Background

The molecular mechanisms underlying non-union bone fractures largely remain elusive. Recently, spatial transcriptomics approaches for musculoskeletal tissue samples have been developed requiring direct placement of histology sections on barcoded slides. However, Formalin-Fixed-Paraffin-Embedded (FFPE) bone sections have been associated with limited RNA quality and read depth compared to soft tissue. Here, we test spatial transcriptomics workflows based on transcriptomic probe transfer to characterize molecular features discriminating non-union and union bone fractures in mice.

Introduction

Immunomodulation represents a novel strategy to improve bone healing in combination with low doses of bone morphogenetic growth factors like BMP-2. This study aims to investigate the effect and timing of monoclonal anti-IL-1ß antibody administration with 1μg BMP-2 on bone healing over 14 weeks in a rat femur segmental defect model.

Aims

Fracture-related infection (FRI) is commonly classified based on the time of onset of symptoms. Early infections (< two weeks) are treated with debridement, antibiotics, and implant retention (DAIR). For late infections (> ten weeks), guidelines recommend implant removal due to tolerant biofilms. For delayed infections (two to ten weeks), recommendations are unclear. In this study we compared infection clearance and bone healing in early and delayed FRI treated with DAIR in a rabbit model.

Aim

The time to onset of symptoms after fracture fixation is still commonly used to classify fracture-related infections (FRI). Early infections (<2 weeks) can often be treated with debridement, systemic antibiotics, irrigation, and implant preservation (DAIR). Late infections (>10 weeks) typically require implant removal as mature, antibiotic-tolerant biofilms have formed. However, the recommendations for delayed infections (2–10 weeks) are not clearly defined. Here, infection healing and bone healing in early and delayed FRI is investigated in a rabbit model with a standardized DAIR procedure.

Orthopedic device-related infection (ODRI) preclinical models are widely used in translational research. Most models require induction of general anesthesia, which frequently results in hypothermia in rodents. This study aimed to evaluate the impact of peri anesthetic hypothermia in rodents on outcomes in preclinical orthopedic device-related infection studies.

A retrospective analysis of all rodents that underwent surgery under general anesthesia to induce an ODRI model with inoculation of Staphylococcus epidermidis between 2016 and 2020 was conducted. A one-way multivariate analysis of covariance was used to determine the fixed effect of peri anesthetic hypothermia (hypothermic defined as rectal temperature <35°C) on the combined harvested tissue and implant colonies forming unit counts, and having controlled for the study groups including treatments received duration of surgery and anesthesia and study period. All animal experiments were approved by relevant ethical committee.

A total of 127 rodents (102 rats and 25 mice) were enrolled in an ODRI and met the inclusion criteria. The mean lowest peri-anesthetic temperature was 35.3 ± 1.5 °C. The overall incidence of peri-anesthetic hypothermia was 41% and was less frequently reported in rats (34% in rats versus 68% in mice). Statistical analysis showed a significant effect of peri anesthetic hypothermia on the post-mortem combined colonies forming unit counts from the harvested tissue and implant(s) (p=0.01) when comparing normo- versus hypothermic rodents. Using Wilks’ Λ as a criterion to determine the contribution of independent variables to the model, peri-anesthetic hypothermia was the most significant, though still a weak predictor, of increased harvested colonies forming unit counts.

Altogether, the data corroborate the concept that bacterial colonization is affected by abnormal body temperature during general anesthesia at the time of bacterial inoculation in rodents, which needs to be taken into consideration to decrease infection data variability and improve experimental reproducibility.

In chronically infected fracture non-unions, treatment requires extensive debridement to remove necrotic and infected bone, often resulting in large defects requiring elaborate and prolonged bone reconstruction. One approach includes the induced membrane technique (IMT), although the differences in outcome between infected and non-infectious aetiologies remain unclear. Here we present a new rabbit humerus model for IMT secondary to infection, and, furthermore, we compare bone healing in rabbits with a chronically infected non-union compared to non-infected equivalents.

A 5 mm defect was created in the humerus and filled with a polymethylmethacrylate (PMMA) spacer or left empty (n=6 per group). After 3 weeks, the PMMA spacer was replaced with a beta-tricalcium phosphate (chronOs, Synthes) scaffold, which was placed within the induced membrane and observed for a further 10 weeks. The same protocol was followed for the infected group, except that four week prior to treatment, the wound was inoculated with Staphylococcus aureus (4×10⁶ CFU/animal) and the PMMA spacer was loaded with gentamicin, and systemic therapy was applied for 4 weeks prior to chronOs application.

