Vitamin E Phosphate Coating Stimulates Bone Deposition in Implant-related Infections in a Rat Model.

BACKGROUND: Implant-related infections are associated with impaired bone healing and osseointegration. In vitro antiadhesive and antibacterial properties and in vivo antiinflammatory effects protecting against bone loss of various formulations of vitamin E have been demonstrated in animal models. However, to the best of our knowledge, no in vivo studies have demonstrated the synergistic activity of vitamin E in preventing bacterial adhesion to orthopaedic implants, thus supporting the bone-implant integration. QUESTIONS/PURPOSES: The purpose of this study was to test whether a vitamin E phosphate coating on titanium implants may be able to reduce (1) the bacterial colonization of prosthetic implants and (2) bone resorption and osteomyelitis in a rat model of Staphylococcus aureus-induced implant-related infection. METHODS: Twelve rats were bilaterally injected in the femurs with S aureus UAMS-1-Xen40 and implanted with uncoated or vitamin E phosphate-coated titanium Kirschner wires without local or systemic antibiotic prophylaxis. Eight rats represented the uninfected control group. A few hours after surgery, two control and three infected animals died as a result of unexpected complications. With the remaining rats, we assessed the presence of bacterial contamination with qualitative bioluminescence imaging and Gram-positive staining and with quantitative bacterial count. Bone changes in terms of resorption and osteomyelitis were quantitatively analyzed through micro-CT (bone mineral density) and semiquantitatively through histologic scoring systems. RESULTS: Six weeks after implantation, we found only a mild decrease in bacterial count in coated versus uncoated implants (Ti versus controls: mean difference [MD], -3.705; 95% confidence interval [CI], -4.416 to -2.994; p < 0.001; TiVE versus controls: MD, -3.063; 95% CI, -3.672 to -2.454; p < 0.001), whereas micro-CT analysis showed a higher bone mineral density at the knee and femoral metaphysis in the vitamin E-treated group compared with uncoated implants (knee joint: MD, -11.88; 95% CI, -16.100 to -7.664; p < 0.001 and femoral metaphysis: MD, -19.87; 95% CI, -28.82 to -10.93; p < 0.001). We found decreased osteonecrosis (difference between medians, 1.5; 95% CI, 1-2; p < 0.002) in the infected group receiving the vitamin E-coated nails compared with the uncoated nails. CONCLUSIONS: These preliminary findings indicate that vitamin E phosphate implant coatings can exert a protective effect on bone deposition in a highly contaminated animal model of implant-related infection. CLINICAL RELEVANCE: The use of vitamin E coatings may open new perspectives for developing coatings that can limit septic loosening of infected implants with bacterial contamination. However, a deeper insight into the mechanism of action and the local release of vitamin E as a coating for orthopaedic implants is required to be used in clinics in the near future. Although this study cannot support the antimicrobial properties of vitamin E, promising results were obtained for bone-implant osseointegration. These preliminary results will require further in vivo investigations to optimize the host response in the presence of antibiotic prophylaxis.

A review on animal models and treatments for the reconstruction of Achilles and flexor tendons.

Tendon is a connective tissue mainly composed of collagen fibers with peculiar mechanical properties essential to functional movements. The increasing incidence of tendon traumatic injuries and ruptures-associated or not with the loss of tissue-falls on the growing interest in the field of tissue engineering and regenerative medicine. The use of animal models is mandatory to deepen the knowledge of the tendon healing response to severe damages or acute transections. Thus, the selection of preclinical models is crucial to ensure a successful translation of effective and safe innovative treatments to the clinical practice. The current review is focused on animal models of tendon ruptures and lacerations or defective injuries with large tissue loss that require surgical approaches or grafting procedures. Data published between 2000 and 2016 were examined. The analyzed articles were compiled from Pub Med-NCBI using search terms, including animal model(s) AND tendon augmentation OR tendon substitute(s) OR tendon substitution OR tendon replacement OR tendon graft(s) OR tendon defect(s) OR tendon rupture(s). This article presents the existing preclinical models - considering their advantages and disadvantages-in which translational progresses have been made by using bioactive sutures or tissue engineering that combines biomaterials with cells and growth factors to efficiently treat transections or large defects of Achilles and flexor tendons.

