Protocol for preparing formalin-fixed paraffin-embedded musculoskeletal tissue samples from mice for spatial transcriptomics.

Here, we present a protocol for using spatial transcriptomics in bone and multi-tissue musculoskeletal formalin-fixed paraffin-embedded (FFPE) samples from mice. We describe steps for tissue harvesting, sample preparation, paraffin embedding, and FFPE sample selection. We detail procedures for sectioning and placement on spatial slides prior to imaging, decrosslinking, library preparation, and final analyses of the sequencing data. The complete protocol takes ca. 18 days for mouse femora with adjacent muscle; of this time, >50% is required for mineralized tissue decalcification. For complete details on the use and execution of this protocol, please refer to Wehrle et al.1 and Mathavan et al.2.

Mouse models of accelerated aging in musculoskeletal research for assessing frailty, sarcopenia, and osteoporosis - A review.

Musculoskeletal aging encompasses the decline in bone and muscle function, leading to conditions such as frailty, osteoporosis, and sarcopenia. Unraveling the underlying molecular mechanisms and developing effective treatments are crucial for improving the quality of life for those affected. In this context, accelerated aging models offer valuable insights into these conditions by displaying the hallmarks of human aging. Herein, this review focuses on relevant mouse models of musculoskeletal aging with particular emphasis on frailty, osteoporosis, and sarcopenia. Among the discussed models, PolgA mice in particular exhibit hallmarks of musculoskeletal aging, presenting early-onset frailty, as well as reduced bone and muscle mass that closely resemble human musculoskeletal aging. Ultimately, findings from these models hold promise for advancing interventions targeted at age-related musculoskeletal disorders, effectively addressing the challenges posed by musculoskeletal aging and associated conditions in humans.

Trabecular bone remodeling in the aging mouse: A micro-multiphysics agent-based in silico model using single-cell mechanomics.

Bone remodeling is regulated by the interaction between different cells and tissues across many spatial and temporal scales. Notably, in silico models are regarded as powerful tools to further understand the signaling pathways that regulate this intricate spatial cellular interplay. To this end, we have established a 3D multiscale micro-multiphysics agent-based (micro-MPA) in silico model of trabecular bone remodeling using longitudinal in vivo data from the sixth caudal vertebra (CV6) of PolgA(D257A/D257A) mice, a mouse model of premature aging. Our in silico model includes a variety of cells as single agents and receptor-ligand kinetics, mechanomics, diffusion and decay of cytokines which regulate the cells' behavior. We highlighted its capabilities by simulating trabecular bone remodeling in the CV6 of five mice over 4 weeks and we evaluated the static and dynamic morphometry of the trabecular bone microarchitecture. Based on the progression of the average trabecular bone volume fraction (BV/TV), we identified a configuration of the model parameters to simulate homeostatic trabecular bone remodeling, here named basal. Crucially, we also produced anabolic, anti-anabolic, catabolic and anti-catabolic responses with an increase or decrease by one standard deviation in the levels of osteoprotegerin (OPG), receptor activator of nuclear factor kB ligand (RANKL), and sclerostin (Scl) produced by the osteocytes. Our results showed that changes in the levels of OPG and RANKL were positively and negatively correlated with the BV/TV values after 4 weeks in comparison to basal levels, respectively. Conversely, changes in Scl levels produced small fluctuations in BV/TV in comparison to the basal state. From these results, Scl was deemed to be the main driver of equilibrium while RANKL and OPG were shown to be involved in changes in bone volume fraction with potential relevance for age-related bone features. Ultimately, this micro-MPA model provides valuable insights into how cells respond to their local mechanical environment and can help to identify critical pathways affected by degenerative conditions and ageing.

Prognostic and therapeutic potential of microRNAs for fracture healing processes and non-union fractures: A systematic review.