All the animals from the infected group were culture positive during the first revision surgery (mean 3×10⁵ CFU/animal, n= 12), while at the second revision, after antibiotic therapy, all the animals were culture negative. The differences in bone healing between the non-infected and infected groups were evaluated by radiography and histology. The initially infected animals showed impaired bone healing at euthanasia, and some remnants of bacteria in histology. The non-infected animals reached bone bridging in both empty and chronOs conditions.

We developed a preclinical in vivo model to investigate how bacterial infection influence bone healing in large defects with the future aim to explore new treatment concepts of infected non-union.

Aim

Focused high energy extracorporeal shockwave therapy (fhESWT) is used to support fracture healing in non-union cases and has been shown to have antibacterial effects. We trialed fhESWT as an adjunct to conventional treatment in a clinically relevant rabbit model of fracture related infection.

Aim

Non-steroidal anti-inflammatory drugs (NSAIDs) are a cornerstone of perioperative pain management in orthopedic trauma surgery, although concerns persist regarding the potential impact of these drugs on fracture healing. Furthermore, NSAIDs may also exert an influence on host immune defenses, which may also be important in the context of infection treatment. However, this has been very much under-investigated in the clinical and scientific literature. The aim of this study was to determine the impact of NSAIDs on the course of an orthopedic device-related infection (ODRI) and its response to antibiotic therapy in a rat model.

Aim

Silver is known for its excellent antimicrobial activity, including activity against multiresistant strains. The aim of the current study was to analyze the biocompatibility and potential influence on the fracture healing process a silver-coating technology for locking plates compared to silver-free locking plates in a rabbit model.

Aim

Open fractures still have a high risk for fracture-related Infection (FRI). The optimal duration of perioperative antibiotic prophylaxis (PAP) for open fractures remains controversial due to heterogeneous guidelines and highly variable prophylactic regimens in clinical practice. In order to provide further evidence with which to support the selection of antibiotic duration for open fracture care, we performed a preclinical evaluation in a contaminated rabbit fracture model.

Aim

Antibiotic prophylaxis is critical for the prevention of fracture related infection (FRI) in trauma patients, particularly those with open wounds. Administration of prophylactic antibiotics prior to arrival at the hospital (e.g. by paramedics) may reduce intraoperative bacterial load and has been recommended; however scientific evidence for pre-hospital administration is scarce.

This longitudinal microCT study revealed the osteolytic response to a Staphylococcus epidermidis-infected implant in vivoand also demonstrates how antibiotics and/or a low bone mass state influence the morphological changes in bone and the course of the infection.

Colonisation of orthopaedic implants with Staphylococcus aureusor S. epidermidisis a major clinical concern, since infection-induced osteolysis can drastically impair implant fixation or integration within bone. High fracture incidence in post-menopausal osteoporosis patients means that this patient group are at risk of implant infection. The low bone mass in these patients may exacerbate infection-induced osteolysis, or alter antibiotic efficacy. Therefore, the aims of this study were to examine the bone changes resulting from a S. epidermidisimplant infection in vivousing microCT imaging, and to determine if a low bone mass stateinfluences the course of the infection and the efficacy of antibiotic therapy. An in vivomodel system using microCT scanning [1], involving the implantation of either a sterile or a S. epidermidis-colonised PEEK screw into the proximal tibia of 24 week-old female Wistar rats, was used to investigate the morphological changes in bone following infection over a 28 day period. In addition, the efficacy of a combination antibiotic therapy (rifampin and cefazolin: administered twice daily from days 7–21 post-screw implantation) for affecting osteolysis was also assessed. A subgroup of animals was subjected to ovariectomy (OVX) at 12 weeks of age, allowing for a 12 week period for bone loss prior to screw implantation at 24 weeks. Bone resorption and formation rates, bone-implant contact and peri-implant bone volume in the proximity of the screw were assessed by microCT scanning at days 0, 3, 6, 9, 14, 20 and 28 days post-surgery. Following euthanasia at day 28, the implanted screw, bone and soft tissues were subjected to quantitative bacteriology as a measure of the efficacy of the antibiotic regimen. In non-OVX animals S. epidermidisinfection induced marked osteolysis, which peaked between 9 and 14 days post-screw implantation. Peak bone resorption was detected at day 6, before recovering to baseline levels at day 14. Infection also resulted in extensive deposition of mineralised tissue, initially within the periosteal region (day 9–14), then subsequently in the osteolytic region at day 20–28. Quantitative bacteriology indicated all non-OVX animals remained infected. Rifampin and cefazolin successfully cleared the infection in 5/6 non-OVX animals group although there was no difference observed in CT-derived bone parameters. OVX resulted in extensive loss of trabecular bone but this did not alter the temporal pattern of infection-induced osteolysis, or mineralised tissue deposition, which was similar to that observed in the non-OVX animals. Similarly, there was no difference in bacterial counts between non-OVX and OVX animals (39,005 colony-forming units (CFU) [range: 3,675–156,800] vs 37,665 CFU [range 3,250–84,000], respectively). Interestingly, antibiotic treatment was less effective in the OVX animals (3/5 remained infected), suggesting that antibiotics have reduced efficacy in OVX animals. This study demonstrates S. epidermidis-induced osteolysis displays a similar temporal pattern in both normal and low bone mass states, with comparable bacterial loads present within the localised infection site.

