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.

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.

Orbital floor (OF) fractures are commonly treated by implanting either bioinert titanium or polyethylene implants, or by autologous grafts. A personalized implant made of biodegradable and osteopromotive poly(trimethylene carbonate) loaded with hydroxyapatite (PTMC-HA) could be a suitable alternative for patients where a permanent implant could be detrimental. A workflow was developed from the implant production using stereolithography (SLA) based on patient CT scan to the implantation and assessment its performance (i.e. implant stability, orbit position, bone formation) compared to personalised titanium implants in a repair OF defect sheep model. Implants fabrication was done using SLA of photo-crosslinkable PTMC mixed with HA [1–3]. Preclinical study: (sheep n=12, ethic number 34_2016) was conducted by first scanning the OF bone of each sheep in order to design and to fabricate patient specific implants (PSI) made of PTMC-HA. The fabricated PSI was implanted after creating OF defect. Bone formation and defect healing was compared to manually shaped titanium mesh using time-laps X-ray analyses, histology (Giemsa-Eosin staining) and sequential fluorochrome staining over 3-months. Additionally, the osteoinductive property of the biomaterials was assessed by intramuscular implantation (IM). In this study, we showed that the composite PTMC-HA allowed for ectopic bone formation after IM implantation, without requiring any biotherapeutics. In addition, we could repair OF defect on sheep using SLA-fabricated PTMC-HA with a good shape fidelity (compared to the virtual implant) and a better bone integration compared to the titanium mesh. This study opens the field of patient-specific implants made of degradable and osteoinductive scaffolds fabricated using additive manufacturing to replace advantageously autologous bone and titanium implants.

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.

Mesenchymal stromal cells (MSCs) have been intensively researched in the orthopaedic field since they hold great promise for aiding the regeneration of musculoskeletal tissues. While there are a range of postulated surface markers to identify MSCs, currently there are no known cell markers that predict in vivo osteochondral potency. Runt-related transcription factor 2 (Runx2) is considered as an essential transcription factor in osteoblast differentiation [1] and has been shown to physically interact with retinoblastoma protein (pRb), which leads the loss of osteoblast proliferation and the activation of genes concerning terminal differentiation of osteoblasts [2]. The aim of this study was to use adenoviral-mediated gene overexpression/knockdown to investigate the interplay between Runx2 and pRb during in vitro osteogenic differentiation of human bone marrow (hBM)-MSCs.

A first generation human adenovirus (hAd) serotype 5 dE/E3 carrying the gene of interest (Runx2 or shRNA-Runx2) were propagated and amplified in AD-293 cells, and purified over successive CsCl gradients. A second generation hAd serotype 5 carrying the gene of interest (Rb1) was generated. High efficiency single or double transduction of undifferentiated hBM-MSCs was achieved using lanthofection [3]. The transduced hBM-MSCs were then differentiated in osteogenic medium (OM) and osteogenic potency was assessed by quantification of alkaline phosphatase (ALP) activity (day 14) and Alizarin red staining (day 28). In addition, cell cultures were assessed for absorbance at OD 450nm, correlating to the refractive index of calcified areas, at days 0, 7, 14, 21 and 28 [4]. Quantitative RT-PCR was used to confirm expression of target genes following viral transduction. Basal medium was used as a control.

Untransduced hBM-MSCs cultures grown in OM demonstrated peak calcium deposition at day 28, while the overexpression of either Runx2 or Rb1 accelerated peak calcium deposition to day 21. Consistent with this, Runx2 overexpression increased ALP activity of hBM-MSCs cultured in OM, while Rb1 overexpression enhanced ALP activity of hBM-MSCs cultured in both basal and osteogenic conditions. Co-expression of Runx2 and Rb1 did not further increase ALP activity compared to cells transduced with Runx2 or Rb1 alone.

Alizarin red staining revealed that overexpression of either Runx2 or Rb1 increased mineral deposition in hBM-MSCs under basal conditions, although mineralisation was not enhanced above that of untransduced cells when cultured in OM. However, mineralisation was markedly enhanced above levels in untransduced cells when Runx2 and Rb1 were co-expressed in hBM-MSCs grown under both basal and osteogenic conditions.

This study demonstrates an important stimulatory role of pRb in enhancing ALP activity of hBM-MSCs in the absence of osteogenic clues. However, pRb overexpression alone is insufficient to enhance mineralisation, requiring the co-expression of Runx2 in hBM-MSCs. The crucial nature of Runx2 for osteogenic differentiation of hBM-MSCs was demonstrated since knockdown of Runx2 prevented both mineral deposition and the increased ALP activity observed in untransduced cells grown in OM. Interestingly, overexpression of Rb1 could not compensate for the knockdown of Runx2 since Rb1 overexpression did not recover either mineral deposition or ALP activity in hBM-MSCs where Runx2 expression was inhibited.

Summary

Carriers for local delivery of stem cells into degenerative intervertebral discs need to be tested under physiological loading since stem cell viability, density and differentiation, as well as carrier stability are strongly affected by loading.