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.

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.

Combining adhesive and nonadhesive injectable hydrogels for intervertebral disc repair in an ovine discectomy model.

Background: Intervertebral disc (IVD) disorders (e.g., herniation) directly contribute to back pain, which is a leading cause of global disability. Next-generation treatments for IVD herniation need advanced preclinical testing to evaluate their ability to repair large defects, prevent reherniation, and limit progressive degeneration. This study tested whether experimental, injectable, and nonbioactive biomaterials could slow IVD degeneration in an ovine discectomy model. Methods: Ten skeletally mature sheep (4-5.5 years) experienced partial discectomy injury with cruciate-style annulus fibrosus (AF) defects and 0.1 g nucleus pulposus (NP) removal in the L1-L2, L2-L3, and L3-L4 lumbar IVDs. L4-L5 IVDs were Intact controls. IVD injury levels received: (1) no treatment (Injury), (2) poly (ethylene glycol) diacrylate (PEGDA), (3) genipin-crosslinked fibrin (FibGen), (4) carboxymethylcellulose-methylcellulose (C-MC), or (5) C-MC and FibGen (FibGen + C-MC). Animals healed for 12 weeks, then IVDs were assessed using computed tomography (CT), magnetic resonance (MR) imaging, and histopathology. Results: All repaired IVDs retained ~90% of their preoperative disc height and showed minor degenerative changes by Pfirrmann grading. All repairs had similar disc height loss and Pfirrmann grade as Injury IVDs. Adhesive AF sealants (i.e., PEGDA and FibGen) did not herniate, although repair caused local endplate (EP) changes and inflammation. NP repair biomaterials (i.e., C-MC) and combination repair (i.e., FibGen + C-MC) exhibited lower levels of degeneration, less EP damage, and less severe inflammation; however, C-MC showed signs of herniation via biomaterial expulsion. Conclusions: All repair IVDs were noninferior to Injury IVDs by IVD height loss and Pfirrmann grade. C-MC and FibGen + C-MC IVDs had the best outcomes, and may be appropriate for enhancement with bioactive factors (e.g., cells, growth factors, and miRNAs). Such bioactive factors appear to be necessary to prevent injury-induced IVD degeneration. Application of AF sealants alone (i.e., PEGDA and FibGen) resulted in EP damage and inflammation, particularly for PEGDA IVDs, suggesting further material refinements are needed.

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.

Alginate microbeads and hydrogels delivering meropenem and bacteriophages to treat Pseudomonas aeruginosa fracture-related infections.

Bacteriophage (phage) therapy has shown promise in treating fracture-related infection (FRI); however, questions remain regarding phage efficacy against biofilms, phage-antibiotic interaction, administration routes and dosing, and the development of phage resistance. The goal of this study was to develop a dual antibiotic-phage delivery system containing hydrogel and alginate microbeads loaded with a phage cocktail plus meropenem and evaluate efficacy against muti-drug resistant Pseudomonas aeruginosa. Two phages (FJK.R9-30 and MK.R3-15) displayed enhanced antibiotic activity against P. aeruginosa biofilms when tested in combination with meropenem. The antimicrobial activity of both antibiotic and phage was retained for eight days at 37 °C in dual phage and antibiotic loaded hydrogel with microbeads (PA-HM). In a mouse FRI model, phages were recovered from all tissues within all treatment groups receiving dual PA-HM. Moreover, animals that received the dual PA-HM either with or without systemic antibiotics had less incidence of phage resistance and less serum neutralization compared to phages in saline. The dual PA-HM could reduce bacterial load in soft tissue when combined with systemic antibiotics, although the infection was not eradicated. The use of alginate microbeads and injectable hydrogel for controlled release of phages and antibiotics, leads to the reduced development of phage resistance and lower exposure to the adaptive immune system, which highlights the translational potential of the dual PA-HM. However, further optimization of phage therapy and its delivery system is necessary to achieve higher bacterial killing activity in vivo in the future.

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.

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.

Arginine concentration in arterial vs venous blood in a bleomycin-induced lung inflammation model in mice.

Pneumonia, always a major malady, became the main public health and economic disaster of historical proportions with the COVID-19 pandemic. This study was based on a premise that pathology of lung metabolism in inflammation may have features invariant to the nature of the underlying cause. Amino acid uptake by the lungs was measured from plasma samples collected pre-terminally from a carotid artery and vena cava in mice with bleomycin-induced lung inflammation (N = 10) and compared to controls treated with saline instillation (N = 6). In the control group, the difference in concentrations between the arterial and venous blood of the 19 amino acids measured reached the level of statistical significance only for arginine (-10.7%, p = 0.0372) and phenylalanine (+5.5%, p = 0.0266). In the bleomycin group, 11 amino acids had significantly lower concentrations in the arterial blood. Arginine concentration was decreased by 21.1% (p<0.0001) and only that of citrulline was significantly increased (by 20.1%, p = 0.0002). Global Arginine Bioavailability Ratio was decreased in arterial blood by 19.5% (p = 0.0305) in the saline group and by 30.4% (p<0.0001) in the bleomycin group. Production of nitric oxide (NO) and citrulline from arginine by the inducible nitric oxide synthase (iNOS) is greatly increased in the immune system's response to lung injury. Deprived of arginine, the endothelial cells downstream may fail to provide enough NO to prevent the activation of thrombocytes. Thrombotic-related vascular dysfunction is a defining characteristic of pneumonia, including COVID-19. This experiment lends further support to arginine replacement as adjuvant therapy in pneumonia.