Systemic and Local Administration of Antimicrobial and Cell Therapies to Prevent Methicillin-Resistant Staphylococcus epidermidis-Induced Femoral Nonunions in a Rat Model.

S. epidermidis is responsible for biofilm-related nonunions. This study compares the response to S. epidermidis-infected fractures in rats systemically or locally injected with vancomycin or bone marrow mesenchymal stem cells (BMSCs) in preventing the nonunion establishment. The 50% of rats receiving BMSCs intravenously (s-rBMSCs) died after treatment. A higher cytokine trend was measured in BMSCs locally injected rats (l-rBMSCs) at day 3 and in vancomycin systemically injected rats (l-VANC) at day 7 compared to the other groups. At day 14, the highest cytokine values were measured in l-VANC and in l-rBMSCs for IL-10. µCT showed a good bony bridging in s-VANC and excellent both in l-VANC and in l-rBMSCs. The bacterial growth was lower in s-VANC and l-VANC than in l-rBMSCs. Histology demonstrated the presence of new woven bone in s-VANC and a more mature bony bridging was found in l-VANC. The l-rBMSCs showed a poor bony bridging of fibrovascular tissue. Our results could suggest the synergic use of systemic and local injection of vancomycin as an effective treatment to prevent septic nonunions. This study cannot sustain the systemic injection of BMSCs due to high risks, while a deeper insight into local BMSCs immunomodulatory effects is mandatory before developing cell therapies in clinics.

Chondrogenic capability of osteoarthritic chondrocytes from the trapeziometacarpal and hip joints.

Osteoarthritis is the most common degenerative disease of joints like the hip and the trapeziometacarpal joint (rhizarthrosis). In this in vitro study, we compared the chondrogenesis of chondrocytes derived from the trapezium and the femoral head cartilage of osteoarthritic patients to have a deeper insight on trapezium chondrocyte behavior as autologous cell source for the repair of cartilage lesions in rhizarthrosis. Chondrocytes collected from trapezium and femoral head articular cartilage were cultured in pellets and analyzed for chondrogenic differentiation, cell proliferation, glycosaminoglycan production, gene expression of chondrogenic and fibrous markers, histological and immunohistochemical analyses. Our results showed a higher cartilaginous matrix deposition and a lower fibrocartilaginous phenotype of the femoral chondrocytes with respect to the trapezium chondrocytes assessed by a higher absolute glycosaminoglycan and type II collagen production, thus demonstrating a superior chondrogenic potential of the femoral with respect to the trapezium chondrocytes. The differences in chondrogenic potential between trapezium and femoral head chondrocytes confirmed a lower regenerative capability in the trapezium than in the femoral head cartilage due to the different environment and loading acting on these joints that affects the metabolism of the resident cells. This could represent a limitation to apply the cell therapy for rhizoarthrosis.

A comparative study of diagnostic and imaging techniques for osteoarthritis of the trapezium.

OBJECTIVES: The aims of this study were to determine whether micro-CT is a reliable investigation method to evaluate the severity of OA in the trapezium and to develop a novel micro-CT scoring system based on a quantitative assessment of the subchondral bone thickness in order to better assess OA through an objective parameter. METHODS: We compared different diagnostic and imaging techniques performed consecutively on each sample: X-ray, visual analysis, micro-CT and histology. OA and healthy trapezia were subjected to semi-quantitative and quantitative analyses to be classified in four degrees of severity in OA (control, OA-2, OA-3 and OA-4). Specifically, samples were analysed using Dell's score for X-ray, Brown's score for visual analysis and Mankin's score for histology. Micro-CT was scored using a novel quantitative scoring system based on subchondral bone thickness measurements. Results obtained with each technique were then compared and correlated. RESULTS: X-ray analysis showed a higher frequency of OA-2 (27%) and OA-3 (32%) compared with OA-4 (5%), whereas visual analysis, micro-CT and histology showed a lower percentage for OA-2 (18%, 18% and 14%) and OA-3 (23%) and increased frequency for OA-4 (45%, 32% and 40%). Only the micro-CT score of subchondral bone thickness correlated significantly with all the other techniques (P < 0.05). CONCLUSION: This is the first comparison of techniques proposing a novel scoring system based on objective and quantitative micro-CT data that can be applied as a useful diagnostic tool for OA, providing a deeper comprehension of the pathophysiology of OA in trapezium.

