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The field of bone regeneration has primarily focused on investigating fracture healing and nonunion in isolated musculoskeletal injuries. Compared to isolated fractures, which frequently heal well, fractures in patients with multiple bodily injuries (polytrauma) may exhibit impaired healing. While some papers have reported the overall cytokine response to polytrauma conditions, significant gaps in our understanding remain in how fractures heal differently in polytrauma patients. We aimed to characterize fracture healing and the temporal local and systemic immune responses to polytrauma in a murine model of polytrauma composed of a femur fracture combined with isolated chest trauma. We collected serum, bone marrow from the uninjured limb, femur fracture tissue, and lung tissue over 3 weeks to study the local and systemic immune responses and cytokine expression after injury. Immune cell distribution was assessed by flow cytometry. Fracture healing was characterized using microcomputed tomography (microCT), histological staining, immunohistochemistry, mechanical testing, and small angle X-ray scattering. We detected more innate immune cells in the polytrauma group, both locally at the fracture site and systemically, compared to other groups. The percentage of B and T cells was dramatically reduced in the polytrauma group 6 h after injury and remained low throughout the study duration. Fracture healing in the polytrauma group was impaired, evidenced by the formation of a poorly mineralized and dysregulated fracture callus. Our data confirm the early, dysregulated inflammatory state in polytrauma that correlates with disorganized and impaired fracture healing.

Bone fracture healing is a complex physiological process influenced by biomechanical and biomolecular factors. Mechanical stability is crucial for successful healing, and disruptions can lead to delayed healing or nonunion. Bone commonly heals itself through secondary fracture healing, which is governed by the mechanical strain at the fracture site. To investigate these phenomena, a validated methodology for capturing the mechanoregulatory process in specimen‐specific models of fracture healing could provide insight into the healing process. This study implemented a prognostic healing simulation framework to predict healing trajectories based on mechanical stimuli. Sixteen sheep were subjected to a 3 mm transverse tibial mid‐shaft osteotomy, stabilized with a custom plate, and equipped with displacement transducer sensors to measure interfragmentary motion over 8 weeks. Computed tomography scans were used to create specimen‐specific bone geometries for finite element analysis. Virtual mechanical testing was performed iteratively to calculate strains in the callus region, which guided tissue differentiation and consequently, healing. The predicted healing outcomes were compared to continuous in vivo sensor data, providing a unique validation data set. Healing times derived from the in vivo sensor and in silico sensor showed no significant differences, suggesting the potential for these predictive models to inform clinical assessments and improve nonunion risk evaluations. This study represents a crucial step towards establishing trustworthy computational models of bone healing and translating these to the preclinical and clinical setting, enhancing our understanding of fracture healing mechanisms. Clinical significance: Prognostic bone fracture healing simulation could assist in non‐union diagnosis and prediction. [ABSTRACT FROM AUTHOR]

Axial micromotion between bone fragments can stimulate callus formation and fracture healing. In this study, we propose a novel mechanically compliant locking plate which achieves up to 0.6 mm of interfragmentary motion as flexures machined into the plate elastically deflect under physiological load. We investigated the biomechanical performance of three compliant plate variations in comparison to rigid control plates with small and large working lengths in a comminuted bridge plating scenario using humeral diaphysis surrogates. Under static axial loading, average interfragmentary motion was 6 times larger at 100 N (0.38 vs. 0.05 mm) and nearly three times larger at 350 N (0.58 vs. 0.2 mm) for compliant plates than rigid plates, respectively. Compliant plates delivered between 2.5 and 3.4 times more symmetric interfragmentary motion than rigid plates (p < 0.01). The bi‐phasic stiffness of compliant pates provided 74%–96% lower initial axial stiffness up to approximately 100 N (p < 0.01), after which compliant plate stiffness was similar to rigid plates with increased working length (p > 0.3). The strength to failure of compliant plates under dynamic loading was on average 48%–55% lower than rigid plate groups (p < 0.01); however, all plates survived cyclic fatigue loading of 100,000 cycles at 350 N. This work characterizes the improvement in interfragmentary motion and the reduction in strength to failure of compliant plates compared to control rigid plates. Compliant plates may offer potential in comminuted fracture healing due to their ability to deliver symmetric interfragmentary motion into the range known to stimulate callus formation while surviving moderate fatigue loading with no signs of failure. [ABSTRACT FROM AUTHOR]

The field of bone regeneration has primarily focused on investigating fracture healing and nonunion in isolated musculoskeletal injuries. Compared to isolated fractures, which frequently heal well, fractures in patients with multiple bodily injuries (polytrauma) may exhibit impaired healing. While some papers have reported the overall cytokine response to polytrauma conditions, significant gaps in our understanding remain in how fractures heal differently in polytrauma patients. We aimed to characterize fracture healing and the temporal local and systemic immune responses to polytrauma in a murine model of polytrauma composed of a femur fracture combined with isolated chest trauma. We collected serum, bone marrow from the uninjured limb, femur fracture tissue, and lung tissue over 3 weeks to study the local and systemic immune responses and cytokine expression after injury. Immune cell distribution was assessed by flow cytometry. Fracture healing was characterized using microcomputed tomography (microCT), histological staining, immunohistochemistry, mechanical testing, and small angle X‐ray scattering. We detected more innate immune cells in the polytrauma group, both locally at the fracture site and systemically, compared to other groups. The percentage of B and T cells was dramatically reduced in the polytrauma group 6 h after injury and remained low throughout the study duration. Fracture healing in the polytrauma group was impaired, evidenced by the formation of a poorly mineralized and dysregulated fracture callus. Our data confirm the early, dysregulated inflammatory state in polytrauma that correlates with disorganized and impaired fracture healing. [ABSTRACT FROM AUTHOR]

Fracture site motion is thought to play an important role in the healing of complex fractures of the distal femur via mechanotransduction. Measuring this motion in vivo is challenging, and this has led researchers to turn to finite element modeling approaches to gain insights into the mechanical environment at the fracture site. Developing a systematic understanding of the effect of different model choices for distal femur fractures may allow more accurate prediction of fracture site motion from these types of simulations. In this study, we aim to assess the effect of four different modeling choices and parameters. We looked at the effect of using bone specific density distributions vs generic values, employing landmark‐based geometry generation, varying fracture alignment within clinically relevant ranges, and determining whether direct apposition of the fracture to the plate was achieved. For validation, five cadaveric femurs had fractures created and repaired with plated constructs, and these were then loaded and fracture site motion was directly measured. We found that using landmark based bone geometry and patient‐specific bone density distributions had a minimal effect on the overall model predictions. Changing the alignment, particularly into varus and procurvatum could have a large (>50%) effect on predicted shear motion, as could direct apposition of the bone to the plate. These findings demonstrate that modeling choices can play an important role in simulating distal femur fracture mechanics, and it is particularly critical that patient customized models attempt to accurately represent alignment of the bone fragments and lateral plate apposition. [ABSTRACT FROM AUTHOR]

Older adults suffer more bone fractures with higher rates of healing complications and increased risk of morbidity and mortality. An improved understanding of the cellular and molecular mechanism of fracture healing and how such processes are perturbed with increasing age may allow for better treatment options to manage fractures in older adults. Macrophages are attractive therapeutics due to their role in several phases of fracture healing. After injury, bone marrow-derived macrophages are recruited to the injury and propagate the inflammatory response, contribute to resolution of inflammation, and promote bone regeneration. A tissue resident population of macrophages named osteal macrophages are present in the periosteum and are directly associated with osteoblasts and these cells contribute to bone formation. Here, we utilized bulk RNA sequencing to analyze the transcriptional activity of osteal macrophages from old and young mice present in primary calvarial cultures. Macrophages demonstrated a diverse transcriptional profile, expressing genes involved in immune function as well as wound healing and regeneration. Periostin was significantly downregulated in macrophages from old mice compared to young. Periostin is an extracellular matrix protein with important functions that promote osteoblast activity during bone regeneration. An age-related decrease of periostin expression was verified in the fracture callus of old mice compared to young. Young periostin knockout mice demonstrated attenuated fracture healing outcomes that reflected what is observed in old mice. This study supports an important role of periostin in fracture healing, and therapeutically targeting the age-related decrease in periostin may improve healing outcomes in older populations.

