Staphylococcus aureus From an Acute Fracture-related Infection Displays Important Bacteriological and Histopathologic Differences From a Chronic Equivalent in a Murine Bone Infection Model.

BACKGROUND: Staphylococcus aureus is the leading pathogen in fracture-related infection. Previous in vitro experiments, in vivo testing in wax moth larvae, and genomic analysis of clinical S. aureu s isolates from fracture-related infection identified low-virulence (Lo-SA5464) and high-virulence (Hi-SA5458) strains. These findings correlated with acute fracture-related infection induced by Hi-SA5458, whereas Lo-SA5464 caused a chronic fracture-related infection in its human host. However, it remains unclear whether and to what extent the causative pathogen is attributable to these disparities in fracture-related infections. QUESTION/PURPOSE: Are there differences in the course of infection when comparing these two different clinical isolates in a murine fracture-related infection model, as measured by (1) clinical observations of weight loss, (2) quantitative bacteriology, (3) immune response, and (4) radiographic and histopathologic morphology? METHODS: Twenty-five (including one replacement animal) female (no sex-specific influences expected), skeletally mature C57Bl/6N inbred mice between 20 and 28 weeks old underwent femoral osteotomy stabilized by titanium locking plates. Fracture-related infection was established by inoculation of high-virulence S. aureus EDCC 5458 (Hi-SA5458) or low-virulence S. aureus EDCC 5464 (Lo-SA5464) in the fracture gap. Each of these groups consisted of 12 randomly assigned animals. Mice were euthanized 4 and 14 days postsurgery, resulting in six animals per group and timepoint. The severity and progression of infection were assessed in terms of clinical observation of weight loss, quantitative bacteriology, quantitative serum cytokine levels, qualitative analysis of postmortem radiographs, and semiquantitative histopathologic evaluation. RESULTS: For clinical observations of weight change, no differences were seen at Day 4 between Hi-SA5458- and Lo-SA5464-infected animals (mean -0.6 ± 0.1 grams versus -0.8 ± 0.2 grams, mean difference -0.2 grams [95% CI -0.8 to 0.5 grams]; p =0.43), while at 14 days, the Hi-SA5458 group lost more weight than the Lo-SA5464 group (mean -1.55 ± 0.2 grams versus -0.8 ± 0.3 grams; mean difference 0.7 grams [95% CI 0.2 to 1.3 grams]; p = 0.02). Quantitative bacteriological results 4 days postoperatively revealed a higher bacterial load in soft tissue samples in Hi-SA5458-infected animals than in the Lo-SA5464-infected cohort (median 6.8 x 10 7 colony-forming units [CFU]/g, range 2.2 x 10 7 to 2.1 x 10 9 CFU/g versus median 6.0 x 10 6 CFU/g, range 1.8 x 10 5 to 1.3 x 10 8 CFU/g; difference of medians 6.2 x 10 7 CFU/g; p = 0.03). At both timepoints, mice infected with the Hi-SA5458 strain also displayed higher proportions of bacterial dissemination into organs than Lo-SA5464-infected animals (67% [24 of 36 organs] versus 14% [five of 36 organs]; OR 12.0 [95% CI 3.7 to 36]; p < 0.001). This was accompanied by a pronounced proinflammatory response on Day 14, indicated by increased serum cytokine levels of interleukin-1ß (mean 9.0 ± 2.2 pg/mL versus 5.3 ± 1.5 pg/mL; mean difference 3.6 pg/mL [95% CI 2.0 to 5.2 pg/mL]; p < 0.001), IL-6 (mean 458.6 ± 370.7 pg/mL versus 201.0 ±89.6 pg/mL; mean difference 257.6 pg/mL [95% CI 68.7 to 446.5 pg/mL]; p = 0.006), IL-10 (mean 15.9 ± 3.5 pg/mL versus 9.9 ± 1.0 pg/mL; mean difference 6.0 pg/mL [95% CI 3.2 to 8.7 pg/mL]; p < 0.001), and interferon-γ (mean 2.7 ± 1.9 pg/mL versus 0.8 ± 0.3 pg/mL; mean difference 1.8 pg/mL [95% CI 0.5 to 3.1 pg/mL]; p = 0.002) in Hi-SA5458-infected compared with Lo-SA5464-infected animals. The semiquantitative histopathologic assessment on Day 4 revealed higher grades of granulocyte infiltration in Hi-SA5458-infected animals (mean grade 2.5 ± 1.0) than in Lo-SA5464-infected animals (mean grade 1.8 ± 1.4; mean difference 0.7 [95% CI 0.001 to 1.4]; p = 0.0498). On Day 14, bone healing at the fracture site was present to a higher extent in Lo-SA5464-infected animals than in Hi-SA5458-infected animals (mean grade 0.2 ± 0.4 versus 1.8 ± 1.2; mean difference -1.6 [95% CI -2.8 to -0.5]; p = 0.008). CONCLUSION: Similar to septic infection in a human host, infection with Hi-SA5458 in this murine model was characterized by a higher bacterial load, more-pronounced systemic dissemination, and stronger systemic and local inflammation. Thus, there is strong support for the idea that pathogenic virulence plays a crucial role in fracture-related infections. To confirm our observations, future studies should focus on characterizing S. aureus virulence at the genomic and transcriptomic levels in more clinical isolates and patients. Comparing knockout and wildtype strains in vitro and in vivo, including the S. aureus strains studied, could confirm our findings and identify the genomic features responsible for S. aureus virulence in fracture-related infections. CLINICAL RELEVANCE: For translational use, virulence profiles of S. aureus may be useful in guiding treatment decisions in the future. Once specific virulence targets are identified, one approach to fracture-related infections with high-virulence strains might be the development of antivirulence agents, particularly to treat or prevent septic dissemination. For fracture-related infections with low virulence, prolonged antimicrobial therapy or exchange of an indwelling implant might be beneficial owing to slower growth and persistence capacity.