Fabrication of multi-well chips for spheroid cultures and implantable constructs through rapid prototyping techniques.

Three-dimensional (3D) culture models are widely used in basic and translational research. In this study, to generate and culture multiple 3D cell spheroids, we exploited laser ablation and replica molding for the fabrication of polydimethylsiloxane (PDMS) multi-well chips, which were validated using articular chondrocytes (ACs). Multi-well ACs spheroids were comparable or superior to standard spheroids, as revealed by glycosaminoglycan and type-II collagen deposition. Moreover, the use of our multi-well chips significantly reduced the operation time for cell seeding and medium refresh. Exploiting a similar approach, we used clinical-grade fibrin to generate implantable multi-well constructs allowing for the precise distribution of multiple cell types. Multi-well fibrin constructs were seeded with ACs generating high cell density regions, as shown by histology and cell fluorescent staining. Multi-well constructs were compared to standard constructs with homogeneously distributed ACs. After 7 days in vitro, expression of SOX9, ACAN, COL2A1, and COMP was increased in both constructs, with multi-well constructs expressing significantly higher levels of chondrogenic genes than standard constructs. After 5 weeks in vivo, we found that despite a dramatic size reduction, the cell distribution pattern was maintained and glycosaminoglycan content per wet weight was significantly increased respect to pre-implantation samples. In conclusion, multi-well chips for the generation and culture of multiple cell spheroids can be fabricated by low-cost rapid prototyping techniques. Furthermore, these techniques can be used to generate implantable constructs with defined architecture and controlled cell distribution, allowing for in vitro and in vivo investigation of cell interactions in a 3D environment.

Exploring an innovative decellularization protocol for porcine nerve grafts: a translational approach to peripheral nerve repair.

Introduction: Peripheral nerves are frequently affected by lesions caused by traumatic or iatrogenic damages, resulting in loss of motor and sensory function, crucial in orthopedic outcomes and with a significant impact on patients' quality of life. Many strategies have been proposed over years to repair nerve injuries with substance loss, to achieve musculoskeletal reinnervation and functional recovery. Allograft have been tested as an alternative to the gold standard, the autograft technique, but nerves from donors frequently cause immunogenic response. For this reason, several studies are focusing to find the best way to decellularize nerves preserving either the extracellular matrix, either the basal lamina, as the key elements used by Schwann cells and axons during the regenerative process. Methods: This study focuses on a novel decellularization protocol for porcine nerves, aimed at reducing immunogenicity while preserving essential elements like the extracellular matrix and basal lamina, vital for nerve regeneration. To investigate the efficacy of the decellularization protocol to remove immunogenic cellular components of the nerve tissue and to preserve the basal lamina and extracellular matrix, morphological analysis was performed through Masson's Trichrome staining, immunofluorescence, high resolution light microscopy and transmission electron microscopy. Decellularized porcine nerve graft were then employed in vivo to repair a rat median nerve lesion. Morphological analysis was also used to study the ability of the porcine decellularized graft to support the nerve regeneration. Results and Discussion: The decellularization method was effective in preparing porcine superficial peroneal nerves for grafting as evidenced by the removal of immunogenic components and preservation of the ECM. Morphological analysis demonstrated that four weeks after injury, regenerating fibers colonized the graft suggesting a promising use to repair severe nerve lesions. The idea of using a porcine nerve graft arises from a translational perspective. This approach offers a promising direction in the orthopedic field for nerve repair, especially in severe cases where conventional methods are limited.

Revolutionizing orthopedic healthcare: a systematic review unveiling recombinant antimicrobial peptides.