Measuring the healing status of a bone fracture is important to determine the clinical care a patient receives. Implantable devices can directly and continuously assess the healing status of fracture fixation constructs, while subject‐specific virtual biomechanical tests can noninvasively determine callus structural integrity at single time points. Despite their potential for objectification, both methods are not yet integrated into clinical practice with further evidence of their benefits required. This study correlated continuous data from an implantable sensor assessing healing status through implant load monitoring with computer tomography (CT) based longitudinal finite element (FE) simulations in a large animal model. Eight sheep were part of a previous preclinical study utilizing a tibial osteotomy model and equipped with such a sensor. Sensor signal was collected over several months, and CT scans were acquired at six interim time points. For each scan, two FE analyses were performed: a virtual torsional rigidity test of the bone and a model of the bone‐implant construct with the sensor. The longitudinal simulation results were compared to the sensor data at corresponding time points and a cohort‐specific empirical healing rule was employed. Healing status predicted by both in silico simulations correlated significantly with the sensor data at corresponding time points and correctly identified a delayed and a nonunion in the cohort. The methodology is readily translatable with the potential to be applied to further preclinical or clinical cohorts to find generalizable healing criteria. Virtual mechanical tests can objectively measure fracture healing progressing using longitudinal CT scans. [ABSTRACT FROM AUTHOR]

In large animal studies, the mechanical reintegration of the bone fragments is measured using postmortem physical testing, but these assessments can only be performed once, after sacrifice. Image‐based virtual mechanical testing is an attractive alternative because it could be used to monitor healing longitudinally. However, the procedures and software required to perform finite element analysis (FEA) on subject‐specific models for virtual mechanical testing can be time consuming and costly. Accordingly, the goal of this study was to determine whether a simpler image‐based geometric measure—the torsion constant, sometimes known as polar moment of inertia—can be reliably used as a surrogate measure of bone healing in large animals. To achieve this, postmortem biomechanical testing and microCT scans were analyzed for a total of 33 operated and 20 intact ovine tibiae. An image‐processing procedure to compute the attenuation‐weighted torsion constant from the microCT scans was developed in MATLAB and this code has been made freely available. Linear regression analysis was performed between the postmortem biomechanical data, the results of virtual mechanical testing using FEA, and the torsion constants measured from the scans. The results showed that virtual mechanical testing is the most reliable surrogate measure of postmortem torsional rigidity, having strong correlations and high absolute agreement. However, when FEA is not practical, the torsion constant is a viable alternative surrogate measure that is moderately correlated with postmortem torsional rigidity and can be readily calculated. [ABSTRACT FROM AUTHOR]

As scientific advancements continue to reshape the world, it becomes increasingly crucial to uphold ethical standards and minimize the potentially adverse impact of research activities. In this context, the implementation of the 3R principles—Replacement, Reduction, and Refinement—has emerged as a prominent framework for promoting ethical research practices in the use of animals. This article aims to explore recent advances in integrating the 3R principles into fracture healing research, highlighting their potential to enhance animal welfare, scientific validity, and societal trust. The review focuses on in vitro, in silico, ex vivo, and refined in vivo methods, which have the potential to replace, reduce, and refine animal experiments in musculoskeletal, bone, and fracture healing research. Here, we review material that was presented at the workshop "Implementing 3R Principles into Fracture Healing Research" at the 2023 Orthopedic Research Society (ORS) Annual Meeting in Dallas, Texas. [ABSTRACT FROM AUTHOR]

Musculoskeletal trauma, specifically fractures, is a leading cause of patient morbidity and disability worldwide. In approximately 20% of cases with fracture and related traumatic muscle loss, bone healing is impaired leading to fracture nonunion. Over the past few years, several studies have demonstrated that bone and the surrounding muscle tissue interact not only anatomically and mechanically but also through biochemical pathways and mediators. Severe damage to the surrounding musculature at the fracture site causes an insufficiency in muscle‐derived osteoprogenitor cells that are crucial for fracture healing. As an endocrine tissue, skeletal muscle produces many myokines that act on different bone cells, such as osteoblasts, osteoclasts, osteocytes, and mesenchymal stem cells. Investigating how muscle influences fracture healing at cellular, molecular, and hormonal levels provides translational therapeutic solutions to this clinical challenge. This review provides an overview about the contributions of surrounding muscle tissue in directing fracture healing. The focus of the review is on describing the interactions between bone and muscle in both healthy and fractured environments. We discuss current progress in identifying the bone–muscle molecular pathways and strategies to harness these pathways as cues for accelerating fracture healing. In addition, we review the existing challenges and research opportunities in the field. [ABSTRACT FROM AUTHOR]

Dynamization, that is, increasing interfragmentary movement (IFM) by reducing fixation stiffness from a rigid to a more flexible state, has been successfully used in clinical practice to promote fracture healing. However, it remains unclear how dynamization timing and degree affect bone healing of different fracture types. Finite element models of tibial fractures based on the OTA/AO classification (Simple: A1‐Spiral, A2‐Oblique, A3‐Transverse; Wedge: B2‐Spiral, B3‐Fragmented; Complex: C2‐Segment, C3‐Irregular), in combination with fuzzy logic‐based mechano‐regulatory tissue differentiation algorithms, were used to simulate the healing process when dynamization of varied degrees (dynamization coefficient or DC = 0‐0.9; 0.9 represents 90% reduction in the fixation stiffness relative to a rigid fixation) were applied at different time points after fracture. The fuzzy logic‐based algorithms have been validated with a preclinical animal model. The results showed that the healing responses of type A fractures were more sensitive to the changes in dynamization degree and timing comparing with type B or C fractures. Additionally, the optimal dynamization regime for each fracture type was different. For type A fractures, a moderate dynamization degree (e.g., DC = 0.5) applied after Week 1 promoted the recovery of biomechanical integrity. For type B and C fractures, the effective dynamization included a greater dynamization degree (DC = 0.7) applied after Week 2. Our results further demonstrated that the fracture morphology affected interfragmentary strain environments within the callus, leading to varied healing results for different fracture types. These results suggest that the effects of dynamization are highly dependent of the fracture types. Therefore, specific dynamization strategies should be chosen for different fracture types to achieve optimal healing outcomes. [ABSTRACT FROM AUTHOR]

While recent studies showed that macrophages are critical for bone fracture healing, and lack of M2 macrophages have been implicated in models of delayed union, functional roles for specific M2 receptors have yet to be defined. Moreover, the M2 scavenger receptor CD163 has been identified as a target to inhibit sepsis following implant‐associated osteomyelitis, but potential adverse effects on bone healing during blockage therapy have yet to be explored. Thus, we investigated fracture healing in C57BL/6 versus CD163−/− mice using a well‐established closed, stabilized, mid‐diaphyseal femur fracture model. While gross fracture healing in CD163−/− mice was similar to that of C57BL/6, plain radiographs revealed persistent fracture gaps in the mutant mice on Day 14, which resolved by Day 21. Consistently, 3D vascular micro‐CT demonstrated delayed union on Day 21, with reduced bone volume (74%, 61%, and 49%) and vasculature (40%, 40%, and 18%) compared to C57BL/6 on Days 10, 14, and 21 postfracture, respectively (p < 0.01). Histology confirmed large amounts of persistent cartilage in CD163−/− versus C57BL/6 fracture callus on Days 7 and 10 that resolves over time, and immunohistochemistry demonstrated deficiencies in CD206+ M2 macrophages. Torsion testing of the fractures confirmed the delayed early union in CD163−/− femurs, which display decreased yield torque on Day 21, and a decreased rigidity with a commensurate increase in rotation at yield on Day 28 (p < 0.01). Collectively, these results demonstrate that CD163 is required for normal angiogenesis, callus formation, and bone remodeling during fracture healing, and raise potential concerns about CD163 blockade therapy. [ABSTRACT FROM AUTHOR]

The small leucine‐rich proteoglycans, decorin and biglycan, are minor components of the tendon extracellular matrix that regulate fibrillogenesis and matrix assembly. Our study objective was to define the temporal roles of decorin and biglycan during tendon healing using inducible knockout mice to include genetic knockdown at specific phases of healing: time of injury, the proliferative phase, and the remodeling phase. We hypothesized that knockdown of decorin or biglycan would adversely affect tendon healing, and that by prescribing the timing of knockdown, we could elucidate the temporal roles of these proteins during healing. Contrary to our hypothesis, decorin knockdown did not affect tendon healing. However, when biglycan was knocked down, either alone or coupled with decorin, tendon modulus was increased relative to wild‐type mice, and this finding was consistent among all induction timepoints. At 6 weeks postinjury, we observed increased expression of genes associated with the extracellular matrix and growth factor signaling in the biglycan knockdown and compound decorin–biglycan knockdown tendons. Interestingly, these groups demonstrated opposing trends in gene expression as a function of knockdown‐induction timepoint, highlighting distinct temporal roles for decorin and biglycan. In summary, this study finds that biglycan plays multiple functions throughout tendon healing, with the most impactful, detrimental role likely occurring during late‐stage healing. Statement of clinical importance: This study helps to define the molecular factors that regulate tendon healing, which may aid in the development of new clinical therapies. [ABSTRACT FROM AUTHOR]