Comparative bone healing with induced membrane technique (IMT) versus empty defects in septic and aseptic conditions in a novel rabbit humerus model.

BACKGROUND: Long bone defects resulting from primary trauma or secondary to debridement of fracture-related infection (FRI) remain a major clinical challenge. One approach often used is the induced membrane technique (IMT). The effectiveness of the IMT in infected versus non-infected settings remains to be definitively established. In this study we present a new rabbit humerus model and compare the IMT approach between animals with prior infection and non-infected equivalents. METHODS: A 5 mm defect was created in the humerus of New Zealand White rabbits (n = 53) and fixed with a 2.5 mm stainless steel plate. In the non-infected groups, the defect was either left empty (n = 6) or treated using the IMT procedure (PMMA spacer for 3 weeks, n = 6). Additionally, both approaches were applied in animals that were inoculated with Staphylococcus aureus 4 weeks prior to defect creation (n = 5 and n = 6, respectively). At the first and second revision surgeries, infected and necrotic tissues were debrided and processed for bacteriological quantification. In the IMT groups, the PMMA spacer was removed 3 weeks post implantation and replaced with a beta-tricalcium phosphate scaffold and bone healing observed for a further 10 weeks. Infected groups also received systemic antibiotic therapy. The differences in bone healing between the groups were evaluated radiographically using a modification of the radiographic union score for tibial fractures (RUST) and by semiquantitative histopathology on Giemsa-Eosin-stained sections. RESULTS: The presence of S. aureus infection at revision surgery was required for inclusion to the second stage. At the second revision surgery all collected samples were culture negative confirming successful treatment. In the empty defect group, bone healing was increased in the previously infected animals compared with non-infected controls as revealed by radiography with significantly higher RUST values at 6 weeks (p = 0.0281) and at the end of the study (p = 0.0411) and by histopathology with increased cortical bridging (80% and 100% in cis and trans cortical bridging in infected animals compared to 17% and 67% in the non-infected animals). With the IMT approach, both infected and non-infected animals had positive healing assessments. CONCLUSION: We successfully developed an in vivo model of bone defect healing with IMT with and without infection. Bone defects can heal after an infection with even better outcomes compared to the non-infected setting, although in both cases, the IMT achieved better healing.

Chronic Periodontal Infection and Not Iatrogenic Interference Is the Trigger of Medication-Related Osteonecrosis of the Jaw: Insights from a Large Animal Study (PerioBRONJ Pig Model).