BACKGROUND: Approximately 10% of all bone fractures result in delayed fracture healing or non-union; thus, the identification of biomarkers and prognostic factors is of great clinical interest. MicroRNAs (miRNAs) are known to be involved in the regulation of the bone healing process and may serve as functional markers for fracture healing. AIMS AND METHODS: This systematic review aimed to identify common miRNAs involved in fracture healing or non-union fractures using a qualitative approach. A systematic literature search was performed with the keywords 'miRNA and fracture healing' and 'miRNA and non-union fracture'. Any original article investigating miRNAs in fracture healing or non-union fractures was screened. Eventually, 82 studies were included in the qualitative analysis for 'miRNA and fracture healing', while 19 were selected for the 'miRNA and fracture non-union' category. RESULTS AND CONCLUSIONS: Out of 151 miRNAs, miR-21, miR-140 and miR-214 were the most investigated miRNAs in fracture healing in general. miR-31-5p, miR-221 and miR-451-5p were identified to be regulated specifically in non-union fractures. Large heterogeneity was detected between studies investigating the role of miRNAs in fracture healing or non-union in terms of patient population, sample types and models used. Nonetheless, our approach identified some miRNAs with the potential to serve as biomarkers for non-union fractures, including miR-31-5p, miR-221 and miR-451-5p. We provide a discussion of involved pathways and suggest on alignment of future research in the field.

Tissue-Level Regeneration and Remodeling Dynamics are Driven by Mechanical Stimuli in the Microenvironment in a Post-Bridging Loaded Femur Defect Healing Model in Mice.

Bone healing and remodeling are mechanically driven processes. While the generalized response to mechanical stimulation in bone is well-understood, much less is known about the mechanobiology-regulating tissue-scale bone formation and resorption during the reparative and remodeling phases of fracture healing. In this study, we combined computational approaches in the form of finite element analysis and experimental approaches by using a loaded femoral defect model in mice to investigate the role of mechanical stimulation in the microenvironment of bone. Specifically, we used longitudinal micro-computed tomography to observe temporal changes in bone at different densities and micro-finite element analysis to map the mechanics of the microenvironment to tissue-scale formation, quiescence (no change in bone presence between time points), and resorption dynamics in the late reparative and remodeling phases (post bridging). Increasing levels of effective strain led to increasing conditional probability of bone formation, while decreasing levels of effective strain led to increasing probability of bone resorption. In addition, the analysis of mineralization dynamics showed both a temporal and effective strain level-dependent behavior. A logarithmic-like response was displayed, where the conditional probability of bone formation or resorption increased rapidly and plateaued or fell rapidly and plateaued as mechanical strain increased.

Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging.

Fracture healing is regulated by mechanical loading. Understanding the underlying mechanisms during the different healing phases is required for targeted mechanical intervention therapies. Here, the influence of individualized cyclic mechanical loading on the remodelling phase of fracture healing was assessed in a non-critical-sized mouse femur defect model. After bridging of the defect, a loading group (n = 10) received individualized cyclic mechanical loading (8-16 N, 10 Hz, 5 min, 3 × /week) based on computed strain distribution in the mineralized callus using animal-specific real-time micro-finite element analysis with 2D/3D visualizations and strain histograms. Controls (n = 10) received 0 N treatment at the same post-operative time-points. By registration of consecutive scans, structural and dynamic callus morphometric parameters were followed in three callus sub-volumes and the adjacent cortex showing that the remodelling phase of fracture healing is highly responsive to cyclic mechanical loading with changes in dynamic parameters leading to significantly larger formation of mineralized callus and higher degree of mineralization. Loading-mediated maintenance of callus remodelling was associated with distinct effects on Wnt-signalling-associated molecular targets Sclerostin and RANKL in callus sub-regions and the adjacent cortex (n = 1/group). Given these distinct local protein expression patterns induced by cyclic mechanical loading during callus remodelling, the femur defect loading model with individualized load application seems suitable to further understand the local spatio-temporal mechano-molecular regulation of the different fracture healing phases.

Real-time finite element analysis allows homogenization of tissue scale strains and reduces variance in a mouse defect healing model.