To determine the optimal position of three cannulated screws in an inverted triangle pattern for internal fixation of femoral neck fractures, including the apex–subchondral bone distance, computed tomography (CT) images of the hip were processed using the MIMICS (Materialize Interactive Medical Image Control System) Research software. Points adjacent to the inferior, anterosuperior, and posterior cortex of the femoral neck were selected on the axial view. Subsequently, the positions of these points were adjusted to maximize the area between them, and splines were drawn in the coronal and sagittal planes to represent the cannulated screws. Finally, the virtual anteroposterior and lateral fluoroscopy images of the proximal femur and splines were created and the parameters calculated. Finite element analysis showed this fixation scheme provides stronger fracture support and stability. Twenty patients with femoral neck fractures (Modified group), with the area and perimeter of the triangle formed on axial postoperative CT images, including the shortest distance from the tip of the screw to the subchondral bone was compared with 23 matched patients for whom the conventional inverted isosceles triangle configuration of screws for internal fixation was used (Conventional group). The area and perimeter of the stabilization screws were larger, with a shorter apex–subchondral bone distance for the Modified group. There was no incidence of screw penetration in the femoral neck or head in either group, and all fractures healed within a follow‐up period of 10 months. The modified screw placement method is simple and safe and provides greater fracture stability than the conventional empirical method of fixation. [ABSTRACT FROM AUTHOR]

The effects of locally applied zinc chloride (ZnCl2) on early and late‐stage parameters of fracture healing were evaluated in a diabetic rat model. Type 1 Diabetes has been shown to negatively impact mechanical parameters of bone as well as biologic markers associated with bone healing. Zinc treatments have been shown to reverse those outcomes in tests of nondiabetic and diabetic animals. This study is the first to assess the efficacy of a noncarrier mediated ZnCl2 on bony healing in diabetic animals. This is a promising basic science approach which may lead to benefits for diabetic patients in the future. Treatment and healing were assessed through quantification of callus zinc, radiographic scoring, microcomputed tomography (µCT), histomorphometry, and mechanical testing. Local ZnCl2 treatment increased callus zinc levels at 1 and 3 days after fracture (p ≤ 0.025). Femur fractures treated with ZnCl2 showed increased mechanical properties after 4 and 6 weeks of healing. Histomorphometry of the ZnCl2‐treated fractures found increased callus cartilage area at Day 7 (p = 0.033) and increased callus bone area at Day 10 (p = 0.038). In contrast, callus cartilage area was decreased (p < 0.01) after 14 days in the ZnCl2‐treated rats. µCT analysis showed increased bone volume in the fracture callus of ZnCl2‐treated rats at 6 weeks (p = 0.0012) with an associated increase in the proportion of µCT voxel axial projections (Z‐rays) spanning the fracture site. The results suggest that local ZnCl2 administration improves callus chondrogenesis leading to greater callus bone formation and improved fracture healing in diabetic rats. [ABSTRACT FROM AUTHOR]

Calcitonin gene‐related peptide (CGRP) is a neuropeptide produced by sensory nerves and functions as a pain sensor. It acts by binding to the calcitonin‐like receptor (CLR, protein; Calcrl, gene). CGRP inhibition has been recently introduced as therapeutic treatment of migraine‐associated pain. Previous studies have shown that CGRP stimulates bone formation. The aim of our study is to determine whether the inhibition of CGRP signaling negatively impacted fracture healing. Using α‐smooth muscle actin (αSMA) Cre animals crossed with Ai9 reporter mice, we showed that CGRP‐expressing nerves are near αSMA + cells in the periosteum. In vitro experiments revealed that periosteal cells express Calcrl and receptor activity modifying protein 1; and CGRP stimulation increased periosteal cell proliferation. Using a tamoxifen‐inducible model αSMACre/CLRfl/fl, we targeted the deletion of CLR to periosteal progenitor cells and examined fracture healing. Microcomputed tomography of fractured femurs showed a reduction in bone mass in αSMACre+/CLRfl/fl female mice relative to controls and callus volume in males. Pharmacological CGRP–CLR inhibition was achieved by subcutaneous delivery of customized pellets with small molecule inhibitor olcegepant (BIBN‐4096) at a dose of 10 μg/day. BIBN‐4096‐treated C57BL/6J mice had a higher latency toward thermal nociception than placebo‐treated mice, indicating impaired sensory function through CGRP inhibition. CGRP inhibition also resulted in reduced callus volume, bone mass, and bone strength compared to placebo controls. These results indicate that inhibiting CGRP by deleting CLR or by using BIBN‐4096, contributes to delayed bone healing. [ABSTRACT FROM AUTHOR]

Since the 1970s, the 2%−10% rule has been used to describe the range of interfragmentary gap closure strains that are conducive for secondary bone healing. Interpreting the available evidence for the association between strain and bone healing remains challenging because interfragmentary strain is impossible to directly measure in vivo. The question of how much strain occurs within and around the fracture gap is also difficult to resolve using bench tests with osteotomy models because these do not reflect the complexity of injury patterns seen in the clinic. To account for these challenges, we used finite element modeling to assess the three‐dimensional interfragmentary strain in a case series of naturally occurring distal femur fractures treated with lateral plating under load conditions representative of the early postoperative period. Preoperative computed tomography scans were used to construct patient‐specific finite element models and plate fixation constructs to match the operative management of each patient. The simulations showed that gap strains were within 2%−10% only for the lowest load application level, 20% static body weight (BW). Moderate loading of 60% static BW and above caused gap strains that far exceeded 10%, but in all cases, strains in the periosteal region external to the fracture line remained low. Comparing these findings with postoperative radiographs suggests that in vivo secondary healing of distal femur fractures may be robust to early gap strains much greater than 10% because formation of new bone is initiated outside the gap where strains are lower, followed by later consolidation within the gap. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is a genetic bone fragility disorder that features frequent fractures. Bone healing outcomes are contingent on a proper balance between bone formation and resorption, and drugs such as bone morphogenetic proteins (BMPs) and bisphosphonates (BPs) have shown to have utility in modulating fracture repair. While BPs are used for OI to increase BMD and reduce pain and fracture rates, there is little evidence for using BMPs as local agents for fracture healing (alone or with BPs). In this study, we examined wild‐type and OI mice (Col1a2+/G610C) in a murine tibial open fracture model with (i) surgery only/no treatment, (ii) local BMP‐2 (10 µg), or (iii) local BMP‐2 and postoperative zoledronic acid (ZA; 0.1 mg/kg total dose). Microcomputed tomography reconstructions of healing fractures indicated BMP‐2 was less effective in an OI setting, however, BMP‐2 +ZA led to considerable increases in bone volume (+193% WT, p < 0.001; +154% OI, p < 0.001) and polar moment of inertia (+125% WT, p < 0.01; +248% OI, p < 0.05). Tissue histology revealed a thinning of the neocortex of the callus in BMP‐2 treated OI bone, but considerable retention of woven bone in the healing callus with BMP + ZA specimens. These data suggest a cautious approach may be warranted with the sole application of BMP‐2 in an OI surgical setting as a bone graft substitute. However, this may be overcome by off‐label BP administration. [ABSTRACT FROM AUTHOR]

The local inflammatory impact of different reaming protocols in intramedullary nailing has been sparsely investigated. We examined the effect of different reaming protocols on fracture hematoma (FH) immunological characteristics in pigs. To do so, a standardized midshaft femur fracture was induced in adult male pigs. Fractures were treated with conventional reamed femoral nailing (group RFN, n = 6); unreamed femoral nailing (group UFN, n = 6); reaming with a Reamer Irrigator Aspirator device (group RIA, n = 12). Animals were observed for 6 h and FH was collected. FH‐cell apoptosis and neutrophil receptor expression (Mac‐1/CD11b and FcγRIII/CD16) were studied by flow cytometry and local temperature changes were analyzed. The study demonstrates that apoptosis‐rates of FH‐immune cells were significantly lower in group RIA (3.50 ± 0.53%) when compared with non‐RIA groups: (group UFN 12.50 ± 5.22%, p = 0.028 UFN vs. RIA), (group RFN 13.30 ± 3.18%, p < 0.001, RFN vs. RIA). Further, RIA‐FH showed lower neutrophil CD11b/CD16 expression when compared with RFN (mean difference of 43.0% median fluorescence intensity (MFI), p = 0.02; and mean difference of 35.3% MFI, p = 0.04, respectively). Finally, RIA induced a transient local hypothermia and hypothermia negatively correlated with both FH‐immune cell apoptosis and neutrophil activation. In conclusion, immunologic changes observed in FH appear to be modified by certain reaming techniques. Irrigation during reaming was associated with transient local hypothermia, decreased apoptosis, and reduced neutrophil activation. Further study is warranted to examine whether the rinsing effect of RIA, specific tissue removal by reaming, or thermal effects predominantly determine local inflammatory changes during reaming. [ABSTRACT FROM AUTHOR]