Background and Objectives: Antiresorptive drugs are widely used in osteology and oncology. An important adverse effect of these drugs is medication-induced osteonecrosis of the jaw (MRONJ). There is scientific uncertainty about the underlying pathomechanism of MRONJ. A promising theory suspects infectious stimuli and local acidification with adverse effects on osteoclastic activity as crucial steps of MRONJ etiology. Clinical evidence showing a direct association between MRONJ and oral infections, such as periodontitis, without preceding surgical interventions is limited. Large animal models investigating the relationship between periodontitis and MRONJ have not been implemented. It is unclear whether the presence of infectious processes without surgical manipulation can trigger MRONJ. The following research question was formulated: is there a link between chronic oral infectious processes (periodontitis) and the occurrence of MRONJ in the absence of oral surgical procedures? Materials and Methods: A minipig large animal model for bisphosphonate-related ONJ (BRONJ) using 16 Göttingen minipigs divided into 2 groups (intervention/control) was designed and implemented. The intervention group included animals receiving i.v. bisphosphonates (zoledronate, n = 8, 0.05 mg/kg/week: ZOL group). The control group received no antiresorptive drug (n = 8: NON-ZOL group). Periodontitis lesions were induced by established procedures after 3 months of pretreatment (for the maxilla: the creation of an artificial gingival crevice and placement of a periodontal silk suture; for the mandible: the placement of a periodontal silk suture only). The outcomes were evaluated clinically and radiologically for 3 months postoperatively. After euthanasia a detailed histological evaluation was performed. Results: Periodontitis lesions could be induced successfully in all animals (both ZOL and NON-ZOL animals). MRONJ lesions of various stages developed around all periodontitis induction sites in the ZOL animals. The presence of MRONJ and periodontitis was proven clinically, radiologically and histologically. Conclusions: The results of this study provide further evidence that the infectious processes without prior dentoalveolar surgical interventions can trigger MRONJ. Therefore, iatrogenic disruption of the oral mucosa cannot be the decisive step in the pathogenesis of MRONJ.

Venous injection of a triphasic calcium-based implant in a sheep model of pulmonary embolism demonstrates minimal acute systemic effects.

PURPOSE: Implant leakage is the most common complication of vertebral augmentation. Alternative injectable materials must demonstrate intravascular safety comparable to or better than polymethyl methacrylate (PMMA). This study assessed the systemic effects of a triphasic calcium-based implant or PMMA injected directly into the femoral vein in a large animal model designed to mimic severe intravascular implant leakage. METHODS: Six skeletally mature female sheep were randomly assigned (n = 3) to either the PMMA or the triphasic implant (AGN1, composition: calcium sulfate, ß-tricalcium phosphate, brushite) treatment group. Femoral veins of each sheep were directly injected with 0.5 mL of implant material to mimic leakage volumes reported during PMMA vertebroplasty. To compare acute systemic effects of the materials, cardiovascular parameters, laboratory coagulation markers, and calcium and sulfate serum levels were monitored for 60 min after implant injection. Thrombotic and embolic events were evaluated by radiologic imaging, necropsy, and histopathology. RESULTS: Heart rate, systemic arterial blood pressure, arterial oxygenation, arterial carbon dioxide content, and coagulation markers remained within physiological range after either AGN1 or PMMA injection. No blood flow interruption in the larger pulmonary vessels was observed in either group. Lung histopathology revealed that the severity of thrombotic changes after AGN1 injection was minimal to slight, while changes after PMMA injection were minimal to massive. CONCLUSION: Acute systemic effects of intravascular AGN1 appeared to be comparable to or less than that of intravascular PMMA. Furthermore, in this preliminary study, the severity and incidence of pulmonary histological changes were lower for AGN1 compared to PMMA.

Influence of screw head diameter on ex vivo fixation of equine lateral condylar fractures with 5.5 mm cortical screws.

