) of TNF-RI, in cell supernatants of PEMF-exposed over PEMF-unexposed cells. Collectively, these results suggest that PEMF may successfully be applied in models of muscle cell trauma to optimise muscle fibre repair, by fine-tuning the release of myokines, promoting myoblast proliferation and myotube formation

Purpose. Gustilo type III open fractures are associated with high infection rates in spite of instituting a standard of care (SOC) consisting of intravenous antibiotics, irrigation and debridement (I&D), and delayed wound closure. Locally-delivered antibiotic has been proven to assist in reducing infection in open fractures. The aims of this study are to determine the effectiveness and safety of a new implantable and biodegradable antibacterial product. 1. in preventing bacterial infections and initiating bone growth in open fractures. Methods. The osteoconductive antibacterial BonyPid. TM. used is a synthetic bone void filler (comprised of ≤1 mm β-tricalcium phosphate granules) coated by a thin layer (≤20 µm) of PolyPid nanotechnology formulation. −. Upon implantation, the coating releases doxycycline at a constant rate for a predetermined period of 30 days. One BonyPid. TM. vial of 10 grams contains 65 mg of formulated doxycycline. After approval, sixteen subjects with Gustilo type III open tibia fractures, were implanted with the BonyPid. TM. immediately on the first surgical intervention (I&D), followed by external fixation. Patients had periodic laboratory, bacteriology and radiology follow-up. Results. Six months results showed that no infection developed and only one BonyPid. TM. implantation was needed with no subsequent I&D, in the target tibia fracture. Immediate soft wound closure was done in 6/16 subjects following implantation. Out of 10 remaining subjects, 3 needed soleus muscle transfer-skin grafting and 7 required delayed primary closure; by skin grafting (5) or suturing (2). Early callus formation was seen at 8–12 weeks post-surgery, followed by bone healing seen from 16 weeks onwards. Safety of implantation was remarkable, with only one deep infection at a fibular open fracture without BonyPid. TM. implantation. One BonyPid. TM. -related adverse event caused delay in skin healing due to excessive granules in the superficial soft tissues. Conclusion. BonyPid. TM. is effective in reducing bone infection and promoting early callus formation, resulting in early bone healing. BonyPid. TM. is safe for immediate implantation into contaminated/infected severe open-bone fractures. Results support that one month release of doxycycline in a controlled manner provides an effective way for treating open fractures. This new local antibiotic delivery system is applicable in unmet medical situations associated with localised infections

The clinical translation of regenerative therapies, whether in the form of mesenchymal cells, macromolecules or small molecules, is hampered by several factors: the poor retention and short biological half-life of the therapeutic agent, the adverse side effects from systemic delivery, and difficulties with the administration of multiple doses to a target site. We report the development and application of a therapeutic reservoir device that enables sustained and repeated administration of small molecules, macromolecules and cells directly to organs and tissues of interest via a polymer-based reservoir connected to a subcutaneous port. In a myocardial infarct rodent model, we show that repeated administration of cells over a four-week period using the reservoir provided functional compared to a single injection of cells and to no treatment. Recent advances of the system include a multi-port and multi-reservoir system that can be tailored to cargo and application need. The pre-clinical use of our therapeutic reservoir as a research model may enable insights into regenerative orthopaedic therapy, particularly those therapies that require multi-dose approaches.