Meteorin‐like protein (Metrnl), homologous to the initially identified neurotrophic factor Meteorin, is a secreted, multifunctional protein. Here we used mouse models to investigate Metrnl's role in skeletal development and bone fracture healing. During development Metrnl was expressed in the perichondrium and primary ossification center. In neonates, single cell RNA‐seq of diaphyseal bone demonstrated strongest expression of Metrnl transcript by osteoblasts. In vitro, Metrnl was osteoinductive, increasing osteoblast differentiation and mineralization in tissue culture models. In vivo, loss of Metrnl expression resulted in no change in skeletal metrics in utero, at birth, or during postnatal growth. Six‐week‐old Metrnl‐null mice displayed similar body length, body weight, tibial length, femoral length, BV/TV, trabecular number, trabecular thickness, and cortical thickness as littermate controls. In 4‐month‐old mice, lack of Metrnl expression did not change structural stiffness, ultimate force, or energy to fracture of femora under 3‐point‐bending. Last, we investigated the role of Metrnl in bone fracture healing. Metrnl expression increased in response to tibial injury, however, loss of Metrnl expression did not affect the amount of bone deposited within the healing tissue nor did it change the structural parameters of healing tissue. This work identifies Metrnl as a dispensable molecule for skeletal development. However, the osteoinductive capabilities of Metrnl may be utilized to modulate osteoblast differentiation in cell‐based orthopedic therapies. [ABSTRACT FROM AUTHOR]

The diagnosis of fracture nonunion following plate osteosynthesis is subjective and frequently ambiguous. Initially following osteosynthesis, loads applied to the bone are primarily transmitted through the plate. However, as callus stiffness increases, the callus is able to bear load proportional to its stiffness while forces through the plate decrease. The purpose of this study was to use a "smart" fracture plate to distinguish between phases of fracture healing by measuring forces transmitted through the plate. A wireless force sensor and small adapter were placed on the outside of a distal femoral locking plate. The adapter converts the slight bending of the plate under axial load into a transverse force which is measurable by the sensor. An osteotomy was created and then plated in the distal femur of biomechanical Sawbones. Specimens were loaded to simulate single‐leg stance first with the osteotomy defect empty (acute healing), then sequentially filled with silicone (early callus) and then polymethyl methacrylate (hard callus). There was a strong correlation between applied axial load and force measured by the "smart" plate. Data demonstrate statistically significant differences between each phase of healing with as little as 150 N of axial load applied to the femur. Forces measured in the plate were significantly different between acute (100%), early callus (66.4%), and hard callus (29.5%). This study demonstrates the potential of a "smart" fracture plate to distinguish between phases of healing. These objective data may enable early diagnosis of nonunion and enhance outcomes for patients. [ABSTRACT FROM AUTHOR]

Bone regeneration and fracture healing are impaired in diabetic patients due to defective functions of associated cells. Thus, the search for molecular causes and new treatment strategies are of particular clinical relevance. We investigated the gene expression profile of bones from type 2 diabetic (db−/db−) mice and wild‐type (wt) mice by comparative microarray analyses before and after placing tibial defects and examined the expression of several osteogenesis‐ and osteoclastogenesis‐related markers by quantitative real‐time polymerase chain reaction. In regenerating wt bones, pathways related to, for example, inhibition of matrix metalloproteases were activated, whereas in db−/db− bones activation of pathways related to, for example, osteoarthritis, transforming growth factor‐beta (Tgfb), or hypoxia‐inducible factor 1a were detected during regeneration. We defined the Tgfb pathway as a potential therapeutic target and locally applied a single dose (0.5 µg) of the Tgfb 1, 2, and 3 neutralizing antibody 1D11 on tibial defects in db−/db− mice (n = 7). Seven days postoperation, histological and immunohistochemical stainings were performed. Decreased bone regeneration, osteogenic differentiation, osteoclast invasion, and angiogenesis in db−/db− mice were significantly restored by local 1D11 application in comparison to the phosphate‐buffered saline controls. Thus, local treatment of db−/db− bony defects with Tgfb neutralizing antibody 1D11 might be considered a good candidate for the successful acceleration of bone regeneration. [ABSTRACT FROM AUTHOR]

There is a dearth of therapies that are safe and effective for the treatment of volumetric muscle loss (VML), defined as the surgical or traumatic loss of muscle tissue, resulting in functional impairment. To address this gap in orthopedic care, we developed a porous sponge‐like scaffold composed of extracellular matrix (ECM) proteins (e.g., gelatin, collagen, and laminin‐111) and an immunosuppressant drug, FK‐506. While the majority of VML injuries occur in orthopedic trauma cases, preclinical models typically study muscle injuries in isolation without a concomitant bone fracture. The goal of this study was to investigate the extent to which FK506 loaded biomimetic sponges support functional muscle regeneration and fracture healing in a composite trauma model involving VML injury to the tibialis anterior muscle and osteotomy (OST) to the tibia. In this model, implantation of the FK‐506 loaded biomimetic sponges limited the extent of inflammation while increasing the total number of myofibers, mean myofiber cross‐sectional area, myosin‐to‐collagen ratio, and peak isometric torque compared to untreated VML+OST muscles on Day 28. Although all tibia fractures were bridged by Day 28 post‐injury, fracture healing was impaired in response to an adjacent VML injury. Sponge treatment increased bone callus volume, yet the bridged mineralized bone volume was not significantly different. Taken together, these results suggest that biomimetic sponges primarily benefitted muscle repair and may provide a promising therapy for traumatized muscle. [ABSTRACT FROM AUTHOR]

Numerous animal and human studies have demonstrated the benefit of controlled interfragmentary motion on fracture healing. In this study, we quantified interfragmentary motion and load transfer in tibial fractures fixed using a novel intramedullary nail (IMN) that allows controlled axial motion. Fifty composite tibias with various fracture patterns were utilized. For all test conditions, two interlocking screws were used to fix the nail in the proximal metaphysis, and two interlocking screws through the distal metaphysis. The nail allowed either no motion (static mode) or 1 mm (dynamic mode) of cyclic axial motion between the two fracture fragments for every fracture pattern tested. As expected, strain shielding was more prominent under static nail conditions. In contrast, specimens tested under dynamic nail conditions transferred axial load between the fracture fragments such that strains near the fracture site were generally similar to those measured on an intact tibia. Maximum shear strains proximal to the fracture were significantly lower in specimens with oblique or butterfly fracture patterns (p < 0.01) compared to intact specimens. This decrease in shear strain indicates that strain shielding effects were likely present due to the implant. However, strain shielding appeared to be reduced in tensile and compressive principal strains. In summary, the novel IMN allowed controlled axial motion between the fragments in a variety of common diaphyseal tibial fracture patterns. Clinical Significance: The present in vitro biomechanical study investigated a novel intramedullary nail capable of controlled axial interfragmentary motion which may potentially enhance fracture healing. [ABSTRACT FROM AUTHOR]

We previously showed that femur fracture in mice caused a reduction in bone volume at distant skeletal sites within 2 weeks post‐fracture. Osteocytes also have the ability to remodel their surrounding bone matrix through perilacunar/canalicular remodeling (PLR). If PLR is altered systemically following fracture, this could affect bone mechanical properties and increase fracture risk at all skeletal sites. In this study, we investigated whether lacunar‐canalicular microstructure and the rate of PLR are altered in the contralateral limb following femoral fracture in mice. We hypothesized that femoral fracture would accelerate PLR by 2 weeks postfracture, followed by partial recovery by 4 weeks. We used histological evaluation and high‐resolution microcomputed tomography to quantify the morphology of the lacunar‐canalicular network at the contralateral tibia, and we used quantitative real‐time polymerase chain reaction (RT‐PCR) and RNA‐seq to measure the expression of PLR‐associated genes in the contralateral femur. We found that at both 2 and 4 weeks postfracture, canalicular width was significantly increased by 18.6% and 16.6%, respectively, in fractured mice relative to unfractured controls. At 3 days and 4 weeks post‐fracture, we observed downregulation of PLR‐associated genes; RNA‐seq analysis at 3 days post‐fracture showed a deceleration of bone formation and mineralization in the contralateral limb. These data demonstrate notable canalicular changes following fracture that could affect bone mechanical properties. These findings expand our understanding of systemic effects of fracture and how biological and structural changes at distant skeletal sites may contribute to increased fracture risk following an acute injury. [ABSTRACT FROM AUTHOR]