Mechanical loading allows both investigation into the mechano-regulation of fracture healing as well as interventions to improve fracture-healing outcomes such as delayed healing or non-unions. However, loading is seldom individualised or even targeted to an effective mechanical stimulus level within the bone tissue. In this study, we use micro-finite element analysis to demonstrate the result of using a constant loading assumption for all mouse femurs in a given group. We then contrast this with the application of an adaptive loading approach, denoted real time Finite Element adaptation, in which micro-computed tomography images provide the basis for micro-FE based simulations and the resulting strains are manipulated and targeted to a reference distribution. Using this approach, we demonstrate that individualised femoral loading leads to a better-specified strain distribution and lower variance in tissue mechanical stimulus across all mice, both longitudinally and cross-sectionally, while making sure that no overloading is occurring leading to refracture of the femur bones.

3D Bioprinting of Human Tissues: Biofabrication, Bioinks, and Bioreactors.

The field of tissue engineering has progressed tremendously over the past few decades in its ability to fabricate functional tissue substitutes for regenerative medicine and pharmaceutical research. Conventional scaffold-based approaches are limited in their capacity to produce constructs with the functionality and complexity of native tissue. Three-dimensional (3D) bioprinting offers exciting prospects for scaffolds fabrication, as it allows precise placement of cells, biochemical factors, and biomaterials in a layer-by-layer process. Compared with traditional scaffold fabrication approaches, 3D bioprinting is better to mimic the complex microstructures of biological tissues and accurately control the distribution of cells. Here, we describe recent technological advances in bio-fabrication focusing on 3D bioprinting processes for tissue engineering from data processing to bioprinting, mainly inkjet, laser, and extrusion-based technique. We then review the associated bioink formulation for 3D bioprinting of human tissues, including biomaterials, cells, and growth factors selection. The key bioink properties for successful bioprinting of human tissue were summarized. After bioprinting, the cells are generally devoid of any exposure to fluid mechanical cues, such as fluid shear stress, tension, and compression, which are crucial for tissue development and function in health and disease. The bioreactor can serve as a simulator to aid in the development of engineering human tissues from in vitro maturation of 3D cell-laden scaffolds. We then describe some of the most common bioreactors found in the engineering of several functional tissues, such as bone, cartilage, and cardiovascular applications. In the end, we conclude with a brief insight into present limitations and future developments on the application of 3D bioprinting and bioreactor systems for engineering human tissue.

Spatio-temporal characterization of fracture healing patterns and assessment of biomaterials by time-lapsed in vivo micro-computed tomography.

Thorough preclinical evaluation of functionalized biomaterials for treatment of large bone defects is essential prior to clinical application. Using in vivo micro-computed tomography (micro-CT) and mouse femoral defect models with different defect sizes, we were able to detect spatio-temporal healing patterns indicative of physiological and impaired healing in three defect sub-volumes and the adjacent cortex. The time-lapsed in vivo micro-CT-based approach was then applied to evaluate the bone regeneration potential of functionalized biomaterials using collagen and bone morphogenetic protein (BMP-2). Both collagen and BMP-2 treatment led to distinct changes in bone turnover in the different healing phases. Despite increased periosteal bone formation, 87.5% of the defects treated with collagen scaffolds resulted in non-unions. Additional BMP-2 application significantly accelerated the healing process and increased the union rate to 100%. This study further shows potential of time-lapsed in vivo micro-CT for capturing spatio-temporal deviations preceding non-union formation and how this can be prevented by application of functionalized biomaterials. This study therefore supports the application of longitudinal in vivo micro-CT for discrimination of normal and disturbed healing patterns and for the spatio-temporal characterization of the bone regeneration capacity of functionalized biomaterials.

Time-lapsed imaging of nanocomposite scaffolds reveals increased bone formation in dynamic compression bioreactors.

