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.

Secondary bone healing is impacted by the extent of interfragmentary motion at the fracture site. It provides mechanical stimulus that is required for the formation of fracture callus. In clinical settings, interfragmentary motion is induced by physiological loading of the broken bone – for example, by weight-bearing. However, there is no consensus about when mechanical stimuli should be applied to achieve fast and robust healing response. Therefore, this study aims to identify the effect of the immediate and delayed application of mechanical stimuli on secondary bone healing.

A partial tibial osteotomy was created in twelve Swiss White Alpine sheep and stabilized using an active external fixator that induced well-controlled interfragmentary motion in form of a strain gradient. Animals were randomly assigned into two groups which mimicked early (immediate group) and late (delayed group) weight-bearing. The immediate group received daily stimulation (1000 cycles/day) from the first day post-op and the delayed group from the 22nd day post-op. Healing progression was evaluated by measurements of the stiffness of the repair tissue during mechanical stimulation and by quantifying callus area on weekly radiographs. At the end of the five weeks period, callus volume was measured on the post-mortem high-resolution computer tomography (HRCT) scan.

Stiffness of the repair tissue (p<0.05) and callus progression (p<0.01) on weekly radiographs were significantly larger for the immediate group compared to the delayed group. The callus volume measured on the HRCT was nearly 3.2 times larger for the immediate group than for the delayed group (p<0.01).

This study demonstrates that the absence of immediate mechanical stimuli delays callus formation, and that mechanical stimulation already applied in the early post-op phase promotes bone healing.

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.

Treatment of bone infection often includes a burdensome two-stage revision. After debridement, contaminated implants are removed and replaced with a non-absorbable cement spacer loaded with antibiotics. Weeks later, the spacer is exchanged with a bone graft aiding bone healing. However, even with this two-stage approach infection persists. In this study, we investigated whether a novel 3D-printed, antibiotic-loaded, osteoinductive calcium phosphate scaffold (CPS) is effective in single-stage revision of an infected non-union with segmental bone loss in rabbits.

A 5 mm defect was created in the radius of female New Zealand White rabbits. The bone fragment was replaced, stabilized with cerclage wire and inoculated with Staphylococcus aureus (MSSA). After 4 weeks, the infected bone fragment was removed, the site debrided and a spacer implanted. Depending on group allocation, rabbits received: 1) PMMA spacer with gentamycin; 2) CPS loaded with rifampin and vancomycin and 3) Non-loaded CPS. These groups received systemic cefazolin for 4 weeks after revision. Group 4 received a loaded CPS without any adjunctive systemic therapy (n=12 group1-3, n=11 group 4). All animals were euthanized 8 weeks after revision and assessed by quantitative bacteriology or histology. Covariance analysis (ANCOVA) and multiple regression were performed.

All animals were culture positive at revision surgery. Half of the animals in all groups had eliminated the infection by end of study. In a historical control group with empty defect and no systemic antibiotic treatment, all animals were infected at euthanasia. There was no significant difference in CFU counts between groups at euthanasia.

Our results show that treating an osteomyelitis with segmental bone loss either with CPS or PMMA has a similar cure rate of infection. However, by not requiring a second surgery, the use of CPS may offer advantages over non-resorbable equivalents such as PMMA.

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.

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.

Serial section electron microscopy (SSEM) was initially developed to map the neural connections in the brain. SSEM eventually led to the term ‘Connectomics’ to be coined to describe process of following a cell or structure through a volume of tissue. This permits the true three-dimensionality to be appreciated and relationships between cells and structures. The purpose of this study was to utilize this methodology to interrogate S. aureus infected bone.

Bone samples were harvested from mice tibia infected with S. aureus and were fixed, decalcified, and osmicated. The samples were paraffin embedded and 5-micron sections were cut to identify regions of bacterial invasion into the osteocyte-lacuna-canalicular-network (OLCN). This area was cut from the paraffin block, deparaffinized, post-fixed and reprocessed into epoxy resin. Serial sections were cut at 60nm and collected onto Kapton tape utilizing the Automated Tape-collecting Ultramicrotome (ATUMtome) system. Samples were mounted onto 4” silicon wafers and post-stained with 2% uranyl acetate followed by 0.3% lead citrate and carbon coated. A ZEISS GeminiSEM 450 scanning electron microscope fitted with an electron backscatter diffusion detector was used to image the sections. The image stack was aligned and segmented using the open-source software, VASTlite.