Infection is a common cause of impaired fracture healing. In the clinical setting, definitive fracture treatment and infection are often treated separately and sequentially, by different clinical specialties. The ability to treat infection while promoting fracture healing will greatly reduce the cost, number of procedures, and patient morbidity associated with infected fractures. In order to develop new therapies, scientists and engineers must understand the clinical need, current standards of care, pathologic effects of infection on fractures, available preclinical models, and novel technologies. One of the main causes of poor fracture healing is infection; unfortunately, bone regeneration and infection research are typically approached independently and viewed as two separate disciplines. Here, we aim to bring these two groups together in an educational workshop to promote research into the basic and translational science that will address the clinical challenge of delayed fracture healing due to infection. Statement of clinical significance: Infection and nonunion are each feared outcomes in fracture care, and infection is a significant driver of nonunion. The impact of nonunions on patie[Q2]nt well‐being is substantial. Outcome data suggests a long bone nonunion is as impactful on health‐related quality of life measures as a diagnosis of type 1 diabetes and fracture‐related infection has been shown to significantly l[Q3]ower a patient's quality of life for over 4 years. Although they frequently are associated with one another, the treatment approaches for infections and nonunions are not always complimentary and cannot be performed simultaneously without accepting tradeoffs. Furthermore, different clinical specialties are often required to address the problem, the orthopedic surgeon treating the fracture and an infectious disease specialist addressing the sources of infection. A sequential approach that optimizes treatment parameters requires more time, more surgeries, and thus confers increased morbidity to the patient. The ability to solve fracture healing and infection clearance simultaneously in a contaminated defect would benefit both the patient and the health care system. Statement of clinical significance: Infection and nonunion are each feared outcomes in fracture care, and infection is a significant driver of nonunion. The impact of nonunions on patient well‐being is substantial. Outcome data suggests a long bone nonunion is as impactful on health‐related quality of life measures as a diagnosis of type 1 diabetes and fracture‐related infection has been shown to significantly lower a patient's quality of life for over 4 years. Although they frequently are associated with one another, the treatment approaches for infections and nonunions are not always complimentary and cannot be performed simultaneously without accepting tradeoffs. Furthermore, different clinical specialties are often required to address the problem, the orthopedic surgeon treating the fracture and an infectious disease specialist addressing the sources of infection. A sequential approach that optimizes treatment parameters requires more time, more surgeries, and thus confers increased morbidity to the patient. The ability to solve fracture healing and infection clearance simultaneously in a contaminated defect would benefit both the patient and the health care system. [ABSTRACT FROM AUTHOR]

Dynamization, increasing the interfragmentary movement (IFM) by reducing the fixation stiffness from a rigid to a more flexible condition, is widely used clinically to promote fracture healing. However, it remains unknown how dynamization degree (relative change in fixation stiffness/IFM from a rigid to a flexible fixation) affects bone healing at various stages. To address this issue, we used a fuzzy logic‐based mechano‐regulated tissue differentiation algorithm on published experimental data from a sheep osteotomy healing model. We applied a varied degree of dynamization, from 0 (fully rigid fixation) to 0.9 (90% reduction in stiffness relative to the rigid fixation) after 1, 2, 3, and 4 weeks of osteotomy (R1wF, R2wF, R3wF, and R4wF) and computationally evaluated bone regeneration and biomechanical integrity over the healing process of 8 weeks. Compared with the constant rigid fixation, early dynamization (R1wF and R2wF) led to delays in bone bridging and biomechanical recovery of the osteotomized bone. However, the effect of early dynamization on healing was dependent of the degree of dynamization. Specifically, a higher dynamization degree (e.g., 0.9 for R1wF) led to a prolonged delay in bone bridging and largely unrecovered bending stiffness (48% relative to the intact bone), whereas a moderate degree of dynamization (e.g., 0.5 or 0.7) significantly enhanced bone formation and biomechanical properties of the osteotomized bone. These results suggest that dynamization degree and timing interactively affect the healing process. A combination of early dynamization with a moderate degree could enhance the ultimate biomechanical recovery of the fractured bone. [ABSTRACT FROM AUTHOR]

This study examined the effect of once‐weekly parathyroid hormone (PTH) combined with alendronate upon osteoporotic fracture healing after long‐term alendronate anti‐osteoporosis therapy. Seventy‐six 12‐week‐old female Sprague–Dawley rats were either sham operated or bilaterally ovariectomized (OVX). Following confirmation of osteoporosis 3 months after OVX, the remaining 64 animals received alendronate therapy. After 3 months of alendronate treatment, all rats underwent unilateral transverse tibial osteotomy. Animals were immediately randomly assigned to one of four groups: (1) alendronate followed by vehicle (ALN‐VEH), (2) continuation of alendronate (ALN‐ALN), (3) alendronate followed by once‐weekly PTH alone (ALN‐PTH), (4) continuation of alendronate combined with once‐weekly PTH (ALN‐ALN + PTH) until collection at 4 or 8 weeks after osteotomy. The fractured tibia was assessed using x‐ray, dual‐energy x‐ray absorptiometry, microcomputed tomography, biomechanical testing, histology, and sequential fluorescence labeling. The ALN‐ALN + PTH treatment significantly increased total callus volume, mineralized callus volume, mineralized callus volume/total callus volume, and biomechanical strength of the callus relative to ALN‐VEH and ALN‐PTH treatments at both 4 and 8 weeks and produced more mature trabecular bone compared with ALN‐ALN treatment at 8 weeks. RANKL/osteoprotegerin (OPG) are osteoclastogenesis markers, while cluster of differentiation 31 (CD31) is an important marker of angiogenesis. Qualitative immunohistochemical analysis revealed that CD31 and OPG expression was was strong after ALN‐ALN + PTH compared with ALN‐ALN treatment, whereas RANKL expression was weak after ALN‐ALN + PTH versus ALN‐PTH treatment. Our study showed that once‐weekly PTH combined with alendronate was beneficial in promoting the healing of fractures acquired after long‐term alendronate therapy in OVX‐induced osteoporotic rats. [ABSTRACT FROM AUTHOR]

Fracture healing is a complex process requiring mechanical stability, an osteoconductive matrix, and osteoinductive and osteogenic biology. This intricate process is easily disrupted by various patient factors such as chronic disease and lifestyle. As the medical complexity and age of patients with fractures continue to increase, the importance of developing relevant experimental models is becoming paramount in preclinical research. The objective of this review is to describe the most common small animal models of systemically impaired fracture healing used in the orthopedic literature including osteoporosis, diabetes mellitus, smoking, alcohol use, obesity, and ageing. This review will provide orthopedic researchers with a summary of current models of systemically impaired fracture healing used in small animals and present an overview of the methods of induction for each condition. [ABSTRACT FROM AUTHOR]

Tendon ruptures heal by forming a mechanically inferior scar. We have shown that male Murphy Roths large (MRL/MpJ) mice exhibit improved tendon healing, suggesting that they can inform biological mechanisms that lead to effective tendon healing. As sex impacts healing, we assessed the effect of sex on tendon healing in MRL/MpJ and normal healer C57BL/6 (B6) mice and compared the associated biological environment with identify genes that may be integral to the improved healing outcome. We hypothesized that (a) male MRL/MpJ mice will heal with improved mechanical properties compared to females; and (b) that regenerative tendon healing will be associated with decreased fibrotic pathways, decreased inflammation, and increased activity of matrix metalloproteinases (MMPs). A midsubstance punch was introduced, and tendons were harvested after (a) 1 or 7 days for profiling of 84 genes; (b) 7 or 14 days for the assessment of MMP‐2 and MMP‐9 activity; and (c) 6 weeks for mechanical assessment. MRL/MpJ tendons healed with the better restoration of mechanical properties than B6 tendons. Sex did not affect the mechanical properties of healing B6 or MRL/MpJ tendons. Comparison of the gene expression profiles in the context of the mechanical outcome revealed several differences between MRL/MpJ and B6 tendon healing, including, lower inflammation, an earlier higher expression of TGF‐β‐related genes that diminish by 7 days, and genes associated with enhanced cell migration in MRL/MpJ in comparison to B6 tendons. We expect that the timecourse and expression levels of these genes in scarless MRL/MpJ tendon healing represent the balanced environment that leads to improved tendon healing. [ABSTRACT FROM AUTHOR]

Finite element analysis with models derived from computed tomography (CT) scans is potentially powerful as a translational research tool because it can achieve what animal studies and cadaver biomechanics cannot—low‐risk, noninvasive, objective assessment of outcomes in living humans who have actually experienced the injury, or treatment being studied. The purpose of this study was to assess the validity of CT‐based virtual mechanical testing with respect to physical biomechanical tests in a large animal model. Three different tibial osteotomy models were performed on 44 sheep. Data from 33 operated limbs and 20 intact limbs was retrospectively analyzed. Radiographic union scoring was performed on the operated limbs and physical torsional tests were performed on all limbs. Morphometric measures and finite element models were developed from CT scans and virtual torsional tests were performed to assess healing with four material assignment techniques. In correlation analysis, morphometric measures and radiographic scores were unreliable predictors of biomechanical rigidity, while the virtual torsion test results were strongly and significantly correlated with measured biomechanical test data, with high absolute agreement. Overall, the results validated the use of virtual mechanical testing as a reliable in vivo assessment of structural bone healing. This method is readily translatable to clinical evaluation for noninvasive assessment of the healing progress of fractures with minimal risk. Clinical significance: virtual mechanical testing can be used to reliably and noninvasively assess the rigidity of a healing fracture using clinical‐resolution CT scans and that this measure is superior to morphometric and radiographic measures. [ABSTRACT FROM AUTHOR]

