Evaluating rearing behaviour as a model-specific pain indicator in mouse osteotomy models.

To assess pain in mouse models of bone fractures, currently applied assessment batteries use combinations of clinical signs with spontaneous behaviours and model-specific behaviours, including walking and weight-bearing behaviour. Rearing behaviour - an upright position on the hindlimbs - has a motivational and an ambulatory component. Thus, rearing behaviour might have the potential to be an indicator for model-specific pain in mouse fracture models. To date, the assessment of rearing behaviour in bone fracture models using mice is only scarcely described. In this study, we aimed to determine whether the duration of rearing behaviour is affected by osteotomy of the femur in male and female C57BL/6N mice with external fixation (rigid vs. flexible) and could be an additional sign for model-specific pain, such as the presence of limping. Rearing duration was significantly decreased after osteotomy in male and female mice at 24 h, 48 h and 72 h, but was not affected by anaesthesia/analgesia alone. In male mice, the relative rearing duration increased over 72 h (both fixations) and at 10 days in the rigid fixation group but remained significantly lower in the flexible fixation group. In contrast, in female mice, no increase in rearing duration was observed within 72 h and at 10 days post-osteotomy, independent of the fixation. We did not identify any association between relative rearing time and presence or absence of limping. In summary, our results do not provide sufficient evidence that altered rearing behaviour might be an indicative sign for pain in this model.

Impact of sensory neuropeptide deficiency on behavioral patterns and gait in a murine surgical osteoarthritis model.

Substance P (SP) and a calcitonin-related gene alpha (αCGRP-/-) are implicated in musculoskeletal pain perception and were shown to have different effects on the pathogenesis of osteoarthritis (OA). However, it has not been investigated, whether deficiency for SP or αCGRP impacts pain-related behavior and well-being as well as gait during development of experimental OA. We induced OA in the right knee of wild-type (WT) mice and mice either deficient for SP (tachykinin 1, Tac-1) or αCGRP (male, n = 8 per genotype) by destabilizing the medial meniscus (DMM). We monitored body weight and food and water intake as indicators of wellbeing, determined nest building and composite pain score, and performed CatWalk gait analysis over 12 weeks. Cartilage degeneration was determined by OARSI scoring. The 12-week post-DMM, cartilage degradation in the medial compartment was significantly reduced in Tac1-/- mice compared to the WT and to αCGRP-/- mice, coinciding with highest unloading of the operated limb in Tac1-/-. Behavioral and gait analysis revealed only minor differences between the genotypes. Paw print area was most prominently reduced in Tac1-/- over the observation period; at 12 weeks, we found a significant reduction in normalized print area in Tac1-/- compared to presurgery and to the WT at the same time-point. Calculated weight bearing was significantly reduced only in Tac1-/-. Overall, we observed minor impact of DMM on gait and behavior in the present study. The reduced cartilage damage in the absence of SP might be in part due to reduced loading, however, the mechanism is not clear yet.

Endothelial SMAD1/5 signaling couples angiogenesis to osteogenesis in juvenile bone.

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone formation in part by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered trabecular and cortical bone formation. SMAD1/5 depletion induced excessive sprouting and disrupting the morphology of the metaphyseal vessels, with impaired anastomotic loop formation at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long-term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops and elevated vascular permeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics in juvenile mouse bone.

Endothelial SMAD1/5 signaling couples angiogenesis to osteogenesis during long bone growth.

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone development by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered cancellous and cortical bone formation. SMAD1/5 depletion induced excessive sprouting, disrupting the columnar structure of the metaphyseal vessels and impaired anastomotic loop morphogenesis at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops, featuring elevated endomucin expression, ectopic tip cell formation, and hyperpermeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics during bone growth.

A buprenorphine depot formulation provides effective sustained post-surgical analgesia for 72 h in mouse femoral fracture models.