Aim

The aim of this study was to define the role of implant material and surface topography on infection susceptibility in a preclinical in vivo model incorporating appropriate fracture biomechanics and bone healing.

The osteointegration of a new three-dimensional reticular titanium material, Trabecular Titanium™, was assessed using a bilateral cancellous (distal femur, proximal tibia) and cortical (tibia diaphysis) bone drill hole model in 18 sheep. TT is a novel Ti6Al4V material characterized by a high open porosity and composed of multi-planar regular hexagonal cells. Two 5.0 mm diameter, 12 mm long cylinders (TT1 & TT2) of two different porosities (TT1:650 μm, TT2:1250 μm) were tested and compared to two solid predicate 5.0 mm diameter, 12 mm long Ti cylinders (PT1 & PT2) coated with porous Ti (PT1: vacuum-plasma spray coating; PT2: inert-gas shielding arc spray coating).

Each implant type was surgically implanted at 4 separate locations in each sheep (16 implants per sheep). Three timepoints of 4, 16 and 52 weeks (n=6 sheep per timepoint) were used. Bone-implant interface was analyzed ex vivo by the determination of: 1) the shear strength (SS) measured during a push out test, 2) the percentage of bone in-growth (%B) using histomorphometry, 3) the bone apposition rate using fluorochrome labelling analysis and 4) the bone-implant contact using backscattered scanning electron microscopy (SEM). An ANOVA with a Bonferroni Post hoc test were used to detect differences between tested and predicate implants. P values 0.05 were considered significant.

At 4 weeks, 5 out of the 6 TT1 could be pushed out of the cortical bone (COB) samples. The remaining TT1 collapsed during testing. All TT1 could be pushed of the cancellous bone (CAB) samples. Four out of the 6 TT2 could be pushed out of CAB and of the COB samples. At 16 and 52 weeks, only one TT1 and one TT2 could be pushed out of the bone samples, the remaining implants collapsed during testing. All the PTs were successfully pushed out at all timepoints.

The mean %B of PT1 and PT2 did not significantly increase over time. For both materials, the mean %B ranged between 1.7% and 4.4% at 4 weeks and between 5.7% and 6.5% at 52 weeks. The mean %B of TT1 significantly increased over time in both COB (10.2% at 4 weeks, 46.2% at 16 weeks, 50.5% at 52 weeks) and CAB (5.8%, 23.9%, 24.3%). Similarly, the mean %B of TT2 significantly increased over time in both COB (7.8%, 48.6%, 65%) and CAB (4.5%, 24.1%, 38.6%). Bone apposition rates for the TT implants remained superior to 2 μm/day for the entire duration of the study. SEM showed an intimate bone-implant contact for all implant types at all timepoints.

At 16 and 52 weeks, histomorphometry revealed an extensive osteointegration of the TT specimens. Bone-implant interface strength was so high for the TT implants that they could not be pushed out of the bone samples. The results of this study would indicate that the TT implants provide a good scaffold for bone in-growth.