Biomaterials with mechanical or biological competence are ubiquitous in musculoskeletal disorders, and understanding the inflammatory response they trigger is key to guide tissue regeneration. While macrophage role has been widely investigated, immune response is regulated by other immune cells, including neutrophils, the most abundant leukocyte in human blood. As first responders to injury, infection or material implantation, neutrophils recruit other immune cells, and therefore influence the onset and resolution of chronic inflammation, and macrophage polarization. This response depends on the physical and chemical properties of the biomaterials, among other factors. In this study we report an in vitro culture model to describe the most important neutrophil functions in relation to tissue repair.

We identified neutrophil survival and death, neutrophils extracellular trap formation, release of reactive oxygen species and degranulation with cytokines release as key functions and introduced a corresponding array of assays. These tests were suitable to identify clear differences in the response by neutrophils that were cultured on material of different origin, stiffness and chemical composition. Overall, substrates from biopolymers of natural origin resulted in increased survival, less neutrophil extracellular trap formation, and more reactive oxygen species production than synthetic polymers. Within the range of mechanical properties explored (storage modulus below 5 k Pa), storage modulus of covalently crosslinked hyaluronic acid hydrogels did not significantly alter neutrophils response, whereas polyvinyl alcohol gels of matching mechanical properties displayed a response indicating increased activation.

Additionally, we present the effect of material stiffness, charge, coating and culture conditions in the measured neutrophils response. Further studies are needed to correlate the neutrophil response to tissue healing.

By deciphering how neutrophils initiate and modulate the immune response to material implantation, we aim at introducing new principles to design immunomodulatory biomaterials for musculoskeletal disorders.

Acknowledgments

This work was supported by the AO Foundation, AO CMF, grant AOCMF-21-04S.

Fracture related infection remains a major challenge in musculoskeletal trauma surgery. Despite best practice, treatment strategies suffer from high failure rates due to antibiotic resistance and tolerance. Bacteriophages represent a promising alternative as they retain activity against such bacteria. However, optimal phage administration protocols remain unknown, although injectable hydrogels, loaded with phage and conventional antibiotics, may support conventional therapy.

In this study we tested the activity of meropenem, and two newly isolated bacteriophages (ϕ9 and ϕ3) embedded within alginate-chitosan microbeads and a hydrogel. Antibiotic and phage stability and activity were monitored in vitro, over a period of 10 days. In vivo, the same material was tested in treatment of a 5-day old Pseudomonas aeruginosa infection of a tibial plate osteotomy in mice. Treatment involved debridement and 5 days of systemic antibiotic therapy plus: i- saline, ii-phages in saline, iii-phages and antibiotics loaded into a hydrogel (n=7 mice/group). To assess the efficacy of the treatments, the infection load was monitored during revision surgery with debridement of the infected tissue after 5,10 and 13 days (euthanasia) by CFU and PFU quantification.

In vitro testing confirmed that the stability of meropenem and activity of ϕ9 and ϕ3, was not affected within the alginate beads or hydrogel over 10 days. The in vivo study showed that all mice receiving phages and antibiotics loaded into a hydrogel survived the infection with a reduction of the bacterial load in the soft tissue. Active phages could be recovered from the infected site at euthanasia (10⁴ PFU/g).

The hydrogel loaded with bacteriophages and meropenem showed a positive result in locally reducing the infection load indicating a synergistic effect of the selected antimicrobials. Overall, our new strategy shows encouraging results for improving the treatment of antibiotic-resistant biofilm infections that are related to medical implants.

Local antimicrobial therapy is an integral aspect of treating orthopaedic device related infection (ODRI), which is conventionally administered via polymethylmethacrylate (PMMA) bone cement. PMMA, however, is limited by a suboptimal antibiotic release profile and a lack of biodegradability.

In this study, we compare the efficacy of PMMA versus an antibioticloaded hydrogel in a single- stage revision for chronic methicillin-resistant Staphylococcus aureus (MRSA) ODRI in

sheep. Antibiofilm activity of the antibiotic combination (gentamicin and vancomycin) was determined in vitro. Swiss alpine sheep underwent a single-stage revision of a tibial intramedullary nail with MRSA infection. Local gentamicin and vancomycin therapy was delivered via hydrogel or PMMA (n = 5 per group), in conjunction with systemic antibiotic therapy. In vivo observations included: local antibiotic tissue concentration, renal and liver function tests, and quantitative microbiology on tissues and hardware post-mortem.

There was a nonsignificant reduction in biofilm with an increasing antibiotic concentration in vitro (p = 0.12), confirming the antibiotic tolerance of the MRSA biofilm. In the in vivo study, four out of five sheep from each treatment group were culture negative. Antibiotic delivery via hydrogel resulted in 10–100 times greater local concentrations for the first 2–3 days compared with PMMA and were comparable thereafter. Systemic concentrations of gentamicin were minimal or undetectable in both groups, while renal and liver function tests were within normal limits.