Adequate pain management is essential for ethical and scientific reasons in animal experiments and should completely cover the period of expected pain without the need for frequent re-application. However, current depot formulations of Buprenorphine are only available in the USA and have limited duration of action. Recently, a new microparticulate Buprenorphine formulation (BUP-Depot) for sustained release has been developed as a potential future alternative to standard formulations available in Europe. Pharmacokinetics indicate a possible effectiveness for about 72 h. Here, we investigated whether the administration of the BUP-Depot ensures continuous and sufficient analgesia in two mouse fracture models (femoral osteotomy) and could, therefore, serve as a potent alternative to the application of Tramadol via the drinking water. Both protocols were examined for analgesic effectiveness, side effects on experimental readout, and effects on fracture healing outcomes in male and female C57BL/6N mice. The BUP-Depot provided effective analgesia for 72 h, comparable to the effectiveness of Tramadol in the drinking water. Fracture healing outcome was not different between analgesic regimes. The availability of a Buprenorphine depot formulation for rodents in Europe would be a beneficial addition for extended pain relief in mice, thereby increasing animal welfare.

Local immune cell contributions to fracture healing in aged individuals - A novel role for interleukin 22.

With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences.

MIF does only marginally enhance the pro-regenerative capacities of DFO in a mouse-osteotomy-model of compromised bone healing conditions.

The initial phase of fracture healing is crucial for the success of bone regeneration and is characterized by an inflammatory milieu and low oxygen tension (hypoxia). Negative interference with or prolongation of this fine-tuned initiation phase will ultimately lead to a delayed or incomplete healing such as non-unions which then requires an effective and gentle therapeutic intervention. Common reasons include a dysregulated immune response, immunosuppression or a failure in cellular adaptation to the inflammatory hypoxic milieu of the fracture gap and a reduction in vascularizing capacity by environmental noxious agents (e.g. rheumatoid arthritis or smoking). The hypoxia-inducible factor (HIF)-1α is responsible for the cellular adaptation to hypoxia, activating angiogenesis and supporting cell attraction and migration to the fracture gap. Here, we hypothesized that stabilizing HIF-1α could be a cost-effective and low-risk prevention strategy for fracture healing disorders. Therefore, we combined a well-known HIF-stabilizer - deferoxamine (DFO) - and a less known HIF-enhancer - macrophage migration inhibitory factor (MIF) - to synergistically induce improved fracture healing. Stabilization of HIF-1α enhanced calcification and osteogenic differentiation of MSCs in vitro. In vivo, only the application of DFO without MIF during the initial healing phase increased callus mineralization and vessel formation in a preclinical mouse-osteotomy-model modified to display a compromised healing. Although we did not find a synergistically effect of MIF when added to DFO, our findings provide additional support for a preventive strategy towards bone healing disorders in patients with a higher risk by accelerating fracture healing using DFO to stabilize HIF-1α.

Systematic review on the reporting accuracy of experimental details in publications using mouse femoral fracture models.

The outcomes of animal experiments can be influenced by a variety of factors. Thus, precise reporting is necessary to provide reliable and reproducible data. Initiatives such as the ARRIVE guidelines have been enrolled during the last decade to provide a road map for sufficient reporting. To understand the sophisticated process of bone regeneration and to develop new therapeutic strategies, small rodents, especially mice, are frequently used in bone healing research. Since many factors might influence the results from those studies, we performed a systematic literature search from 2010 to 2019 to identify studies involving mouse femoral fracture models (stable fixation) and evaluated the reporting of general and model-specific experimental details. 254 pre-selected publications were systematically analyzed, showing a high reporting accuracy for the used mouse strain, the age or developmental stage and sex of mice as well as model-specific information on fixation methods and fracturing procedures. However, reporting was more often insufficient in terms of mouse substrains and genetic backgrounds of genetically modified mice, body weight, hygiene monitoring/immune status of the animal, anesthesia, and analgesia. Consistent and reliable reporting of experimental variables in mouse fracture surgeries will improve scientific quality, enhance animal welfare, and foster translation into the clinic.