Progress in bone scaffold development relies on cost-intensive and hardly scalable animal studies. In contrast to in vivo, in vitro studies are often conducted in the absence of dynamic compression. Here, we present an in vitro dynamic compression bioreactor approach to monitor bone formation in scaffolds under cyclic loading. A biopolymer was processed into mechanically competent bone scaffolds that incorporate a high-volume content of ultrasonically treated hydroxyapatite or a mixture with barium titanate nanoparticles. After seeding with human bone marrow stromal cells, time-lapsed imaging of scaffolds in bioreactors revealed increased bone formation in hydroxyapatite scaffolds under cyclic loading. This stimulatory effect was even more pronounced in scaffolds containing a mixture of barium titanate and hydroxyapatite and corroborated by immunohistological staining. Therefore, by combining mechanical loading and time-lapsed imaging, this in vitro bioreactor strategy may potentially accelerate development of engineered bone scaffolds and reduce the use of animals for experimentation.

Optimization of mechanical stiffness and cell density of 3D bioprinted cell-laden scaffolds improves extracellular matrix mineralization and cellular organization for bone tissue engineering.

Bioprinting is an emerging technology in which cell-laden biomaterials are precisely dispersed to engineer artificial tissues that mimic aspects of the anatomical and structural complexity of relatively soft tissues such as skin, vessels, and cartilage. However, reproducing the highly mineralized and cellular diversity of bone tissue is still not easily achievable and is yet to be demonstrated. Here, an extrusion-based 3D bioprinting strategy is utilized to fabricate 3D bone-like tissue constructs containing osteogenic cellular organization. A simple and low-cost bioink for 3D bioprinting of bone-like tissue is prepared based on two unmodified polymers (alginate and gelatin) and combined with human mesenchymal stem cells (hMSCs). To form 3D bone-like tissue and bone cell phenotype, the influence of different scaffold stiffness and cell density of 3D bioprinted cell-laden porous scaffolds on osteogenic differentiation and bone-like tissue formation was investigated over time. Our results showed that soft scaffolds (0.8%alg, 0.66 ± 0.08 kPa) had higher DNA content, enhanced ALP activity and stimulated osteogenic differentiation than stiff scaffolds (1.8%alg, 5.4 ± 1.2 kPa). At day 42, significantly more mineralized tissue was formed in soft scaffolds than in stiff scaffolds (43.5 ± 7.1 mm3 vs. 22.6 ± 6.0 mm3). Importantly, immunohistochemistry staining demonstrated more osteocalcin protein expression in high mineral compared to low mineral regions. Additionally, cells in soft scaffolds exhibited osteoblast- and early osteocyte-related gene expression and 3D cellular network within the mineralized matrix at day 42. Furthermore, the results showed that cell density in 15 M cells/ml can promote cell-cell connections at day 7 and mineral formation at day 14, while 5 M cells/ml had the significantly higher mineral formation rate than 15 M cells/ml from day 14 to day 21. In summary, this work reports the formation of 3D bioprinted bone-like tissue using a simple and low-cost cell-laden bioink, which was optimized for stiffness and cell density, showing great promise for bone tissue engineering applications. STATEMENT OF SIGNIFICANCE: In this study, we presented for the first time a framework combining 3D bioprinting, bioreactor system and time-lapsed micro-CT monitoring to provide in vitro scaffold fabrication, maturation, and mineral visualization for bone tissue engineering. 3D bone-like tissue constructs have been formed via optimizing scaffold stiffness and cell density. The soft scaffolds had higher cell proliferation, enhanced alkaline phosphatase activity and stimulated osteogenic differentiation with 3D cellular network foramtion than stiff scaffolds. Significantly more mineralized bone-like tissue was formed in soft scaffolds than stiff scaffolds at day 42. Meanwhile, cell density in 15 M cells/ml can promote cell-cell connections and mineral formation in 14 days, while the higher mineral formation rate was found in 5 M cells/ml from day 14 to day 21.

Hallmarks of frailty and osteosarcopenia in prematurely aged PolgA(D257A/D257A) mice.