The increasing demand for orthopedic surgeries, including joint replacements, is driven by an aging population and improved diagnosis of joint conditions. Orthopedic surgeries carry a risk of infection, especially in patients with comorbidities. The rise of antibiotic resistance exacerbates this issue, necessitating alternatives like in vitro bioengineered antimicrobial peptides (AMPs), offering broad-spectrum activity and multiple action mechanisms. This review aimed to assess the prevalence of antimicrobial potential and the yield after purification among recombinant AMP families. The antimicrobial potential was evaluated using the Minimum Inhibitory Concentration (MIC) values against the most common bacteria involved in clinical infections. This systematic review adhered to PRISMA guidelines, focusing on in vitro studies of recombinant AMPs. The search strategy was run on PubMed, Scopus and Embase up to 30th March 2023. The Population, Exposure and Outcome model was used to extract the data from studies and ToxRTool for the risk of bias analysis. This review included studies providing peptide production yield data and MIC values against pathogenic bacteria. Non-English texts, reviews, conference abstracts, books, studies focusing solely on chemical synthesis, those reporting incomplete data sets, using non-standard MIC assessment methods, or presenting MIC values as ranges rather than precise concentrations, were excluded. From 370 publications, 34 studies on AMPs were analyzed. These covered 46 AMPs across 18 families, with Defensins and Hepcidins being most common. Yields varied from 0.5 to 2,700 mg/L. AMPs were tested against 23 bacterial genera, with MIC values ranging from 0.125 to >1,152 µg/mL. Arenicins showed the highest antimicrobial activity, particularly against common orthopedic infection pathogens. However, AMP production yields varied and some AMPs demonstrated limited effectiveness against certain bacterial strains. This systematic review emphasizes the critical role of bioengineered AMPs to cope infections and antibiotic resistance. It meticulously evaluates recombinant AMPs, focusing on their antimicrobial efficacy and production yields. The review highlights that, despite the variability in AMP yields and effectiveness, Arenicins and Defensins are promising candidates for future research and clinical applications in treating antibiotic-resistant orthopedic infections. This study contributes significantly to the understanding of AMPs in healthcare, underscoring their potential in addressing the growing challenge of antibiotic resistance. Systematic review registration:https://osf.io/2uq4c/.

Exploring multielement nanogranular coatings to forestall implant-related infections.

Introduction: As we approach the post-antibiotic era, the development of innovative antimicrobial strategies that carry out their activities through non-specific mechanisms could limit the onset and spread of drug resistance. In this context, the use of nanogranular coatings of multielement nanoparticles (NPs) conjugated to the surface of implantable biomaterials might represent a strategy to reduce the systemic drawbacks by locally confining the NPs effects against either prokaryotic or eukaryotic cells. Methods: In the present study, two new multielement nanogranular coatings combining Ag and Cu with either Ti or Mg were synthesized by a gas phase physical method and tested against pathogens isolated from periprosthetic joint infections to address their potential antimicrobial value and toxicity in an in vitro experimental setting. Results: Overall, Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli displayed a significantly decreased adhesion when cultured on Ti-Ag-Cu and Mg-Ag-Cu coatings compared to uncoated controls, regardless of their antibiotic resistance traits. A dissimilar behavior was observed when Pseudomonas aeruginosa was cultured for 30 and 120 minutes upon the surface of Ti-Ag-Cu and Mg-Ag-Cu-coated discs. Biofilm formation was mainly reduced by the active effect of Mg-Ag-Cu compared to Ti-Ag-Cu and, again, coatings had a milder effect on P. aeruginosa, probably due to its exceptional capability of attachment and matrix production. These data were further confirmed by the evaluation of bacterial colonization on nanoparticle-coated discs through confocal microscopy. Finally, to exclude any cytotoxic effects on eukaryotic cells, the biocompatibility of NPs-coated discs was studied. Results demonstrated a viability of 95.8% and 89.4% of cells cultured in the presence of Ti-Ag-Cu and Mg-Ag-Cu discs, respectively, when compared to negative controls. Conclusion: In conclusion, the present study demonstrated the promising anti-adhesive features of both Ti-Ag-Cu and Mg-Ag-Cu coatings, as well as their action in hampering the biofilm formation, highlighting the safe use of the tested multi-element families of nanoparticles as new strategies against bacterial attachment to the surface of biomedical implants.

Antibiotic-Loaded Hyperbranched Polyester Embedded into Peptide-Enriched Silk Fibroin for the Treatment of Orthopedic or Dental Infections.