This study shows that a single-stage revision with hydrogel or PMMA is equally effective, although the hydrogel offers certain practical benefits over PMMA, which make it an attractive proposition for clinical use.

Introduction

Current cell-based treatments and marrow stimulating techniques to repair articular cartilage defects are limited in restoring the tissue in its native composition. Despite progress in cartilage tissue engineering and chondrogenesis in vitro, the main limitation of this approach is the progression towards hypertrophy during prolonged culture in pellets or embedded in biomaterials. The objectives of this study were (A) to compare human bone marrow-derived mesenchymal stromal cells (hMSC) chondrogenesis and hypertrophy in pellet culture from single cells or cell spheroids and (B) to investigate the effect of tyramine-modified hyaluronic acid (THA) and collagen I (Col) content in composite hydrogels on the chondrogenesis and hypertrophy of encapsulated hMSC spheroids.

Orthopedic device-related bone infection is one of the most distressing complications of the surgical fixation of fractures. Despite best practice in medical and surgical interventions, the rate of infection remains stubbornly persistent, and current estimates indicate that treatment failure rates are also significant. As we approach the limit of the effectiveness of current anti-infective preventative and therapeutic strategies, novel approaches to infection management assume great importance. This presentation will describe our efforts to develop and test various hydrogels to serve as customized antibiotic delivery vehicles for infection prevention and treatment. Hydrogels offer solutions for many of the challenges faced by complex trauma wounds as they are not restricted spatially within a poorly defined surgical field, they often degrade rapidly with no compatibility issues, and releases 100% of the loaded antibiotic. The preliminary data set proving efficacy in preventing and treating infection in both rabbit and sheep studies will be described, including local antibiotic concentrations in the intramedullary canal over time, compared to that of the more conventional antibiotic loaded bone cement. These two technologies show potential for the prevention and treatment of infection in trauma patients, with a clear focus on optimized antibiotic delivery tailored for complex wounds.

Aim

Implant-associated osteomyelitis is a devastating complication with poor outcomes following treatment, especially when caused by antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). A large animal model of a two-stage revision to treat MRSA implant-associated osteomyelitis has been developed to assess novel treatments. A bioresorbable, thermo-responsive hyaluronan hydrogel (THH) loaded with antibiotics has been developed and our aim was to investigate it´s in vivo efficacy as a local antibiotic carrier compared to the current standard of care i.e. antibiotic-loaded polymethylmethacrylate (PMMA) bone cement.

Collagen and hyaluronic acid are two major components of intervertebral disc (IVD). They give resistance and hydration to Nucleus Pulposus. In this study, we assessed the impact of Collagen (COLL) and Hyaluronic acid-Tyramine (THA) contents on the mechanical properties and the structure of composite hydrogels. For this purpose, a range of composites were obtained using a 4 mg/mL collagen concentration and different COLL/THA ratios from 8:1 to 1:5 (w/w). Composite gelling was performed by pH increase, triggering collagen fibrillogenesis and oxidative coupling of tyramine moieties in THA catalyzed by H₂O₂ and horseradish peroxidase (HRP). To modulate the THA gelling kinetic, different HRP concentrations (0.05; 0.1 and 0.5 U/mL) were used. Composites with a low THA content exhibited a fibrillar structure and possessed mechanical properties close to those of pure collagen hydrogels (200 Pa). From the ratio 1:1, the storage modulus increased to reach c.a 1200 Pa for the ratio 1:5. From the ratio 1:2, the fibrillar structure disappeared and sheets, characteristic of THA hydrogels, were observed. The HRP activity dramatically impacted the physical properties. A rapid THA gelling associated with a high THA content tended to destabilize collagen fibrils and promoted the formation of covalent bond between collagen and THA. On the opposite a slow gelling kinetic favored collagen fibril formation up to the COLL/THA ratio 1:2. Taken together, these results show that a slow gelling and an 8 mg/mL THA concentration are the appropriate conditions to obtain biomimetic biomaterials for the treatment of Nucleus Pulposus.

Aim

The treatment of chronic orthopedic device-related infection (ODRI) often requires multiple surgeries and prolonged antibiotic therapy. In a two-stage exchange procedure, the treatment protocol includes device removal and placement of an antibiotic-loaded bone cement spacer to achieve high local antibiotic concentrations. At the second stage, further surgery is required to remove the spacer and replace it with the definitive device. We have recently developed a thermo-responsive hyaluronan hydrogel (THH) that may be loaded with antibiotics and used as delivery system. Since the material is bio-resorbable, it does not require surgical removal and may therefore be suitable for use as treatment strategy in a single-stage exchange.

This aim of this study was to evaluate gentamicin sulphate (Genta)-loaded THH (THH-Genta) for treating a chronic Staphylococcus aureus ODRI in sheep using a single-stage procedure.