OBJECTIVE: To determine the influence of screw head diameter on equine condylar fracture fixation with 5.5 mm cortical screws. STUDY DESIGN: Ex vivo, biomechanical study, blinded, matched-pair design. SAMPLE POPULATION: Fifteen pairs of equine third metacarpal (MC3) bones. METHODS: Lateral condylar fractures were simulated by parasagittal osteotomies and repaired pairwise by 2 × 5.5 mm cortical screws of 8 mm (standard) or 10 mm (modified) head diameter. Interfragmentary compression at maximum screw insertion torque was measured. The instrumented specimens were pairwise stratified for biomechanical testing under the following modalities (n = 5): (1) screw insertion torque to failure, (2) quasi-static axial load to failure, and (3) cyclic axial load to 2 mm displacement followed by failure. Tests (1) and (2) were analyzed for yield, maximum, and failure torque/angle and load/displacement, respectively. Number of cycles to 2 mm displacement and failure was assessed from test (3). RESULTS: Maximum insertion torque was greater, and failure angle smaller, when constructs repaired with modified screws were tested (8.1 ± 0.5 vs. 7.4 ± 0.5 Nm; P = .0047 and 550 ± 104 vs. 1130 ± 230; P = .008). Axial yield (7118 ± 707 vs. 5740 ± 2267 N; P = .043) and failure load (12 347 ± 3359 vs. 8695 ± 2277 N; P = .043) were greater for specimens repaired with modified screws. No difference was detected between constructs in the number of cycles to 2 mm displacement. CONCLUSION: Condylar MC3 osteotomies repaired with modified 5.5 mm cortical screws sustained greater maximal hand torque insertion, smaller insertion failure angle and 1.4 fold greater quasi-static failure forces than constructs repaired with standard 5.5 mm screws. CLINICAL SIGNIFICANCE: Use of modified screws with larger heads may improve the fixation of condylar fractures in horses. These results provide evidence to justify clinical evaluation in horses undergoing fracture repair.

Computed Tomography-Based Investigation on the Effects of Intravenous Bisphosphonate Administration on Tooth Growth in a Minipig Animal Model.

Background and Objectives: The objective of this study was to evaluate the effects of bisphosphonate (BP) administration on tooth growth, using CT-data of a minipig animal model investigation. Materials and Methods: Tooth growth was evaluated in minipigs, with eight animals receiving weekly zoledronate (ZOL) and three animals serving as the control group. Tooth growth was evaluated at the right 2nd molar (M2) in the maxilla. A computed tomography-based measuring method was applied to evaluate tooth growth in the coronal-apical, buccal-oral and mesial-distal axis. Results: ZOL-administration was found to impact tooth growth in all evaluated measuring axes, with the highest effect observed in the coronal-apical axis. Conclusions: Detrimental effects of BP administration on growing teeth have been reported by a number of investigators. The results of this investigation demonstrate that intravenous ZOL affects the growth of the whole tooth within a short period of administration. With BPs being administered to a growing number of pediatric patients, further studies should be conducted to qualify and quantify the effects of BPs on developing teeth.

Continuous Implant Load Monitoring to Assess Bone Healing Status-Evidence from Animal Testing.

Background and Objectives: Fracture healing is currently assessed through qualitative evaluation of radiographic images, which is highly subjective in nature. Radiographs can only provide snapshots in time, which are limited due to logistics and radiation exposure. We recently proposed assessing the bone healing status through continuous monitoring of the implant load, utilizing an implanted sensor system, the Fracture Monitor. The device telemetrically transmits statistically derived implant parameters via the patient's mobile phone to assist physicians in diagnostics and treatment decision-making. This preclinical study aims to systematically investigate the device safety and performance in an animal setting. Materials and Methods: Mid-shaft tibial osteotomies of different sizes (0.6-30 mm) were created in eleven Swiss mountain sheep. The bones were stabilized with either a conventional Titanium or stainless-steel locking plate equipped with a Fracture Monitor. Data were continuously collected over the device's lifetime. Conventional radiographs and clinical CT scans were taken longitudinally over the study period. The radiographs were systematically scored and CTs were evaluated for normalized bone volume in the defect. The animals were euthanized after 9 months. The sensor output was correlated with the radiologic parameters. Tissue samples from the device location were histologically examined. Results: The sensors functioned autonomously for 6.5-8.4 months until energy depletion. No macroscopic or microscopic adverse effects from device implantation were observed. The relative implant loads at 4 and 8 weeks post-operation correlated significantly with the radiographic scores and with the normalized bone volume metric. Conclusions: Continuous implant load monitoring appears as a relevant approach to support and objectify fracture healing assessments and carries a strong potential to enable patient-tailored rehabilitation in the future.