The development of innovative osteoconductive matrices, which are enriched with antibiotic delivery nanosystems, has the invaluable potential to achieve both local contaminant eradication and the osseointegration of implanted devices. With the aim of producing safe, bioactive materials that have osteoconductive and antibacterial properties, novel, antibiotic-loaded, functionalized nanoparticles (AFN)-based on carboxylic acid functionalized hyperbranched aliphatic polyester (CHAP) that can be integrated into peptide-enriched silk fibroin (PSF) matrices with osteoconductive properties-were successfully synthesized. The obtained AFNPSF sponges were first physico-chemically characterized and then tested in vitro against eukaryotic cells and bacteria involved in orthopedic or oral infections. The biocompatibility and microbiological tests confirmed the promising characteristics of the AFN-PSF products for both orthopedic and dental applications. These preliminary results encourage the establishment of AFN-PSF-based preventative strategies in the fight against implant-related infections.

Vancomycin-nanofunctionalized peptide-enriched silk fibroin to prevent methicillin-resistant Staphylococcus epidermidis-induced femoral nonunions in rats.

Introduction: Implant-related infections and infected fractures are significant burdens in orthopedics. Staphylococcus epidermidis is one of the main causes of bone infections related to biofilm formation upon implants. Current antibiotic prophylaxis/therapy is often inadequate to prevent biofilm formation and results in antibiotic resistance. The development of bioactive materials combining antimicrobial and osteoconductive properties offers great potential for the eradication of microorganisms and for the enhancement of bone deposition in the presence of infections. The purpose of this study is to prevent the development of methicillin-resistant S. epidermidis (MRSE)-infected nonunion in a rat model. Methods: To this end, a recently developed in our laboratories bioactive material consisting of antibiotic-loaded nanoparticles based on carboxylic acid functionalized hyperbranched aliphatic polyester (CHAP) that are integrated into peptide-enriched silk fibroin sponges with osteoconductive properties (AFN-PSF) was employed, whose biocompatibility and microbiological tests provided proof of its potential for the treatment of both orthopedic and dental infections. In particular, non-critical femoral fractures fixed with plates and screws were performed in Wistar rats, which were then randomly divided into three groups: 1) the sham control (no infection, no treatment); 2) the control group, infected with MRSE and treated with peptide-enriched silk fibroin sponges incorporating non-drug-loaded functionalized nanoparticles (PSF); 3) the treated group, infected with MRSE and treated with peptide-enriched silk fibroin sponges incorporating vancomycin-loaded functionalized nanoparticles (AFN-PSF). After 8 weeks, bone healing and osteomyelitis were clinically assessed and evaluated by micro-CT, microbiological and histological analyses. Results: The sham group showed no signs of infection and complete bone healing. The PSF group failed to repair the infected fracture, displaying 75% of altered bone healing and severe signs of osteomyelitis. The AFN-PSF treated group reached 70% of fracture healing in the absence of signs of osteomyelitis, such as abscesses in the cortical and intraosseous compartments and bone necrosis with sequestra. Discussion: AFN-PSF sponges have proven effective in preventing the development of infected nonunion in vivo. The proposed nanotechnology for local administration of antibiotics can have a significant impact on patient health in the case of orthopedic infections.

Independent, Controllable Stretch-Perfusion Bioreactor Chambers to Functionalize Cell-Seeded Decellularized Tendons.

Tissue-engineered decellularized matrices can progress clinical replacement of full-thickness ruptures or tendon defects. This study develops and validates a custom-made automated bioreactor, called oscillating stretch-perfusion bioreactor (OSPB), consisting of multiple, independent culture chambers able to combine a bidirectional perfusion with a programmable, uniaxial strain to functionalize cell-seeded decellularized tendons. Decellularized tendon matrices were seeded on their surfaces and within the tendon fibers with mesenchymal stem cells. Then, they were subjected to a bidirectional perfusion and programmed stretching cycles of 15-30-60 min on-off two times per day for 7 days of culture. In vitro analyses showed viable cells, homogenously distributed on the surface of the constructs. More importantly, cell-seeded decellularized tendon grafts undergoing cyclic load in our bioreactor had a superior production and organization of newly formed collagen matrix compared to static cultured constructs. The coherency and local alignment of the new collagen deposition within the inner injected channels quantitatively supported histological findings. The designed OSPB could be considered a unique, cost-effective system able to involve multiple independently controlled chambers in terms of biological and mechanical protocols. This system allows parallel processing of several customized tendon constructs to be used as grafts to enhance the surgical repair of large tendon defects.