Infection is a devastating complication following an open fracture. We investigated whether local rifampin‐loaded hydrogel can combat infection and improve healing in a murine model of methicillin‐resistant Staphylococcus aureus (MRSA) osteomyelitis. A transverse fracture was made at the tibia midshaft of C57BL/6J mice aged 10–12 weeks and stabilized with an intramedullary pin. A total of 1 × 106 colony‐forming units (CFU) of MRSA was inoculated. A collagen‐based hydrogel containing low‐dose (60 μg) and high‐dose (300 μg) rifampin was applied before closure. Postoperative treatment response was assessed through bacterial CFU counts from tissue and hardware, tibial radiographs and microcomputed tomography (μCT), immunohistochemistry, and histological analyses. All untreated MRSA‐infected fractures progressed to nonunion by 28 days with profuse MRSA colonization. Infected fractures demonstrated decreased soft callus formation on safranin O stain compared to controls. Areas of dense interleukin‐1β stain were associated with poor callus formation. High‐dose rifampin hydrogels reduced the average MRSA load in tissue (p < 0.0001) and implants (p = 0.041). Low‐dose rifampin hydrogels reduced tissue bacterial load by 50% (p = 0.021). Among sterile models, 88% achieved union compared to 0% of those infected. Mean radiographic union scale in tibia scores improved from 6 to 8.7 with high‐dose rifampin hydrogel (p = 0.024) and to 10 with combination local/systemic rifampin therapy (p < 0.0001). μCT demonstrated reactive bone formation in MRSA infection. Histology demonstrated restored fracture healing with bacterial elimination. Rifampin‐loaded hydrogels suppressed osteomyelitis, prevented implant colonization, and improved healing. Systemic rifampin was more effective at eliminating infection and improving fracture healing. Further investigation into rifampin‐loaded hydrogels is required to correlate these findings with clinical efficacy. [ABSTRACT FROM AUTHOR]

Currently, there are no standardized methods for quantitatively measuring fracture repair. Physicians rely on subjective physical examinations and qualitative evaluation of radiographs to detect mineralized tissue. Since most fractures heal indirectly through a cartilage intermediate, these tools are limited in their diagnostic utility of early repair. Prior to converting to the bone, cartilage undergoes hypertrophic maturation, characterized by the deposition of a provisional collagen X matrix. The objective of this study was to characterize the kinetics of a novel collagen X biomarker relative to other biological measurements of fracture healing using a murine model of endochondral fracture repair in which a closed, mid‐shaft tibia fracture was created using the classic drop‐weight technique. Serum was collected 5 to 42 days post‐fracture in male and female mice and compared to uninjured controls (n = 8‐12). Collagen X in the serum was quantified using a recently validated ELISA‐based bioassay ("Cxm")1 and compared to genetic and histological markers of fracture healing and inflammation. We found the Cxm biomarker reliably increased from baseline to a statistically unique peak 14 days post‐fracture that then resolved to pre‐fracture levels by 3 weeks following injury. The shape and timing of the Cxm curve followed the genetic and histological expression of collagen X but did not show a strong correlation with local inflammatory states. Assessment of fracture healing progress is crucial to making correct and timely clinical decisions for patients. This Cxm bioassay represents a minimally invasive, inexpensive technique that could provide reliable information on the biology of the fracture to significantly improve clinical care. [ABSTRACT FROM AUTHOR]

Fracture healing is a complex process that relies heavily on the carefully orchestrated expansion and differentiation of periosteal mesenchymal progenitor cells (MSC). Identification of new markers for periosteal MSCs is essential for the development of fracture therapeutics. Expression of the matricellular protein thrombospondin‐2 (TSP2) increases during early fracture healing; however, it is currently unknown what cell population expresses TSP2. Using a TSP2 GFP reporter mouse and a stabilized murine fracture model, we characterized the expression of TSP2 during the inflammatory, soft callus formation, and hard callus formation phases of fracture healing. In addition, using TSP2 GFP positive cells harvested from reporter mouse cells, we characterized the cell population using flow cytometry and colony formation assays. In uninjured diaphyseal bone, we observed TSP2 expression in the cells located along the inner periosteum. We also observed a population of TSP2 expressing cells in undifferentiated regions of early fracture callus and along the periphery of the callus. Later in callus development, TSP2 cells were broadly distributed in the undifferentiated callus, but GFP was not expressed by chondrocytes. Flow cytometry confirmed that the majority of TSP2 expressing cells were positive for traditional murine MSC markers. Our in vitro assays further supported these findings by demonstrating all adherent and colony‐forming cells expressed TSP2. Taken together, our results suggest that TSP2 is expressed by undifferentiated MSCs, but downregulated in chondrocytes. Clinical significance: expression of the matricellular protein TSP2 is a promising new marker to identify MSCs in early fracture healing. [ABSTRACT FROM AUTHOR]

To assess whether far‐cortical locking (FCL) screws alter the fracture site strain environment and allow shorter bridge plate constructs for supracondylar femoral fractures, we tested the fracture site displacement under force of synthetic left femora with a 5‐cm metaphyseal fracture gap, modeling comminution. Five models of nine constructs were tested (three types of diaphyseal screws [nonlocking, locking, and FCL] and two plate lengths [13 holes and 5 holes]). Long plate models using three or four diaphyseal screws (working length 13.5 or 7.5 cm, respectively) were compared with short plates with three diaphyseal screws (working length 7.5 cm). Models were loaded axially and torsionally; 100 cycles in random order. Primary outcome measures were axial and torsional fracture site stiffness. FCL screws decreased rotational stiffness 19% (P <.01) compared with baseline nonlocking screws in the same plate and working length construct, mirroring the effect (20% decrease in stiffness, P <.01) of nearly doubling the nonlocking construct working length (7.5‐13.5 cm). Similarly, FCL screws decreased axial stiffness 23% (P <.01) in the same baseline comparison. Fracture site displacement under loading comparable to a long working length nonlocked plate construct was achieved using a shorter FCL plate construct. By closely replicating the biomechanical properties of a long plate construct, a fracture site strain environment considered favorable in promoting fracture healing might still be achievable using a shorter plate length. Clinical Significance: It might be possible to optimize fracture site strain environment and displacement under loading using shorter FCL plate constructs. Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 00:00–00, 2020. [ABSTRACT FROM AUTHOR]

Fracture healing involves interactions of different cell types, driven by various growth factors, and signaling cascades. Periosteal mesenchymal progenitor cells give rise to the majority of osteoblasts and chondrocytes in a fracture callus. Notch signaling has emerged as an important regulator of skeletal cell proliferation and differentiation. We investigated the effects of Notch signaling during the fracture healing process. Increased Notch signaling in osteochondroprogenitor cells driven by overexpression of Notch1 intracellular domain (NICD1) (αSMACreERT2 mice crossed with Rosa‐NICD1) during fracture resulted in less cartilage, more mineralized callus tissue, and stronger and stiffer bones after 3 weeks. Periosteal cells overexpressing NICD1 showed increased proliferation and migration in vitro. In vivo data confirmed that increased Notch1 signaling caused expansion of alpha‐smooth muscle actin (αSMA)‐positive cells and their progeny including αSMA‐derived osteoblasts in the callus without affecting osteoclast numbers. In contrast, anti‐NRR1 antibody treatment to inhibit Notch1 signaling resulted in increased callus cartilage area, reduced callus bone mass, and reduced biomechanical strength. Our study shows a positive effect of induced Notch1 signaling on the fracture healing process, suggesting that stimulating the Notch pathway could be beneficial for fracture repair. [ABSTRACT FROM AUTHOR]

The majority of fractures, especially in elderly and osteoporotic patients, occurs in metaphyseal bone. However, only a few experimental models exist to study metaphyseal bone healing in mice. Currently used mouse models of metaphyseal fracture healing are either based on drill hole defects, lacking adequate biomechanical stimulation at the site of fracture and therefore endochondral ossification in the fracture callus, or are introduced into the distal part of the mouse femur stabilized by a locking plate, which is challenging due to the small specimen size. Therefore, the aim of the current study was to develop a new mouse model to study metaphyseal fracture healing of the proximal femur. We chose a combination between an open osteotomy and a closed intramedullary stabilization. A 24 G needle was inserted into the femur in a closed manner, then an osteotomy was made with a 0.4‐mm Gigli wire saw between the third and the lesser trochanter of the femur using an open approach. Fractured femurs were analyzed using microcomputed tomography and histology at days 14 and 21 after surgery. No animals were lost due to surgery or anesthesia. All animals displayed normal limb loading and a physiological gait pattern within the first three days after fracture. We found robust endochondral ossification during the fracture healing process with high expression of late chondrocyte and early osteogenic markers at day 14 (d14). By day 21 (d21), all fractures had a bony bridging score of 3 or more, indicating successful healing. Callus volume significantly decreased from d14 to d21, whereas high numbers of osteoclasts appeared at the fracture callus until d21, indicating that callus remodeling had already started at d21. In conclusion, we successfully developed a novel mouse model to study endochondral fracture healing of the proximal femur. This model might be useful for future studies using transgenic animals to unravel molecular mechanisms of osteoporotic metaphyseal fracture healing. [ABSTRACT FROM AUTHOR]