A Hyaluronic Acid Hydrogel Loaded with Gentamicin and Vancomycin Successfully Eradicates Chronic Methicillin-Resistant Staphylococcus aureus Orthopedic Infection in a Sheep Model.

Implantable orthopedic devices have had an enormously positive impact on human health; however, despite best practice, patients are prone to developing orthopedic device-related infections (ODRI) that have high treatment failure rates. One barrier to the development of improved treatment options is the lack of an animal model that may serve as a robust preclinical assessment of efficacy. We present a clinically relevant large animal model of chronic methicillin-resistant Staphylococcus aureus (MRSA) ODRI that persists despite current clinical practice in medical and surgical treatment at rates equivalent to clinical observations. Furthermore, we showed that an injectable, thermoresponsive, hyaluronic acid-based hydrogel loaded with gentamicin and vancomycin outperforms current clinical practice treatment in this model, eliminating bacteria from all animals. These results confirm that local antibiotic delivery with an injectable hydrogel can dramatically increase treatment success rates beyond current clinical practice, with efficacy proven in a robust animal model.

An Exopolysaccharide Produced by Bifidobacterium longum 35624® Inhibits Osteoclast Formation via a TLR2-Dependent Mechanism.

The probiotic Bifidobacterium longum subsp. longum 35624® (B. longum 35624®), with its surface exopolysaccharide (EPS624), has previously been demonstrated to induce immunoregulatory responses in the host and may, therefore, be a novel approach to prevent bone loss in inflammatory conditions such as post-menopausal osteoporosis (PMO). The aim of this study was to investigate the effect of EPS624 on osteoclast and osteoblast differentiation and to assess the potential of B. longum 35624® to prevent bone loss in vivo. In vitro cell assays were used to assess the impact of EPS624 on osteoclast and osteoblast differentiation. The potential of two probiotic B. longum 35624® strains, including an EPS-deficient strain, for preventing ovariectomy (Ovx)-induced bone loss was assessed in a murine model. EPS624 prevented osteoclast formation from murine bone marrow precursors under both normal and TNFα-induced inflammatory conditions and modestly increased mineralized matrix deposition in osteogenic cell cultures. However, in the presence of an anti-TLR2 blocking antibody, or in MyD88-/- osteoclast precursors, the inhibitory effect of EPS624 on osteoclast formation was diminished or completely prevented, respectively. Moreover, EPS624 induced IL-10 production in osteoclast precursors in a TLR2-dependent manner, although IL-10 was dispensable in the EPS624-mediated inhibition of osteoclast formation. In addition, EPS624-producing B. longum 35624® partially prevented bone loss in Ovx mice when administered by oral gavage. This study introduced EPS624 as a potential anti-resorptive therapy, although optimal in vivo delivery of the probiotic strain for treating low-grade inflammatory diseases such as PMO remains to be determined.

Butyrate Inhibits Osteoclast Activity In Vitro and Regulates Systemic Inflammation and Bone Healing in a Murine Osteotomy Model Compared to Antibiotic-Treated Mice.

Short-chain fatty acids (SCFAs) produced by the gut microbiota have previously been demonstrated to play a role in numerous chronic inflammatory diseases and to be key mediators in the gut-bone signaling axis. However, the role of SCFAs in bone fracture healing and its impact on systemic inflammation during the regeneration process has not been extensively investigated yet. The aim of this study was to first determine the effects of the SCFA butyrate on key cells involved in fracture healing in vitro, namely, osteoclasts and mesenchymal stromal cells (MSCs), and second, to assess if butyrate supplementation or antibiotic therapy impacts bone healing, systemic immune status, and inflammation levels in a murine osteotomy model. Butyrate significantly reduced osteoclast formation and resorption activity in a dose-dependent manner and displayed a trend for increased calcium deposits in MSC cultures. Numerous genes associated with osteoclast differentiation were differentially expressed in osteoclast precursor cells upon butyrate exposure. In vivo, antibiotic-treated mice showed reduced SCFA levels in the cecum, as well as a distinct gut microbiome composition. Furthermore, circulating proinflammatory TNFα, IL-17a, and IL-17f levels, and bone preserving osteoprotegerin (OPG), were increased in antibiotic-treated mice compared to controls. Antibiotic-treated mice also displayed a trend towards delayed bone healing as revealed by reduced mineral apposition at the defect site and higher circulating levels of the bone turnover marker PINP. Butyrate supplementation resulted in a lower abundance of monocyte/macrophages in the bone marrow, as well as reduced circulating proinflammatory IL-6 levels compared to antibiotic- and control-treated mice. In conclusion, this study supports our hypothesis that SCFAs, in particular butyrate, are important contributors to successful bone healing by modulating key cells involved in fracture healing as well as systemic inflammation and immune responses.