Phenotypic Modulation of Biofilm Formation in a Staphylococcus epidermidis Orthopedic Clinical Isolate Grown Under Different Mechanical Stimuli: Contribution From a Combined Proteomic Study.

One of the major causes of prosthetic joint failure is infection. Recently, coagulase negative Staphylococcus epidermidis has been identified as an emergent, nosocomial pathogen involved in subclinical prosthetic joint infections (PJIs). The diagnosis of PJIs mediated by S. epidermidis is usually complex and difficult due to the absence of acute clinical signs derived from the host immune system response. Therefore, analysis of protein patterns in biofilm-producing S. epidermidis allows for the examination of the molecular basis of biofilm formation. Thus, in the present study, the proteome of a clinical isolate S. epidermidis was analyzed when cultured in its planktonic or sessile form to examine protein expression changes depending on culture conditions. After 24 h of culture, sessile bacteria exhibited increased gene expression for ribosomal activity and for production of proteins related to the initial attachment phase, involved in the capsular polysaccharide/adhesin, surface associated proteins and peptidoglycan biosynthesis. Likewise, planktonic S. epidermidis was able to aggregate after 24 h, synthesizing the accumulation associate protein and cell-wall molecules through the activation of the YycFG and ArlRS, two component regulatory pathways. Prolonged culture under vigorous agitation generated a stressful growing environment triggering aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. Further studies will be essential to support these findings in order to further delineate the complex mechanisms of biofilm formation of S. epidermidis and they could provide the groundwork for the development of new drugs against biofilm-related infections, as well as the identification of novel biomarkers of subclinical or chronic infections mediated by these emerging, low virulence pathogens.

Cellulose nanocrystals: a multimodal tool to enhance the targeted drug delivery against bone disorders.

Aim: We investigated the use of cellulose nanocrystals (CNCs) as drug nanocarriers combining an anti-osteoporotic agent, alendronate (ALN), and an anti-cancer drug, doxorubicin (DOX). Materials & methods: CNC physicochemical characterization, in vivo imaging coupled with histology and in vitro uptake and toxicity assays were carried out. Results:In vivo CNC-ALN did not modify bone tropism and lung penetration, whereas its liver and kidney accumulation was slightly higher compared with CNCs alone. In vitro studies showed that CNC-ALN did not impair ALN's effect on osteoclasts, whereas CNC-DOX confirmed the therapeutic potential against bone metastatic cancer cells. Conclusions: This study provides robust proof of the potential of CNCs as easy, flexible and specific carriers to deliver compounds to the bone.

Peptide-Enriched Silk Fibroin Sponge and Trabecular Titanium Composites to Enhance Bone Ingrowth of Prosthetic Implants in an Ovine Model of Bone Gaps.

Osteoarthritis frequently requires arthroplasty. Cementless implants are widely used in clinics to replace damaged cartilage or missing bone tissue. In cementless arthroplasty, the risk of aseptic loosening strictly depends on implant stability and bone-implant interface, which are fundamental to guarantee the long-term success of the implant. Ameliorating the features of prosthetic materials, including their porosity and/or geometry, and identifying osteoconductive and/or osteoinductive coatings of implant surfaces are the main strategies to enhance the bone-implant contact surface area. Herein, the development of a novel composite consisting in the association of macro-porous trabecular titanium with silk fibroin (SF) sponges enriched with anionic fibroin-derived polypeptides is described. This composite is applied to improve early bone ingrowth into the implant mesh in a sheep model of bone defects. The composite enables to nucleate carbonated hydroxyapatite and accelerates the osteoblastic differentiation of resident cells, inducing an outward bone growth, a feature that can be particularly relevant when applying these implants in the case of poor osseointegration. Moreover, the osteoconductive properties of peptide-enriched SF sponges support an inward bone deposition from the native bone towards the implants. This technology can be exploited to improve the biological functionality of various prosthetic materials in terms of early bone fixation and prevention of aseptic loosening in prosthetic surgery.