BACKGROUND: Frailty is a geriatric syndrome characterized by increased susceptibility to adverse health outcomes. One major determinant thereof is the gradual weakening of the musculoskeletal system and the associated osteosarcopenia. To improve our understanding of the underlying pathophysiology and, more importantly, to test potential interventions aimed at counteracting frailty, suitable animal models are needed. METHODS: To evaluate the relevance of prematurely aged PolgA(D257A/D257A) mice as a model for frailty and osteosarcopenia, we quantified the clinical mouse frailty index in PolgA(D257A/D257A) and wild-type littermates (PolgA(+/+) , WT) with age and concertedly assessed the quantity and quality of bone and muscle tissue. Lastly, the anabolic responsiveness of skeletal muscle, muscle progenitors, and bone was assessed. RESULTS: PolgA(D257A/D257A) accumulated health deficits at a higher rate compared with WT, resulting in a higher frailty index at 40 and 46 weeks of age (+166%, +278%, P < 0.0001), respectively, with no differences between genotypes at 34 weeks. Concomitantly, PolgA(D257A/D257A) displayed progressive musculoskeletal deterioration such as reduced bone and muscle mass as well as impaired functionality thereof. In addition to lower muscle weights (-14%, P < 0.05, -23%, P < 0.0001) and fibre area (-20%, P < 0.05, -22%, P < 0.0001) at 40 and 46 weeks, respectively, PolgA(D257A/D257A) showed impairments in grip strength and concentric muscle forces (P < 0.05). PolgA(D257A/D257A) mutation altered the acute response to various anabolic stimuli in skeletal muscle and muscle progenitors. While PolgA(D257A/D257A) muscles were hypersensitive to eccentric contractions as well as leucine administration, shown by larger downstream signalling response of the mechanistic target of rapamycin complex 1, myogenic progenitors cultured in vitro showed severe anabolic resistance to leucine and robust impairments in cell proliferation. Longitudinal micro-computed tomography analysis of the sixth caudal vertebrae showed that PolgA(D257A/D257A) had lower bone morphometric parameters (e.g. bone volume fraction, trabecular, and cortical thickness, P < 0.05) as well as reduced remodelling activities (e.g. bone formation and resorption rate, P < 0.05) compared with WT. When subjected to 4 weeks of cyclic loading, young but not aged PolgA(D257A/D257A) caudal vertebrae showed load-induced bone adaptation, suggesting reduced mechanosensitivity with age. CONCLUSIONS: PolgA(D257A/D257A) mutation leads to hallmarks of age-related frailty and osteosarcopenia and provides a powerful model to better understand the relationship between frailty and the aging musculoskeletal system.

The association between mineralised tissue formation and the mechanical local in vivo environment: Time-lapsed quantification of a mouse defect healing model.

An improved understanding of how local mechanical stimuli guide the fracture healing process has the potential to enhance clinical treatment of bone injury. Recent preclinical studies of bone defect in animal models have used cross-sectional data to examine this phenomenon indirectly. In this study, a direct time-lapsed imaging approach was used to investigate the local mechanical strains that precede the formation of mineralised tissue at the tissue scale. The goal was to test two hypotheses: 1) the local mechanical signal that precedes the onset of tissue mineralisation is higher in areas which mineralise, and 2) this local mechanical signal is independent of the magnitude of global mechanical loading of the tissue in the defect. Two groups of mice with femoral defects of length 0.85 mm (n = 10) and 1.45 mm (n = 9) were studied, allowing for distinct distributions of tissue scale strains in the defects. The regeneration and (re)modelling of mineralised tissue was observed weekly using in vivo micro-computed tomography (micro-CT), which served as a ground truth for resolving areas of mineralised tissue formation. The mechanical environment was determined using micro-finite element analysis (micro-FE) on baseline images. The formation of mineralised tissue showed strong association with areas of higher mechanical strain (area-under-the-curve: 0.91 ± 0.04, true positive rate: 0.85 ± 0.05) while surface based strains could correctly classify 43% of remodelling events. These findings support our hypotheses by showing a direct association between the local mechanical strains and the formation of mineralised tissue.