Differences between auto-fluorescence and tetracycline-fluorescence in medication-related osteonecrosis of the jaw-a preclinical proof of concept study in the mini-pig.

OBJECTIVES: Fluorescence-guided bone surgery is a well-established technique in the treatment of medication-related osteonecrosis of the jaw. No histopathological evidence for bone auto-fluorescence is currently available, and thus, any differences from tetracycline-fluorescence remain unclear. Therefore, the goals of this study were to find out if macroscopic and histological differences occur between the auto- and tetracycline-fluorescence in the delineation of viable and necrotic jawbone in the mini-pig. MATERIALS AND METHODS: According to the proof of concept, osteonecrosis was provoked in eight Göttingen minipigs. Pigs were divided into two groups (AF group: no fluorochrome label; TF group: tetracycline label). Delineation of necrosis and viable bone was evaluated in vivo and in vitro macro-/microscopically, correlated to fluorescence properties and compared between the two study groups. RESULTS: No macroscopic and microscopic clinical differences were seen in fluorescence between the AF and TF groups. Macroscopic and microscopic viable bone fluoresced green, whereas necrotic bone showed no or only pale fluorescence in both groups. The auto-fluorescence was attributable to the arrangements and structure of collagen and the cell-filled bone lacunae. CONCLUSION: Neither in vivo nor in vitro macroscopically differences are apparent between the auto-fluorescence and the tetracycline-fluorescence of bone. The auto-fluorescence is attributable to the arrangements and structure of collagen and the cell-filled bone lacunae. Tetracycline-fluorescence is a mixture of tetracycline (at the bone edges with increased bone formation) and large components of auto-fluorescence. CLINICAL RELEVANCE: Because auto-fluorescence is easy to apply, reproducible, and does not rely on the subjective impression of the surgeon, it promises to be an important standardized alternative to tetracycline-labeled MRONJ therapy.

Programable Active Fixator System for Systematic In Vivo Investigation of Bone Healing Processes.

This manuscript introduces a programable active bone fixator system that enables systematic investigation of bone healing processes in a sheep animal model. In contrast to previous systems, this solution combines the ability to precisely control the mechanical conditions acting within a fracture with continuous monitoring of the healing progression and autonomous operation of the system throughout the experiment. The active fixator system was implemented on a double osteotomy model that shields the experimental fracture from the influence of the animal's functional loading. A force sensor was integrated into the fixator to continuously measure stiffness of the repair tissue as an indicator for healing progression. A dedicated control unit was developed that allows programing of different loading protocols which are later executed autonomously by the active fixator. To verify the feasibility of the system, it was implanted in two sheep with different loading protocols, mimicking immediate and delayed weight-bearing, respectively. The implanted devices operated according to the programmed protocols and delivered seamless data over the whole course of the experiment. The in vivo trial confirmed the feasibility of the system. Hence, it can be applied in further preclinical studies to better understand the influence of mechanical conditions on fracture healing.

Development of a novel murine delayed secondary fracture healing in vivo model using periosteal cauterization.

INTRODUCTION: Delayed union and nonunion development remain a major clinical problematic complication during fracture healing, with partially unclear pathophysiology. Incidences range from 5 to 40% in high-risk patients, such as patients with periosteal damage. The periosteum is essential in adequate fracture healing, especially during soft callus formation. In this study, we hypothesize that inducing periosteal damage in a murine bone healing model will result in a novel delayed union model. MATERIALS AND METHODS: A mid-shaft femoral non-critically sized osteotomy was created in skeletally mature C57BL/6 mice and stabilized with a bridging plate. In half of the mice, a thin band of periosteum adjacent to the osteotomy was cauterized. Over 42 days of healing, radiographic, biomechanical, micro-computed tomography and histological analysis was performed to assess the degree of fracture healing. RESULTS: Analysis showed complete secondary fracture healing in the control group without periosteal injury. Whereas the periosteal injury group demonstrated less than half as much maximum callus volume (p < 0.05) and bridging, recovery of stiffness and temporal expression of callus growth and remodelling was delayed by 7-15 days. CONCLUSION: This paper introduces a novel mouse model of delayed union without a critically sized defect and with standardized biomechanical conditions, which enables further investigation into the molecular biological, biomechanical, and biochemical processes involved in (delayed) fracture healing and nonunion development. This model provides a continuum between normal fracture healing and the development of nonunions.