Animal models of orthopaedic infections. A review of rabbit models used to induce long bone bacterial infections.

The development of infections is one of the main complications in orthopaedics, especially in the presence of implants for the osteosynthesis of compound fractures and joint prosthesis. Indeed, foreign materials and implants act as substrates for the adhesion and proliferation of bacterial strains able to produce biofilm, causing peri-implant osteomyelitis. The eradication of biofilm remains a great challenge for the host immune system, as well as for medical and surgical approaches, thus imposing the need for new prophylactic and/or therapeutic strategies in which animal models have an essential role. In vivo orthopaedic models have mainly been used to study the pathogenesis of infections, biofilm behaviour and the efficacy of antimicrobial strategies, to select diagnostic techniques and test the efficacy of novel materials or surface modifications to impede both the establishment of bone infections and the associated septic loosening of implants. Among several models of osteomyelitis and implant-related infections described in small rodents and large animals, the rabbit has been widely used as a reliable and reproducible model of orthopaedic infections. This review examines the relevance of rabbits for the development of clinically representative models by analysing the pros and cons of the different approaches published in the literature. This analysis will aid in increasing our knowledge concerning orthopaedic infections by using this species. This review will be a tool for researchers who need to approach pre-clinical studies in the field of bone infection and have to identify the most appropriate animal model to verify their scientific hypothesis.

Bone Marrow-Derived Cell Therapies to Heal Long-Bone Nonunions: A Systematic Review and Meta-Analysis-Which Is the Best Available Treatment?

Nonunions represent one of the major indications for clinical settings with stem cell-based therapies. The objective of this research was to systematically assess the current evidence for the efficacy of bone marrow-derived cell-based approaches associated or not with bone scaffolds for the treatment of nonunions. We searched MEDLINE (PubMed) and CENTRAL up to July 2019 for clinical studies focused on the use of cell-based therapies and bone marrow derivatives to treat bone nonunions. Three investigators independently extracted the data and appraised the risk of bias. We analysed 27 studies including a total number of 347 participants exposed to four interventions: bone marrow concentrate (BMAC), BMAC combined with scaffold (BMAC/Scaffold), bone marrow-derived mesenchymal stromal cells (BMSCs), and BMSC combined with scaffold (BMSC/Scaffold). Two controlled studies showed a positive trend in bone healing in favour of BMAC/Scaffold or BMSC/Scaffold treatment against bone autograft, although the difference was not statistically significant (RR 0.11, 95% CI -0.05; 0.28). Among single cohort studies, the highest mean pooled proportion of healing rate was reported for BMAC (77%; 95% CI 63%-89%; 107 cases, n = 8) and BMAC/Scaffold treatments with (71%; 95% CI 50%-89%; 117 cases, n = 8) at 6 months of follow-up. At 12 months of follow-up, an increasing proportion of bone healing was observed in all the treatment groups, ranging from 81% to 100%. These results indicate that BMAC or BMAC/Scaffold might be considered as the primary choice to treat nonunions with a successful healing rate at a midterm follow-up. Moreover, this meta-analysis highlighted that the presence of a scaffold positively influences the healing rate at a long-term follow-up. More case-control studies are still needed to support the clinical improvement of cell-based therapies against autografts, up to now considered as the gold standard for the treatment of nonunions.

A Precautionary Approach to Guide the Use of Transition Metal-Based Nanotechnology to Prevent Orthopedic Infections.

The increase of multidrug-resistant bacteria remains a global concern. Among the proposed strategies, the use of nanoparticles (NPs) alone or associated with orthopedic implants represents a promising solution. NPs are well-known for their antimicrobial effects, induced by their size, shape, charge, concentration and reactive oxygen species (ROS) generation. However, this non-specific cytotoxic potential is a powerful weapon effective against almost all microorganisms, but also against eukaryotic cells, raising concerns related to their safe use. Among the analyzed transition metals, silver is the most investigated element due to its antimicrobial properties per se or as NPs; however, its toxicity raises questions about its biosafety. Even though it has milder antimicrobial and cytotoxic activity, TiO2 needs to be exposed to UV light to be activated, thus limiting its use conjugated to orthopedic devices. By contrast, gold has a good balance between antimicrobial activity as an NP and cytocompatibility because of its inability to generate ROS. Nevertheless, although the toxicity and persistence of NPs within filter organs are not well verified, nowadays, several basic research on NP development and potential uses as antimicrobial weapons is reported, overemphasizing NPs potentialities, but without any existing potential of translation in clinics. This analysis cautions readers with respect to regulation in advancing the development and use of NPs. Hopefully, future works in vivo and clinical trials will support and regulate the use of nano-coatings to guarantee safer use of this promising approach against antibiotic-resistant microorganisms.