264 serial sections were imaged, representing approximately 40 × 45 × 15-micron (x, y, z) volume of tissue. 70% of the canaliculi demonstrated infiltration by S. aureus.

This study demonstrates that SSEM can be applied to the skeletal system and provide a new solution to investigate the OLCN system. It is feasible that this methodology could be implemented to investigate why some canaliculi are resistant to colonization and potentially opens up a new direction for the prevention of chronic osteomyelitis. In order to make this a realistic target, automated segmentation methodologies utilizing machine learning must be developed and applied to the bone tissue datasets.

Autologous cancellous bone graft is the gold standard in large bone defect repair. However, studies using autologous bone grafting in rats are rare and donor sites as well as harvesting techniques vary. The aim of this study was to determine the feasibility of autologous cancellous bone graft harvest from 5 different anatomical sites in rats and compare their suitability as donor sites for autologous bone graft.

13 freshly euthanised rats were used to describe the surgical approaches for autologous bone graft harvest from the humerus, iliac crest, femur, tibia and tail vertebrae (n=4), determine the cancellous bone volume and microstructure of those five donor sites using µCT (n=5), and compare their cancellous bone collected qualitatively by looking at cell outgrowth and osteogenic differentiation using an ALP assay and Alizarin Red S staining (n=4).

It was feasible to harvest cancellous bone graft from all 5 anatomical sites with the humerus and tail being more surgically challenging. The microstructural analysis showed a significantly lower bone volume fraction, bone mineral density, and trabecular thickness of the humerus and iliac crest compared to the femur, tibia, and tail vertebrae. The harvested volume did not differ between the donor sites. All donor sites apart from the femur yielded primary osteogenic cells confirmed by the presence of ALP and Alizarin Red S stain. Bone samples from the iliac crest showed the most consistent outgrowth of osteoprogenitor cells.

The tibia and iliac crest may be the most favourable donor sites considering the surgical approach. However, due to the differences in microstructure of the cancellous bone and the consistency of outgrowth of osteoprogenitor cells, the donor sites may have different healing properties, that need further investigation in an in vivo study.

Introduction and Objective

It is widely accepted that interfragmentary strain stimulus promotes callus formation during secondary bone healing. However, the impact of the temporal variation of mechanical stimulation on fracture healing is still not well understood. Moreover, the minimum strain value that initiates callus formation is unknown. The goal of this study was to develop an active fixation system that allows for in vivo testing of varying temporal distribution of mechanical stimulation and that enables detection of the strain limit that initiates callus formation.

The course of secondary fracture healing typically consists of four major phases including inflammation, soft and hard callus formation, and bone remodeling. Callus formation is promoted by mechanical stimulation, yet little is known about the healing tissue response to strain stimuli over shorter timeframes on hourly and daily basis. The aim of this study was to explore the hourly, daily and weekly variations in bone healing progression and to analyze the short-term response of the repair tissue to well-controlled mechanical stimulation.

A system for continuous monitoring of fracture healing was designed for implantation in sheep tibia. The experimental model was adapted from Tufekci et al. 2018 and consisted of 3 mm transverse osteotomy and 30 mm bone defect resulting in an intermediate mobile bone fragment in the tibial shaft. Whereas the distal and proximal parts of the tibia were fixed with external fixator, the mobile fragment was connected to the proximal part via a second, active fixator. A linear actuator embedded in the active fixator moved the mobile fragment axially, thus stimulating mechanically the tissue in the osteotomy gap via well-controlled displacement being independent from the sheep's functional weightbearing. A load sensor was integrated in the active fixation to measure the force acting in the osteotomy gap. During each stimulation cycle the displacement and force magnitudes were recorded to determine in vivo fracture stiffness. Following approval of the local ethics committee, experiments were conducted on four skeletally mature sheep. Starting from the first day after surgery, the daily stimulation protocols consisted of 1000 loading events equally distributed over 12 hours from 9:00 to 21:00 resulting in a single loading event every 44 seconds. No stimulation was performed overnight.