Subchondral screw abutment: does it harm the joint cartilage? An in vivo study on sheep tibiae.

PURPOSE: Subchondral screw abutment in osteosynthesis of joint fractures is an effective method to achieve sufficient screw grip. In this study we investigated if subchondral screw placement is possible without harming the overlying subchondral plate and joint cartilage iatrogenic. MATERIALS AND METHODS: A 3.5-mm conventional steel screw was placed in the tibia of ten sheep in distances between 1 and 7 mm beneath the joint cartilage. After a follow up of two and four months, evaluation of the subchondral bone and joint cartilage was performed by means of a histological osteoarthritis score, HRpQCT imaging and determination of the glycosaminoglycan content in the cartilage. The control group was the contralateral knee of the same animal. RESULTS: Histomorphometric evaluation of the Mankin osteoarthritis score revealed no significant difference compared to the control after two (p = 0.102) and four months (p = 0.429). No correlation between distance of the screw to the cartilage and histological scoring was found (p = 0.658, R2 = 0.04 after two months and p = 0.171, R2 = 0.18 after four months). HRpQCT measurements of the subchondral thickness between screw and cartilage after two (p = 0.05) and four months (p = 0.424) showed no significant difference. Mean glycosaminoglycan content in the treatment group compared to the control after two months (p = 0.25) and four months (p = 0.523) was not significant different. CONCLUSION: In conclusion subchondral screw abutment did not damage the joint cartilage after a two- and four-month follow up in this sheep model.

Evaluation of a press-fit osteochondral poly(ester-urethane) scaffold in a rabbit defect model.

The purpose of this study was to evaluate the impact on osteochondral healing of press-fitted multiphasic osteochondral scaffolds consisting of poly(ester-urethane) (PUR) and hydroxyapatite into a cylindric osteochondral defect in the distal non-weight bearing femoral trochlear ridge of the rabbit. Two scaffolds were investigated, one with and one without an intermediate microporous membrane between the cartilage and the bone compartment of the scaffold. A control group without a scaffold placed into the defect was included. After 12 weeks macroscopic and histomorphological analyses were performed. The scaffold was easily press-fitted and provided a stable matrix for tissue repair. The membrane did not demonstrate a detrimental effect on tissue healing compared with the scaffold without membrane. However, the control group had statistically superior healing as reflected by histological differences in the cartilage and subchondral bone compartment between control group and each scaffold group. A more detailed analysis revealed that the difference was localized in the bone compartment healing. The present study demonstrates that an elastomeric PUR scaffold can easily be press-fitted into an osteochondral defect and provides a stable matrix for tissue repair. However, the multi-phasic scaffold did not provide a clear advantage for tissue healing. Future investigations should refine especially the bone phase of the implant to increase its stiffness, biocompatibility and osteoconductive activity. A more precise fabrication technique would be necessary for the matching of tissue organisation.

Restoring implant fixation strength in osteoporotic bone with a hydrogel locally delivering zoledronic acid and bone morphogenetic protein 2. A longitudinal in vivo microCT study in rats.