Evaluation of longitudinal time-lapsed in vivo micro-CT for monitoring fracture healing in mouse femur defect models.

Longitudinal in vivo micro-computed tomography (micro-CT) is of interest to non-invasively capture the healing process of individual animals in preclinical fracture healing studies. However, it is not known whether longitudinal imaging itself has an impact on callus formation and remodeling. In this study, a scan group received weekly micro-CT measurements (week 0-6), whereas controls were only scanned post-operatively and at week 5 and 6. Registration of consecutive scans using a branching scheme (bridged vs. unbridged defect) combined with a two-threshold approach enabled assessment of localized bone turnover and mineralization kinetics relevant for monitoring callus remodeling. Weekly micro-CT application did not significantly change any of the assessed callus parameters in the defect and periosteal volumes. This was supported by histomorphometry showing only small amounts of cartilage residuals in both groups, indicating progression towards the end of the healing period. Also, immunohistochemical staining of Sclerostin, previously associated with mediating adverse radiation effects on bone, did not reveal differences between groups. The established longitudinal in vivo micro-CT-based approach allows monitoring of healing phases in mouse femur defect models without significant effects of anesthesia, handling and radiation on callus properties. Therefore, this study supports application of longitudinal in vivo micro-CT for healing-phase-specific monitoring of fracture repair in mice.

Alginate dependent changes of physical properties in 3D bioprinted cell-laden porous scaffolds affect cell viability and cell morphology.

Three-dimensional (3D) cell-laden scaffolds are becoming more prevalent in bone tissue repair and regeneration. However, the influence of physical scaffold properties on cell behavior is still unclear. In this study, we fabricated four different alginate concentration (0.8, 1.3, 1.8 and 2.3%alg) composite cell-laden porous scaffolds using a 3D bioprinting technique. The aim was to investigate the changes of physical properties affected by the alginate concentration and the influences on cell behavior. The study showed that the different alginate concentration scaffolds had uniform macropores (500-600 µm) with compressive moduli ranging from 1.5 kPa (0.8%alg) to 14.2 kPa (2.3%alg). Long-term structural integrity of the printed scaffolds was achieved when cultured in cell culture media, but not when cultured in phosphate buffered saline (PBS). Scaffold structure, swelling behavior, and compressive moduli decreased with culturing time and higher alginate concentration lead to more stable physical scaffold properties. Meanwhile, human mesenchymal stem cell (hMSCs) laden non-printed and bioprinted composite scaffolds were fabricated. Bioprinting did not affect cell viability, but alginate concentration had a significant influence on cell viability and cell morphology. Lower alginate concentration scaffolds (0.8%alg) showed higher cell viability (84% ± 0.7% versus 68% ± 1.3%) compared to higher alginate concentration scaffolds (2.3%alg) at day 14. Live cell image in the 0.8%alg scaffolds demonstrated the formation of a 3D interconnected cellular network, while cells in the 1.8 and 2.3%alg scaffolds formed spheroids. In conclusion, this study broadens the design space for alginate-based bioinks for 3D bioprinting. Higher alginate concentration preserved better scaffold fidelity and mechanical properties. Better cell viability and cell spreading morphology was achieved in lower alginate concentration scaffolds, which is relevant for potential applications in bone tissue engineering.

Bone mechanobiology in mice: toward single-cell in vivo mechanomics.