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.

Homing of vertebral-delivered mesenchymal stromal cells for degenerative intervertebral discs repair - an in vivo proof-of-concept study.

Introduction: Cell transplantation shows promising results for intervertebral disc (IVD) repair, however, contemporary strategies present concerns regarding needle puncture damage, cell retention, and straining the limited nutrient availability. Mesenchymal stromal cell (MSC) homing is a natural mechanism of long-distance cellular migration to sites of damage and regeneration. Previous ex vivo studies have confirmed the potential of MSC to migrate over the endplate and enhance IVD-matrix production. In this study, we aimed to exploit this mechanism to engender IVD repair in a rat disc degeneration model. Methods: Female Sprague Dawley rats were subjected to coccygeal disc degeneration through nucleus pulposus (NP) aspiration. In part 1; MSC or saline was transplanted into the vertebrae neighboring healthy or degenerative IVD subjected to irradiation or left untouched, and the ability to maintain the IVD integrity for 2 and 4 weeks was assessed by disc height index (DHI) and histology. For part 2, ubiquitously GFP expressing MSC were transplanted either intradiscally or vertebrally, and regenerative outcomes were compared at days 1, 5, and 14 post-transplantation. Moreover, the homing potential from vertebrae to IVD of the GFP+ MSC was assessed through cryosection mediated immunohistochemistry. Results: Part 1 of the study revealed significantly improved maintenance of DHI for IVD vertebrally receiving MSC. Moreover, histological observations revealed a trend of IVD integrity maintenance. Part 2 of the study highlighted the enhanced DHI and matrix integrity for discs receiving MSC vertebrally compared with intradiscal injection. Moreover, GFP rates highlighted MSC migration and integration in the IVD at similar rates as the intradiscally treated cohort. Conclusion: Vertebrally transplanted MSC had a beneficial effect on the degenerative cascade in their neighboring IVD, and thus potentially present an alternative administration strategy. Further investigation will be needed to determine the long-term effects, elucidate the role of cellular homing versus paracrine signaling, and validate our observations on a large animal model.

Surgical technique and comparison of autologous cancellous bone grafts from various donor sites in rats.

Autologous cancellous bone graft is the gold standard in large bone defect repair. However, studies using autologous bone grafting in rats are rare. To determine the feasibility of autologous cancellous bone graft harvest from different anatomical donor sites (humerus, ilium, femur, tibia, and tail vertebrae) in rats and compare their suitability as donor sites, a total of 13 freshly euthanized rats were used to describe the surgical technique, determine the cancellous bone volume and microstructure, and compare the cancellous bone collected quantitatively and qualitatively. It was feasible to harvest cancellous bone grafts from all five anatomical sites with the humerus and tail being more surgically challenging. The microstructural analysis using micro-computed tomography 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 weight and volume did not differ between the donor sites. All donor sites apart from the femur yielded primary osteogenic cells confirmed by the presence of alkaline phosphatase and Alizarin Red S stain. Bone samples from the iliac crest showed the most consistent outgrowth of osteoprogenitor cells. In conclusion, the tibia and iliac crest may be the most favorable 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.

Standard in vitro evaluations of engineered bone substitutes are not sufficient to predict in vivo preclinical model outcomes.

Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non-unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, human bone marrow - derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with relevant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the second and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1rnu rats for 8 weeks or cell free in an immunocompetent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced upregulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone formation. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies. STATEMENT OF SIGNIFICANCE: Preclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor correlation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm.

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.

Protocol for a systematic review of good surgical practice guidelines for experimental rodent surgery.

Objective: Surgery is an integral part of many experimental studies. Aseptic and minimal invasive surgical technique and optimal perioperative and post-operative care are prerequisites to achieve surgical success and best possible animal welfare outcomes. Good surgical practice cannot only improve the animal's postoperative recovery, but also study outcome and validity. There seems to be a lack of implementation of good surgical practice during rodent surgery. The aim of this systematic review is to identify, critically evaluate and compare the currently recommended standards and underlying guidelines for rodent surgery-and finally to compile a comprehensive guideline of good surgical practice for rodent surgery. Search strategy: PubMed, Embase and Web of Science were searched to identify guidelines published in peer-reviewed journals. To identify grey literature and unpublished guidelines, we will perform a Google search for published guidelines and search laboratory animal sciences books for relevant book chapters. Additionally, we will conduct a survey among animal researchers enquiring about the guidelines they use. Screening and study selection: For publications retrieved by the systematic search, unique references are screened by two reviewers, first for eligibility based on title and abstract and subsequently for final inclusion based on full text. Eligibility of books is based on title and content, final inclusion based on chapter full text. Guidelines are either retrieved by Google searches or a survey. Google searches will be conducted by at least four of the authors. Thereafter, guidelines will be screened by two of the authors. Data extraction and synthesis: We will extract data from publications, book chapters and guidelines. Based on the extracted data, we will perform a descriptive synthesis of the bibliographical details, guideline development and endorsement, and the prevalence of individual recommendations, including subgroup analysis of the guidance per continent or country and differences between peer-reviewed versus non-peer-reviewed guidance.