Osteoporosis poses a major public health challenge, and it is characterized by low bone mass, deterioration of the microarchitecture of bone tissue, causing a consequent increase in bone fragility and susceptibility to fractures and complicating bone fixation, particularly screw implantation. In the present study, our aim was to improve implant stability in osteoporotic bone using a thermoresponsive hyaluronan hydrogel (HA-pNIPAM) to locally deliver the bisphosphonate zoledronic acid (ZOL) to prevent bone resorption and bone morphogenetic protein 2 (BMP2) to induce bone formation. Adult female Wistar rats (n = 36) were divided into 2 treatment groups: one group of SHAM-operated animals and another group that received an ovariectomy (OVX) to induce an osteoporotic state. All animals received a polyetheretherketone (PEEK) screw in the proximal tibia. In addition, subgroups of SHAM or OVX animals received either the HA-pNIPAM hydrogel without or with ZOL/BMP2, placed into the defect site prior to screw implantation. Periprosthetic bone and implant fixation were monitored using longitudinal in vivo microCT scanning post-operatively and at 3, 6, 9, 14, 20 and 28 days. Histological assessment was performed post-mortem. Our data showed that pure hydrogel has no impact of implant fixation The ZOL/BMP2-hydrogel significantly increased bone-implant contact and peri-implant bone fraction, primarily through reduced resorption. STATEMENT OF CLINICAL SIGNIFICANCE: Local delivery of ZOL and BMP2 using a biocompatible hydrogel improved implant stability in osteoporotic bone. This approach could constitute a potent alternative to systemic drug administration and may be useful in avoiding implant loosening in clinical settings.

Implant retention in a rabbit model of fracture-related infection.

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. Methods: Staphylococcus aureus was inoculated into a humeral osteotomy in 17 rabbits after plate osteosynthesis. Infection developed for one week (early group, n = 6) or four weeks (delayed group, n = 6) before DAIR (systemic antibiotics: two weeks, nafcillin + rifampin; four weeks, levofloxacin + rifampin). A control group (n = 5) received revision surgery after four weeks without antibiotics. Bacteriology of humerus, soft-tissue, and implants was performed seven weeks after revision surgery. Bone healing was assessed using a modified radiological union scale in tibial fractures (mRUST). Results: Greater bacterial burden in the early group compared to the delayed and control groups at revision surgery indicates a retraction of the infection from one to four weeks. Infection was cleared in all animals in the early and delayed groups at euthanasia, but not in the control group. Osteotomies healed in the early group, but bone healing was significantly compromised in the delayed and control groups. Conclusion: The duration of the infection from one to four weeks does not impact the success of infection clearance in this model. Bone healing, however, is impaired as the duration of the infection increases.

Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice.

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Δpvl), or complemented mutant (Δpvl+pvl). Three days post-surgery, Δpvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Δpvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR+ monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Δpvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

Peri-anesthetic hypothermia in rodents: A factor to consider for accurate and reproducible outcomes in orthopedic device-related infection studies.

Orthopedic device-related infection (ODRI) preclinical models are widely used in translational research. Most ODRI 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 ODRI 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 (one-way MANCOVA) 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 colony-forming unit (CFU) counts, and having controlled for the study groups including treatments received, duration of surgery and anesthesia, and study period. The results showed a significant effect of peri-anesthetic hypothermia on the post-mortem combined CFU 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 CFU 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.

The absence of immediate stimulation delays bone healing.

AIM: Secondary bone healing requires an adequate level of mechanical stimulation expressed by the extent of interfragmentary motion in the fracture. However, there is no consensus about when the mechanical stimulation should be initiated to ensure a timely healing response. Therefore, this study aims to compare the effect of the immediate and delayed application of mechanical stimulation in a large animal model. METHODS: Twelve Swiss White Alpine sheep underwent partial osteotomy of a tibia that was stabilised with an active fixator inducing well-controlled mechanical stimulation. Animals were randomly assigned into two groups with different stimulation protocols. The immediate group received daily stimulation (1000 cycles/day) from the first day post-operation, while in the delayed group, stimulation began only on the 22nd day post-operation. Healing progression was evaluated daily by measuring the in vivo stiffness of the repair tissue and by quantifying callus area on weekly radiographs. All animals were euthanised five weeks post-op. Post-mortem callus volume was determined from high-resolution computer tomography (HRCT). RESULTS: Fracture stiffness (p < 0.05) and callus area (p < 0.01) were significantly larger for the immediate group compared to the delayed stimulation group. In addition, the callus volume measured on the post-mortem HRCT showed 319 % greater callus volume for the immediate stimulation group (p < 0.01). CONCLUSIONS: This study demonstrates that a delay in the onset of mechanical stimulation retards fracture callus development and that mechanical stimulation already applied in the early post-op phase promotes bone healing.