One animal had to be excluded due to inconsistencies in the load sensor data. The onset of tissue stiffening was detected around the eleventh day post-op. However, on a daily basis, the stiffness was not steadily increasing, but instead, an abrupt drop was observed in the beginning of the daily stimulations. Following this initial drop, the stiffness increased until the last stimulation cycle of the day.

The continuous measurements enabled resolving the tissue response to strain stimuli over hours and days. The presented data contributes to the understanding of the influence of patient activity on daily variations in tissue stiffness and can serve to optimize rehabilitation protocols post fractures.

Fracture fixation has advanced significantly with the introduction of locked plating and minimally invasive surgical techniques. However, healing complications occur in up to 10% of cases, of which a significant portion may be attributed to unfavorable mechanical conditions at the fracture. Moreover, state-of-the-art plates are prone to failure from excessive loading or fatigue. A novel biphasic plating concept has been developed to create reliable mechanical conditions for timely bone healing and simultaneously improve implant strength. The goal of this study was to test the feasibility and investigate the robustness of fracture healing with a biphasic plate in a large animal experiment. Twenty-four sheep underwent a 2mm mid-diaphyseal tibia osteotomy stabilized with either the novel biphasic plate or a control locking plate.

Different fracture patterns in terms of defect location and orientation were investigated. Animals were free to fully bear weight during the post-operative period. After 12 weeks, the healing fractures were evaluated for callus formation using micro-computer tomography and strength and stiffness using biomechanical testing. No plate deformation or failures were observed under full weight bearing with the biphasic plate. Osteotomies stabilized with the biphasic plate demonstrated robust callus formation. Torsion tests after plate removal revealed no statistical difference in peak torsion to failure and stiffness for the different fracture patterns stabilized with the biphasic plate. However, the biphasic plate group specimens were 45% stronger (p=0.002) and 48% stiffer (p=0.007) than the controls.

The results of this large animal study demonstrate the clinical potential of this novel stabilization concept.

In the course of uneventful secondary bone healing, a fracture gap is progressively overgrown by callus which subsequently calcifies and remodels into new bone. It is widely accepted that callus formation is promoted by mechanical stimulation of the tissue in the fracture gap. However, the optimal levels of the interfragmentary motion's amplitude, frequency and timing remain unknown. The aim of this study was to develop an active fixation system capable of installing a well-controlled mechanical environment in the fracture gap with continuous monitoring of the bone healing progression.

The experimental model was adapted from Tufekci et al. 2018 and required creation of a critical size defect and an osteotomy in a sheep tibia. They were separated by a mobile bone fragment. The distal and proximal parts of the tibia were fixed with an external fixator, whereas the mobile fragment was connected to the proximal part with an active fixator equipped with a linear actuator to move it axially for mechanical stimulation of the tissue in the fracture gap. This configuration installed well-controlled mechanical conditions in the osteotomy, dependent only on the motion of the active fixator and shielded from the influence of the sheep's functional weightbearing. A load sensor was integrated to measure the force acting in the fracture gap during mechanical stimulation. The motion of the bone fragment was controlled by means of a custom-made controller allowing to program stimulation protocols of various profiles, amplitudes and frequencies of loading events. Following in vitro testing, the system was tested in two Swiss White Alpine Sheep. It was configured to simulate immediate weightbearing for one of the animals and delayed weightbearing for the other. The applied loading protocol consisted of 1000 loading events evenly distributed over 12 hours resulting in in a single loading event every 44 seconds.

Bench testing confirmed the ability of the system to operate effectively with frequencies up to 1Hz over a range of stimulation amplitudes from 0.1 to 1.5 mm. Continuous measurements of in vivo callus stiffness revealed progressive fracture consolidation in the course of each experiment. A delayed onset of fracture healing was observed in the sheep with simulated delayed weightbearing.

The conducted preclinical experiments demonstrated its robustness and reliability. The system can be applied for further preclinical research and comprehensive in-depth investigation of fracture healing.