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.

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.

Interleukin-1 receptor antagonist enhances the therapeutic efficacy of a low dose of rhBMP-2 in a weight-bearing rat femoral defect model.

In the clinical treatment of fractures, rhBMP-2 administration is associated with a well-established profile of side-effects, including osteolysis and ectopic bone formation, which are driven by pro-inflammatory processes triggered by the use of high doses. Immunomodulatory strategies could minimize the incidence of side-effects by enabling the use of lower, and safer, rhBMP-2 doses. This study investigated whether interleukin-1 receptor antagonist (IL-1Ra) can enhance the therapeutic efficacy of a low dose of rhBMP-2 in a weight-bearing femoral fracture healing model. Exogenous IL-1Ra, in combination with rhBMP-2, was delivered using a collagen-hydroxyapatite scaffold (CHA) to attenuate IL-1ß produced in response to fracture. Femoral defects were treated with CHA scaffolds alone, or loaded with IL-1Ra (2.5 µg), rhBMP-2 (1 µg), IL-1Ra (2.5 µg) in combination with rhBMP-2 (1 µg). Bone healing was assessed over 14 weeks in comparison to control groups, empty defect, and a higher dose of rhBMP-2 (5 µg), which were recently demonstrated to lead to non-union, and successful bridging of the defect, respectively. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, at both week 4 and 6, compared to a low dose of rhBMP-2 alone. By 14 weeks, the combination of IL-1Ra and a rhBMP-2 promoted full bridging of femurs, which were 3-fold more mechanically reliable compared to the femurs treated with a low dose of rhBMP-2 alone. Taken together, this study demonstrates that IL-1Ra can significantly enhance femoral bone healing when used in combination with a low dose of rhBMP-2. STATEMENT OF SIGNIFICANCE: Enabling the use of lower and safer doses of rhBMP-2, a potent inducer of bone formation, is of clinical relevance in orthopaedic medicine. In this study, the immunomodulatory interleukin-1 receptor antagonist (IL-1Ra) was investigated for its capacity to enhance the therapeutic efficacy of rhBMP-2 when used at lower doses in a weight-bearing femoral fracture healing model. The combination of IL-1Ra and rhBMP-2 led to significantly faster early bone formation, and resulted in more mechanically reliable healed femurs, compared to a low dose of rhBMP-2 alone. This demonstrates for the first time in a rat long bone healing model that IL-1Ra can significantly enhance bone healing when used in combination with a low dose of rhBMP-2.

Comparison and optimization of sheep in vivo intervertebral disc injury model.

Background: The current standard of care for intervertebral disc (IVD) herniation, surgical discectomy, does not repair annulus fibrosus (AF) defects, which is partly due to the lack of effective methods to do so and is why new repair strategies are widely investigated and tested preclinically. There is a need to develop a standardized IVD injury model in large animals to enable comparison and interpretation across preclinical study results. The purpose of this study was to compare in vivo IVD injury models in sheep to determine which annulus fibrosus (AF) defect type combined with partial nucleus pulposus (NP) removal would better mimic degenerative human spinal pathologies. Methods: Six skeletally mature sheep were randomly assigned to one of the two observation periods (1 and 3 months) and underwent creation of 3 different AF defect types (slit, cruciate, and box-cut AF defects) in conjunction with 0.1 g NP removal in three lumbar levels using a lateral retroperitoneal surgical approach. The spine was monitored by clinical CT scans pre- and postoperatively, at 2 weeks and euthanasia, and by magnetic resonance imaging (MRI) and histology after euthanasia to determine the severity of degeneration (disc height loss, Pfirrmann grading, semiquantitative histopathology grading). Results: All AF defects led to significant degenerative changes detectable on CT and MR images, produced bulging of disc tissue without disc herniation and led to degenerative and inflammatory histopathological changes. However, AF defects were not equal in terms of disc height loss at 3 months postoperatively; the cruciate and box-cut AF defects showed significantly decreased disc height compared to their preoperative height, with the box-cut defect creating the greatest disc height loss, while the slit AF defect showed restoration of normal preoperative disc height. Conclusions: The tested IVD injury models do not all generate comparable disc degeneration but can be considered suitable IVD injury models to investigate new treatments. Results of the current study clearly indicate that slit AF defect should be avoided if disc height is used as one of the main outcomes; additional confirmatory studies may be warranted to generalize this finding.

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.