Proteomic Analysis Reveals a Biofilm-Like Behavior of Planktonic Aggregates of Staphylococcus epidermidis Grown Under Environmental Pressure/Stress.

Prosthetic joint replacement failure has a huge impact on quality of life and hospitalization costs. A leading cause of prosthetic joint infection is bacteria-forming biofilm on the surface of orthopedic devices. Staphylococcus epidermidis is an emergent, low-virulence pathogen implicated in chronic infections, barely indistinguishable from aseptic loosening when embedded in a mature matrix. The literature on the behavior of quiescent S. epidermidis in mature biofilms is scarce. To fill this gap, we performed comparative analysis of the whole proteomic profiles of two methicillin-resistant S. epidermidis strains growing in planktonic and in sessile form to investigate the molecular mechanisms underlying biofilm stability. After 72-h culture of biofilm-forming S. epidermidis, overexpression of proteins involved in the synthesis of nucleoside triphosphate and polysaccharides was observed, whereas planktonic bacteria expressed proteins linked to stress and anaerobic growth. Cytological analysis was performed to determine why planktonic bacteria unexpectedly expressed proteins typical of sessile culture. Images evidenced that prolonged culture under vigorous agitation can create a stressful growing environment that triggers microorganism aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. The choice of a unique late time point provided an important clue for future investigations into the biofilm-like behavior of planktonic cells. Our preliminary results may inform comparative proteomic strategies in the study of mature bacterial biofilm. Finally, there is an increasing number of studies on the aggregation of free-floating bacteria embedded in an extracellular matrix, prompting the need to gain further insight into this mode of bacterial growth.

Achilles Tendon Repair by Decellularized and Engineered Xenografts in a Rabbit Model.

Tendon tissue ruptures often require the replacement of damaged tissues. The use of auto- or allografts is notoriously limited due to the scarce supply and the high risks of immune adverse reactions. To overcome these limitations, tissue engineering (TE) has been considered a promising approach. Among several biomaterials, decellularized xenografts are available in large quantity and could represent a possible solution for tendon reconstruction. The present study is aimed at evaluating TE xenografts in Achilles tendon defects. Specifically, the ability to enhance the biomechanical functionality, while improving the graft interaction with the host, was tested. The combination of decellularized equine-derived tendon xenografts with or without the matrix repopulation with autologous bone marrow mesenchymal stem cells (BMSCs) under stretch-perfusion dynamic conditions might improve the side-to-side tendon reconstruction. Thirty-six New Zealand rabbits were used to create 2 cm long segmental defects of the Achilles tendon. Then, animals were implanted with autograft (AG) as the gold standard control, decellularized graft (DG), or in vitro tissue-engineered graft (TEG) and evaluated postoperatively at 12 weeks. After sacrifice, histological, immunohistochemical, biochemical, and biomechanical analyses were performed along with the matrix metalloproteinases. The results demonstrated the beneficial role of undifferentiated BMSCs loaded within decellularized xenografts undergoing a stretch-perfusion culture as an immunomodulatory weapon reducing the inflammatory process. Interestingly, AG and TEG groups exhibited similar results, behaved similarly, and showed a significant superior tissue healing compared to DG in terms of newly formed collagen fibres and biomechanical parameters. Whereas, DG demonstrated a massive inflammatory and giant cell response associated with graft destruction and necrosis, absence of type I and III collagen, and a higher amount of proteoglycans and MMP-2, thus unfavourably affecting the biomechanical response. In conclusion, this in vivo study suggests a potential use of the proposed tissue-engineered constructs for tendon reconstruction.