Platelet‐rich plasma (PRP) is an increasingly widespread treatment for joint pathologies. Its characteristics and administration route are variables that may influence the clinical outcome. The aim of this in vivo study was to analyze in aged rats the biological and structure effects of intraosseous infiltrations of two different types of PRP obtained from young and old donors. During 6 months intraosseous infiltrations were performed and 4 days after the last infiltration, animals were sacrificed, and bones were extracted for micro‐computed tomography (micro‐CT) and histological analysis. Molecular composition of the PRP of aged donors presented higher levels of proinflammatory molecules. The histological studies showed a greater cellularity of bone marrow in groups treated with PRP. Concerning micro‐CT analysis, young PRP showed a better femoral bone structure according to values of percentage of trabecular bone, trabecular space, trabecular density, and subchondral bone plate volume. In summary, this study has demonstrated that intraosseous infiltrations of PRP from young donors prevent from age‐related bone degeneration. This treatment could stimulate the biological processes that maintain homeostasis and bone structure and avoid osteoarticular pathologies. [ABSTRACT FROM AUTHOR]

Focused high‐energy extracorporeal shockwave therapy (fhESWT) is used to improve fracture healing in cases of nonunion. In addition, it has been shown to have direct antibacterial effects. We evaluated fhESWT as an adjunct to conventional treatment in a clinically relevant rabbit model of fracture‐related infection (FRI). A humeral osteotomy in 31 rabbits was fixed with a seven‐hole locking compression plate. FRI was established with a clinical Staphylococcus aureus isolate. After 2 weeks, a revision surgery was performed with debridement, irrigation, and implant retention. Rabbits then received: no further treatment (controls); shockwaves (4000 impulses with 23 kV at days 2 and 6 after revision); systemic antibiotics (rifampin and nafcillin); or the combination of antibiotics and shockwaves. Treatments were applied over 1 week. Blood cultures were taken before and after shockwave sessions. After another week without treatment, rabbits were euthanized and quantitative bacteriology was performed on implants and tissues to determine infection burden. Indicator organs (brain, heart, liver, lungs, kidneys, and spleen) were cultured to assess possible bacteremia. All the rabbits were infected at revision surgery as determined by the bacteriological culture of debrided materials. fhESWT in combination with antibiotic treatment lowered the bacterial burden 100‐fold compared with antibiotic treatment alone in all samples (P =.38). This effect was most prevalent for the implant sample (P =.08). No significant effect was seen for fhESWT alone compared with untreated controls. No signs of bacteremia occurred in blood cultures and organs. fhESWT appears safe and could be a helpful adjunct to conventional treatment in certain difficult‐to‐treat FRIs. [ABSTRACT FROM AUTHOR]

C1q/TNF‐related protein 3 (CTRP3) is a cytokine known to regulate a variety of metabolic processes. Though previously undescribed in the context of bone regeneration, high throughput gene expression experiments in mice identified CTRP3 as one of the most highly upregulated genes in fracture callus tissue. Hypothesizing a positive regulatory role for CTRP3 in bone regeneration, we phenotyped skeletal development and fracture healing in CTRP3 knockout (KO) and CTRP3 overexpressing transgenic (TG) mice relative to wild‐type (WT) control animals. CTRP3 KO mice experienced delayed endochondral fracture healing, resulting in abnormal mineral distribution, the presence of periosteal marrow compartments, and a nonunion‐like state. Decreased osteoclast number was also observed in CTRP3 KO mice, whereas CTRP3 TG mice underwent accelerated callus remodeling. Gene expression profiling revealed a broad impact on osteoblast/osteoclast lineage commitment and metabolism, including arrested progression toward mature skeletal lineages in the KO group. A single systemic injection of CTRP3 protein at the time of fracture was insufficient to phenocopy the chronic TG healing response in WT mice. By associating CTRP3 levels with fracture healing progression, these data identify a novel protein family with potential therapeutic and diagnostic value. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:00‐19966, 2020 [ABSTRACT FROM AUTHOR]

Conditional deletion of the transcription factor Runt‐related transcription factor 1 (Runx1) in myeloid osteoclast precursors promotes osteoclastogenesis and subsequent bone loss. This study posits whether Runx1 regulates clastic cell‐mediated bone and cartilage resorption in the fracture callus. We first generated mice, in which Runx1 was conditionally abrogated in osteoclast precursors (LysM‐Cre;Runx1F/F; Runx1 cKO). Runx1 cKO and control mice were then subjected to experimental mid‐diaphyseal femoral fractures. Our study found differential resorption of bony and calcified cartilage callus matrix by osteoclasts and chondroclasts within Runx1 cKO calluses, with increased early bony callus resorption and delayed calcified cartilage resorption. There was an increased number of osteoclasts and chondroclasts in the chondro‐osseous junction of Runx1 cKO calluses starting at day 11 post‐fracture, with minimal woven bone occupying the callus at day 18 post‐fracture. LysM‐Cre;Runx1F/F mutant mice had increased bone compliance at day 28, but their strength and work to failure were comparable with controls. Taken together, these results indicate that Runx1 is a critical transcription factor in controlling osteoclastogenesis that negatively regulates bone and cartilage resorption in the fracture callus. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1007‐1015, 2020 [ABSTRACT FROM AUTHOR]

Impaired fracture healing continues to be a significant public health issue. This is more frequently observed in aging populations and patients with co‐morbidities that can directly influence bone repair. Tremendous progress has been made in the development of biologics to enhance and accelerate the healing process; however, side‐effects persist that can cause significant discomfort and tissue damage. This has been the impetus for the development of safe and natural strategies to hasten natural bone healing. Of the many possible approaches, nutrition represents a safe, affordable, and non‐invasive strategy to positively influence each phase of fracture repair. However, our understanding of how healing can be hindered by malnutrition or enhanced with nutritional supplementation has lagged behind the advancements in both surgical management and the knowledge of molecular and cellular drivers of skeletal fracture repair. This review serves to bridge this knowledge gap as well as define the importance of nutrition during fracture healing. The extant literature clearly indicates that pre‐existing nutritional deficiencies should be corrected, and nutritional status should be carefully monitored to prevent the development of malnutrition for the best possible healing outcome. It remains unclear, however, whether the provision of nutrients beyond sufficiency has any benefit on fracture repair and patient outcomes. The combined body of pre‐clinical studies using a variety of animal models suggests a promising role of nutrition as an adjuvant therapy to facilitate fracture repair, but extensive research is needed, specifically at the clinical level, to clarify the utility of nutritional interventions in orthopedics. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:695‐707, 2020 [ABSTRACT FROM AUTHOR]

Neither allograft nor commercially available bone graft substitutes provide the same quality of bone healing as autograft. Incorporation of bioactive molecules like parathyroid hormone (PTH) within bone graft substitute materials may provide similar, if not better treatment options to grafting. The goal of this work was to develop a biomaterial system for the local delivery of PTH to large bone defects for promoting bone regeneration. PTH was loaded in a thiol‐ene hydrogel at several concentrations and polymerized in and around an osteoconductive poly(propylene fumarate) (PPF) scaffold. PTH was shown to be bioactive when released from the hydrogel for up to 21 days. Eighty percent of the PTH was released by day 3 with the remaining 20% released by day 14. Bone healing was quantified in rat critical size femoral defects that were treated with hydrogel/PPF and 0, 1, 3, 10, or 30 µg of PTH. Although complete osseous healing was not observed in all samples in any one treatment group, all samples in the 10 µg PTH group were bridged fully by bone or a combination of bone and cartilage containing hypertrophic chondrocytes and endochondral ossification. Outcome measures indicated improved defect bridging by a combination of bony and cartilaginous tissue in the 10 μg treatment group compared with empty bone defects and defects treated with only hydrogel/PPF (i.e., without PTH). Given the tailorability of the hydrogel, future studies will investigate the effects of prolonged gradual PTH release on bone healing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:536–544, 2020 [ABSTRACT FROM AUTHOR]

Sarcopenia is highly prevalent in fragility fracture patients and is associated with delayed healing. In this study, we investigated the effect of low‐magnitude high‐frequency vibration (LMHFV) on osteoporotic fracture with sarcopenia and the potential role of myostatin. Osteoporotic fractures created in sarcopenic SAMP8, non‐sarcopenic SAMR1 were randomized to control or LMHFV (SAMP8, SAMR1, SAMP8‐V, or SAMR1‐V) groups. Healing and myostatin expression were evaluated at 2, 4, and 6 weeks post‐fracture. In vitro, conditioned‐media were collected from myofibers isolated from aged and young SAMP8 or C2C12 myoblasts with or without LMHFV. Osteoblastic MC3T3‐E1 under osteogenic differentiation were treated with plain or conditioned‐medium (±myostatin propeptide). LMHFV significantly enhanced callus formation was in non‐sarcopenic SAMR1 mice; but the enhancement effect was not significant in SAMP8 mice at week 2. Myostatin expressions in callus and biceps femoris of SAMP8 group were significantly higher all groups with significant negative correlation with callus size (R2 = 0.7256; p = 0.0004). Mechanical properties (week 4) and callus remodeling (week 6) were inferior in SAMP8 versus SAMR1 and were significantly enhanced by LMHFV. Alkaline Phosphatase (ALP) and Runx2 expression of MC3T3‐E1 was lower in aged myofiber compared with young, but upregulated by LMHFV or myostatin inhibition; also confirmed with C2C12. LMHFV enhanced early callus formation, microarchitecture, callus remodeling and mechanical properties of fracture healing in both SAMP8 and SAMR1; however, more effective in non‐sarcopenic SAMR1 mice. Impaired fracture healing in sarcopenic SAMP8 mice is attributed by elevated myostatin expression in callus and muscle, which correlated negatively with callus formation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:277‐287, 2020 [ABSTRACT FROM AUTHOR]

Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Plasminogen activator inhibitor 1 (PAI‐1/SERPINE1), a master suppressor of fibrinolysis and protease activity, is associated with adhesions. Here, we used next‐generation RNA sequencing (RNA‐Seq) to assess genome‐wide differences in messenger RNA expression due to PAI‐1 deficiency after zone II flexor tendon injury. We used the ingenuity pathway analysis to characterize molecular pathways and biological drivers associated with differentially expressed genes (DEG). Analysis of hundreds of overlapping and DEG in PAI‐1 knockout (KO) and wild‐type mice (C57Bl/6J) during tendon healing revealed common and distinct biological processes. Pathway analysis identified cell proliferation, survival, and senescence, as well as chronic inflammation as potential drivers of fibrotic healing and adhesions in injured tendons. Importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1‐associated biological processes as unique transcriptional signatures of the healing tendon in the PAI‐1/Serpine1 KO mice. Further, transcriptomic differences due to the genetic deletion of PAI‐1 were mechanistically linked to PI3K/Akt/mTOR, PKC, and MAPK signaling cascades. These transcriptional observations provide novel insights into the biological roles of PAI‐1 in tendon healing and could identify therapeutic targets to achieve scar‐free regenerative healing of tendons. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:43–58, 2020 [ABSTRACT FROM AUTHOR]

There is clinical evidence that patient‐specific comorbidities like osteoporosis, concomitant tissue injury, and ischemia may strongly interfere with bone regeneration. However, underlying mechanisms are still unclear. To study these mechanisms in detail, appropriate animal models are needed. For decades, bone healing has been studied in large animals, including dogs, rabbits, pigs, or sheep. However, large animal models display a limited ability to study molecular pathways and cellular functions. Therefore in recent years, mice and rats have become increasingly popular as a model organism for fracture healing research due to the availability of molecular analysis tools and transgenic models. Both large and small animals can be used to study comorbidities and risk factors, modelling the human clinical situation. However, attention has to be paid when choosing an appropriate model due to species differences between large animals, rodents, and humans. This review focuses on large and small animal models for the common comorbidities ischemic injury/reduced vascularization, osteoporosis, and polytrauma, and critically discusses the translational and molecular aspects of these models. Here, we review material which was presented at the workshop "Animal Models of Comorbidities in Fracture Healing Research" at the 2019 ORS Annual Meeting in Austin Texas. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2491–2498, 2019 [ABSTRACT FROM AUTHOR]

Owing to the central role of osteoclasts in bone physiology and remodeling, manipulation of their maturation process provides a potential therapeutic strategy for treating bone diseases. To investigate this, we genetically inhibited the Notch signaling pathway in the myeloid lineage, which includes osteoclast precursors, using a dominant negative form of MAML (dnMAML) that inhibits the transcriptional complex required for downstream Notch signaling. Osteoclasts derived from dnMAML mice showed no significant differences in early osteoclastic gene expression compared to the wild type. Further, these demonstrated significantly lowered resorption activity using bone surfaces while retaining their osteoblast stimulating ability using ex vivo techniques. Using in vivo approaches, we detected significantly higher bone formation rates and osteoblast gene expression in dnMAML cohorts. Further, these mice exhibited increased bone/tissue mineral density compared to wild type and larger bony calluses in later stages of fracture healing. These observations suggest that therapeutic suppression of osteoclast Notch signaling could reduce, but not eliminate, osteoclastic resorption without suppression of restorative bone remodeling and, therefore, presents a balanced paradigm for increasing bone formation, regeneration, and healing. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2089–2103, 2019 [ABSTRACT FROM AUTHOR]

Current diagnostic modalities, such as radiographs or computed tomography, exhibit limited ability to predict the outcome of bone fracture healing. Failed fracture healing after orthopaedic surgical treatments are typically treated by secondary surgery; however, the negative correlation of time between primary and secondary surgeries with resultant health outcome and medical cost accumulation drives the need for improved diagnostic tools. This study describes the simultaneous use of multiple (n = 5) implantable flexible substrate wireless microelectromechanical (fsBioMEMS) sensors adhered to an intramedullary nail (IMN) to quantify the biomechanical environment along the length of fracture fixation hardware during simulated healing in ex vivo ovine tibiae. This study further describes the development of an antenna array for interrogation of five fsBioMEMS sensors simultaneously, and quantifies the ability of these sensors to transmit signal through overlaying soft tissues. The ex vivo data indicated significant differences associated with sensor location on the IMN (p < 0.01) and fracture state (p < 0.01). These data indicate that the fsBioMEMS sensor can serve as a tool to diagnose the current state of fracture healing, and further supports the use of the fsBioMEMS as a means to predict fracture healing due to the known existence of latency between changes in fracture site material properties and radiographic changes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1873–1880, 2019 [ABSTRACT FROM AUTHOR]

A significant number of fractures develop non‐union. Mesenchymal stem cell (MSC) therapy may be beneficial, however, this requires cell acquisition, culture and delivery. Endogenous mobilization of stem cells offers a non‐invasive alternative. The hypothesis was administration of VEGF and the CXCR4 antagonist AMD3100 would increase the circulating pool of available MSCs and improve fracture healing. Ex‐breeder female wistar rats received VEGF followed by AMD3100, or sham PBS. Blood prepared for culture and colonies were counted. P3 cells were analyzed by flow cytometry, bi‐differentiation. The effect of mobilization on fracture healing was evaluated with 1.5 mm femoral osteotomy stabilized with an external fixator in 12–14 week old female Wistars. The mobilized group had significantly greater number of cfus/ml compared to controls, p = 0.029. The isolated cells expressed 1.8% CD34, 35% CD45, 61% CD29, 78% CD90, and differentiated into osteoblasts but not into adipocytes. The fracture gap in animals treated with VEGF and AMD3100 showed increased bone volume; 5.22 ± 1.7 µm3 and trabecular thickness 0.05 ± 0.01 µm compared with control animals (4.3 ± 3.1 µm3, 0.04 ± 0.01 µm, respectively). Radiographic scores quantifying fracture healing (RUST) showed that the animals in the mobilization group had a higher healing score compared to controls (9.6 vs. 7.7). Histologically, mobilization resulted in significantly lower group variability in bone formation (p = 0.032) and greater amounts of bone and less fibrous tissue than the control group. Clinical significance: This pre‐clinical study demonstrates a beneficial effect of endogenous MSC mobilization on fracture healing, which may have translation potential to prevent or treat clinical fractures at risk of delayed or non‐union fractures. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1294–1302, 2019. [ABSTRACT FROM AUTHOR]

The study reports the prospective outcome of treating severe recalcitrant fracture nonunion in patients with autologous bone marrow‐derived mesenchymal stromal cells (BMSC) from 2003 to 2010 and analyze predictors of union. Autologous BMSC were culture expanded and inserted at nonunion site with or without carriers in addition to surgical stabilization of the fracture. Radiological union was ascertained by musculoskeletal radiologists on plain radiographs and/or CT scans. A logistic regression analysis was performed with cell‐expansion parameters (cell numbers, cell doubling time) and known clinical factors (e.g., smoking and diabetes) as independent variables and fracture union as the dependent variable to identify the factors that influence bony healing. An Eq5D index score assessed the effect of treatment on general quality of health. A total of 35 patients (mean age 51+/−13 years) with established nonunion (median 2.9 years, 1–33) and, at least one failed nonunion surgery (median 4,1–14) received treatment. Fracture union was achieved in 21 patients (60%; 95%CI 44–75) at 2.6 years. Multiple penalized logistic regression revealed faster cell doubling time (p = 0.07), absence of diabetes (p = 0.003), less previous surgeries (p = 0.008), and lower age at cell implantation (p = 0.02) were significant predictors for fracture union. A significant increase in Eq5D index (p = 0.01) was noted with a mean rise of the score by 0.34 units (95%CI 0.11–0.58) at 1 year following the study. In summary, the study revealed cell doubling time as a novel in vitro parameter in conjunction with age, multiple surgeries, and diabetes as being significant predictors of the fracture union. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1303–1309, 2019. [ABSTRACT FROM AUTHOR]