Volume and density of fracture callus are important outcome parameters in fracture healing studies. These values provide an indication for the recovery of the mechanical function of the bone. Traditionally, fracture callus’ have been evaluated from radiographs, which represent 2D projections of the three-dimensional structures, therefore such an analysis can be affected by many artefacts. With the availability of Computer Tomography (CT) scanners for the evaluation of healing bones, it is now possible to perform precise, three-dimensional reconstructions of the fracture callus and therefore to evaluate true three-dimensional callus volumes and bone mineral densities. We wanted to make use of this technology in the evaluation of a study looking at the healing of a multifragmentary fracture in sheep after 4 and 8 weeks of healing time (Wullschleger et al, ANZORS, 2006). Our goal was to develop a protocol that would allow for the standardised calculation of cortical bone and callus tissue volumes with minimal user influence. Here, we report on the development of this evaluation protocol and some early results. A clinical CT scanner was used to scan the experimental limbs, immediately after the sheep had been euthanized. Further analysis of the CT dataset was accomplished with the commercial computer software Amira. The region of interest was cropped to a 9 cm section of the bone shaft, guaranteed to comprise the entire fracture callus. Next, the cortical bone and the callus tissue were segmented by choosing appropriate threshold values for the measured grey levels. The volume of the segmented regions was then calculated by the software. The application of this protocol to six CT scans from our experimental study resulted in average callus volumes of 12.21 ± 1.96 (standard deviation) cm2 after 4 weeks healing time and 14.28 ± 1.58 cm2 after 8 weeks healing time. In conclusion, we demonstrated the feasibility of using CT data for a quantitative 3D analysis of callus volumes. While this technique is undoubtedly superior to the estimation of callus volumes from two-dimensional radiographs, the absolute accuracy of the results will need to be determined by comparison with histological data

Volume and density of fracture callus are important outcome parameters in fracture healing studies. These values provide an indication for the recovery of the mechanical function of the bone. Traditionally, fracture callus’ have been evaluated from radiographs, which represent 2D projections of the three-dimensional structures; therefore such an analysis can be affected by many artefacts. With the availability of Computer Tomography (CT) scanners for the evaluation of healing bones, it is now possible to perform precise, three-dimensional reconstructions of the fracture callus and therefore to evaluate true three-dimensional callus volumes and bone mineral densities. We wanted to make use of this technology in the evaluation of a study looking at the healing of a multifragmentary fracture in sheep after 4 and 8 weeks of healing time (Wullschleger et al, ANZORS, 2006). Our goal was to develop a protocol that would allow for the standardised calculation of cortical bone and callus tissue volumes with minimal user influence. Here, we report on the development of this evaluation protocol and some early results. A clinical CT scanner was used to scan the experimental limbs, immediately after the sheep had been euthanized. Further analysis of the CT dataset was accomplished with the commercial computer software Amira. The region of interest was cropped to a 9 cm section of the bone shaft, guaranteed to comprise the entire fracture callus. Next, the cortical bone and the callus tissue were segmented by choosing appropriate threshold values for the measured grey levels. The volume of the segmented regions was then calculated by the software. The application of this protocol to six CT scans from our experimental study resulted in average callus volumes of 12.21 ± 1.96 (standard deviation) cm2 after 4 weeks healing time and 14.28 ± 1.58 cm2 after 8 weeks healing time. In conclusion, we demonstrated the feasibility of using CT data for a quantitative 3D analysis of callus volumes. While this technique is undoubtedly superior to the estimation of callus volumes from two-dimensional radiographs, the absolute accuracy of the results will need to be determined by comparison with histological data

Aims. Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy. Methods. Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high-fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analysis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq). Results. Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA-Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation. Conclusion. These findings suggest that elevated or prolonged inflammation contributes to delayed fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients. Cite this article: Bone Joint Res 2023;12(10):657–666