Introduction

For many years, minimally invasive joint-preserving regenerative therapy has been desired for the early stages of osteonecrosis of the femoral head (ONFH). In an animal study using adult rabbits, we reported that a single local injection of rhFGF-2-impregnated gelatin hydrogel, which has superior slow-release characteristics, suppresses the progression of femoral head necrosis. The purpose of this study was to evaluate the safety and clinical outcomes of a single local administration of rhFGF-2-impregnated gelatin hydrogel for the precollapse stage of ONFH. Patients and Methods: Ten patients with femoral heads up to precollapse stage 2 underwent a single local administration of 800-µg rhFGF-2-impregnated gelatin hydrogel and were followed up for two years. The eligibility criteria were age between 20 and 80 years and presence of ONFH at precollapse stage 1 or 2 according to the classification system for ONFH developed by the Japanese Investigation Committee of Health and Welfare. Primary outcomes included adverse events and complications. Secondary outcomes included changes in Harris Hip Scores (HHS), visual analog scale pain scores (VAS), the University of California, Los Angeles (UCLA) activity rating scores, radiological changes as determined via radiographs, computed tomography (CT) scans, and magnetic resonance imaging (MRI) of the hip joint. Results: We included five men (five hips) and five women (five hips), with a mean age of 39.8 years (range: 29–53 years) at the time of surgery. Eight patients had bilateral ONFH, three had already undergone THA on the contralateral side. Eight patients were receiving treatment with corticosteroid therapy, and two patients overused alcohol. Stage 1 and 2 disease was present in one and nine patients, respectively. One patient each had type A, type B, and type C1 disease, whereas seven patients had a type C2 lesion. All Adverse events were recovered without problem. The surgery was performed with a minimally invasive technique based core decompression (1 cm of skin incision), and walking was allowed from the day after surgery. Mean clinical scores improved significantly after three year compared with before surgery (before vs. after: VAS for pain, 21.2 vs. 5.3 mm; UCLA activity score 5.5 vs. 6.6; HHS, 81.0 vs. 98.4 points, respectively). There was only one case of femoral head collapse, and it had the greatest necrosis volume fraction and was considered to be in the early collapse stage at the time of operation. The other nine cases did not involve ONFH stage progression, and collapse was prevented. CT images and recent MRI postoperatively confirmed bone regeneration and reduction of the necrotic area. Conclusion: Clinical application of rhFGF-2-impregnated gelatin hydrogel for patients with precollapse stage of ONFH was feasible and safe. Our research is ongoing, further phase II multiple center study has been started in January 2016.

Summary. Management of metal on metal hip replacements can be accomplished with a simple algorithm including easily available metal ion levels and hip MRI with metal artifact reducing software. After revision serum metal ion levels can be expected to fall rapidly. Introduction. Metallic ion release may be related to bearing surface wear and thus serves as an indicator of the in-vivo performance of metal on metal articulations. The purpose of this prospective, controlled study was to compare new large head metal on metal hip components with established modular metal on metal and metal on polyethylene and to determine their effects on serum metal levels before and after revision. Patients & Methods. We performed a multi-surgeon, prospective, controlled trial to compare clinical, radiographic, and metal ion concentration in serum (cobalt and chrome) results across multiple devices including the Large Head ASR XL System (MoM-1), the Ultamet Advanced Modularity System (MoM-2), and as the control the Pinacle Acetabular Cup System with polyethylene liner (MoP). One hundred and fifty-one consecutive patients undergoing THA were enrolled in the study: MoM-1 n=97; MoM-2 n=22; MoP n=32. Clinical, radiographic, and venous blood assessments were performed pre-operatively, and post-operatively at 6 months, 1 year and 2 years, and after revision (1,3,6,12 months). All serum ion concentrations are reported in nmol/L. We are following metal ion levels after revision and have developed an algorithm to diagnose and manage patients with MoM THA. Results. MoM-1 patients had significantly increased average cobalt and chromium levels. Clinical scores improved after surgery in all groups and continued to improve in MoM-2 and MoP patients after 2 years but decreased slightly in the MoM-1 patients at 2 years. Average cup inclination angle did not differ significantly between the groups: MoM-1 50.2, MoM-2 47.8, and MoP 51.7. In the MoM-1 group 11 patients (11%) had significantly elevated ion levels (MoM-1 Outliers). Nine hips (9.3%) in 8 MoM-1 outlier patients required revision. Metal ion levels were not significantly different between MoM-2 and MoP groups. Metal ion levels after revision in the MoM-1 group decreased rapidly but at one year post-operatively have still not returned to an equivalent baseline comparable to the MoM-2 and MoP groups. All revisions were in the MoM-1 group. Chromium levels decreased more slowly than Cobalt levels. Discussion. To our knowledge this is the only data in the literature prospectively comparing ion levels among groups and reporting post revision ion levels. Average serum ion levels were elevated at all post-operative samples in the MoM-1 group but this was due to significantly elevated levels in a subset of outliers who required revision. Excluding the outliers there is not a significant difference in post-operative ion levels between the groups. There was no radiographic evidence of component malposition or aseptic loosening in any of the groups. Control groups (MoM-2, MoP) performed comparatively across all variables. We present an algorithm to diagnose and manage patients with metal on metal THA and offer evidence that metal ion levels do decrease after revision but still remain abnormally elevated at one-year post revision compared to the control group. A significant portion of MoM-1 performs comparatively to the controls in terms of ions