Mechanically driven bone (re)modeling is a multiscale process mediated through complex interactions between multiple cell types and their microenvironments. However, the underlying mechanisms of how cells respond to mechanical signals are still unclear and are at the focus of the field of bone mechanobiology. Traditionally, this complex process has been addressed by reducing the system to single scales and cell types. It is only recently that more integrative approaches have been established to study bone mechanobiology across multiple scales in which mechanical load at the organ level is related to molecular responses at the cellular level. The availability of mouse loading models and imaging techniques with improved spatial and temporal resolution has made it possible to track dynamic bone (re)modeling at the tissue and cellular level in vivo. Coupled with advanced computational models, the (re)modeling activities at the tissue scale can be associated with the mechanical microenvironment. However, methods are lacking to link the molecular responses of different cell types to their local mechanical microenvironment and bone (re)modeling activities occurring at the tissue scale. With recent improvements in "omics" technologies and single-cell molecular biology, it is now possible to sequence the complete genome and transcriptome of single cells. These technologies offer unique opportunities to comprehensively investigate the cellular transcriptional profiles within their specific microenvironment. By combining single-cell "omics" technologies with well-established tissue-scale models of bone mechanobiology, we propose a mechanomics approach to locally analyze the transcriptome of single cells with respect to their local 3D mechanical in vivo environment.

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice.

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 G: peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (µCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (-81%) and bone formation (-80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor ß (ERß, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased ß-catenin, suggesting that ERß might mediate these negative effects through inhibition of osteoanabolic Wnt/ß-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERß was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.

Distinct frequency dependent effects of whole-body vibration on non-fractured bone and fracture healing in mice.

Low-magnitude high-frequency vibration (LMHFV) provokes anabolic effects in non-fractured bone; however, in fracture healing, inconsistent results were reported and optimum vibration conditions remain unidentified. Here, we investigated frequency dependent effects of LMHFV on fracture healing. Twelve-week-old, female C57BL/6 mice received a femur osteotomy stabilized using an external fixator. The mice received whole-body vibrations (20 min/day) with 0.3g peak-to-peak acceleration and a frequency of either 35 or 45 Hz. After 10 and 21 days, the osteotomized femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, µ-computed tomography, and histomorphometry. In non-fractured trabecular bone, vibration with 35 Hz significantly increased the relative amount of bone (+28%) and the trabecular number (+29%), whereas cortical bone was not influenced. LMHFV with 45 Hz failed to provoke anabolic effects in trabecular or cortical bone. Fracture healing was not significantly influenced by whole-body vibration with 35 Hz, whereas 45 Hz significantly reduced bone formation (-64%) and flexural rigidity (-34%) of the callus. Although the exact mechanisms remain open, our results suggest that small vibration setting changes could considerably influence LMHFV effects on bone formation in remodeling and repair, and even disrupt fracture healing, implicating caution when treating patients with impaired fracture healing.

Conversion from external fixator to intramedullary nail causes a second hit and impairs fracture healing in a severe trauma model.

In poly-traumatic patients, second hits are known to potentiate the posttraumatic systemic inflammatory response, thus increasing the risk of multi-organ dysfunction. In accordance with "damage control orthopaedic surgery" principles, fractures are initially treated with external fixators, which are replaced by internal osteosynthesis once the immunological status of the patient is considered stable. Recently, we demonstrated that a severe trauma impaired the healing of fractures stabilized by external fixation during the entire healing period. The question arose, whether switching to intramedullary nailing increases the inflammatory response in terms of a second hit, leading to a further impairment of bone healing. Wistar rats received a femoral osteotomy stabilized by an external fixator. Simultaneously half of the rats underwent an additional thoracic trauma. After 4 days, the external fixator was replaced by an intramedullary nail in half of the rats of the two groups. The inflammatory response was evaluated by measuring serum C5a levels. Fracture healing was determined by three-point-bending, µCT, and histomorphometry. The thoracic trauma significantly increased C5a concentrations 6, 24, and 72 h after the second surgical intervention. After 40 days, conversion to intramedullary nailing considerably decreased the flexural rigidity of the callus, with no significant differences between rats with or without thoracic trauma. After 47 days, flexural rigidity in rats subjected to conversion remained decreased compared to animals solely treated by external fixation, particularly in combination with blunt chest trauma. The results indicate that accumulation of second hits after multiple injuries could lead to aggravation of the fracture healing outcome.