Infections regularly complicate orthopaedic procedures loosing implant stability and impairing bone union. Nevertheless, the question whether infection is caused by pathogens transposed intraoperatively, infiltrating the implant with blood stream or lymph, or dwelling in clinically healthy tissues, remains unanswered. The AIM of our study was to validate the hypothesis that pathogens may residue deep tissue. Material and Methods: Skin, subcutaneous fat, muscle and fracture gap callus were obtained from 155 adult patients operated on due to closed comminuted fractures of tibia or femur, 75 because of non-alignment of bone axis and 80 due to delayed fracture healing. Results: Aerobic bacteria were isolated from gap callus of 12% healing and 31% non-healing fractures, but also from deep soft tissues. No anaerobic bacteria were detected. PCR amplifications of 16s rRNA were found positive in 40% of callus specimens proving presence of bacterial DNA even when no isolates were found. The 95% similarity of the genetic pattern of some strains from foot skin and callus, estimated with RAPD technique, suggested their foot skin origin. Conclusions: The colonizing bacteria and their DNA were detected in fracture callus and deep soft tissues. Contamination was precluded by lack of isolates in disinfected skin and materials used for sampling cultured after surgery. Our results point out that bacterial cells residing clinically non-infected deep tissues may be a source of infection, when activated by mechanical trauma and/or orthopaedic implant insertion

Aims: The use of vitamin K was proposed in the treatment of osteoporosis. Some experimental studies suggested that vitamin K might promote mesenchymal stem cells (MSCs) differentiation into osteoblasts progenitors and inhibit osteoclasts formation. In the present study we analysed the effects of vitamin K at different concentrations on human mesenchymal stem cells derived from fracture callus. Methods: MSCs were obtained from the fracture’s site of three patients during surgical operation of osteosynthesis. Cells were grown on plastic plates in DMEM, 10% foetal bovine serum (FBS), 1% penicillin-streptomycin, 1% fungizone, 5mM beta-glycerophosphate and 50 microg/ml ascorbic acid. Half of the samples was incubated with vitamin D (10 nm) and K at different concentrations (1, 3, 10 microM). Proliferation rate (MTT colorimetric assay) and cell differentiation (FACSCalibur flow cytometry) were assessed at 3, 10 and 20 days. Immunocytochemical analysis (not-carboxylated osteocalcin and carboxylated osteocalcin) was also performed. Results: MSCs stimulated with vitamin K and D expressed higher levels of osteoblastic markers than controls at 3, 10 and 20 days of colture. Conclusions: This study confirmed the results obtained in previous in vitro experiments: vitamin K has osteoinductive properties on MSCs derived from fracture callus and it could play a role in reparative osteogenesis in vivo

The multifunctional adhesion molecule CD44 is a major cell-surface receptor for hyaluronic acid (HUA). Recent data suggest that it may also bind the ubiquitous bone-matrix protein, osteopontin (OPN). Because OPN has been shown to be a potentially important protein in bone remodelling, we investigated the hypothesis that OPN interactions with the CD44 receptor on bone cells participate in the regulation of the healing of fractures. We examined the spatial and temporal patterns of expression of OPN and CD44 in healing fractures of rat femora by in situ hybridisation and immunohistochemistry. We also localised HUA in the fracture callus using biotinylated HUA-binding protein. OPN was expressed in remodelling areas of the hard callus and was found in osteocytes, osteoclasts and osteoprogenitor cells, but not in cuboidal osteoblasts which were otherwise shown to express osteocalcin. The OPN signal in osteocytes was not uniformly distributed, but was restricted to specific regions near sites where OPN mRNA-positive osteoclasts were attached to bone surfaces. In the remodelling callus, intense immunostaining for CD44 was detected in osteocyte lacunae, along canaliculi, and on the basolateral plasma membrane of osteoclasts, but not in the cuboidal osteoblasts. HUA staining was detected in fibrous tissues but little was observed in areas of hard callus where bone remodelling was progressing. Our findings suggest that OPN, rather than HUA, is the major ligand for CD44 on bone cells in the remodelling phase of healing of fractures. They also raise the possibility that such interactions may be involved in the communication of osteocytes with each other and with osteoclasts on bone surfaces. The interactions between CD44 and OPN may have important clinical implications in the repair of skeletal tissues

1. Autografts, isografts and homografts of fibrocartilaginous callus were observed in the anterior chamber of the eye in rats. Proliferation of cartilage ceased, endochondral ossification followed, and the end-product was a new and complete ossicle with a cortex and a marrow cavity. The size and shape of the ossicle was determined by the size and shape of the sample of callus. Thus the callus in the eye performed the function of a cartilage model like that of the developing epiphysis or a healing fracture of a long bone.

2. Fibrocartilaginous callus, heavily labelled with ³H-thymidine, was transplanted to the eye twenty-four hours after the last injection, when there was little if any radioactive thymidine circulating in the blood. A few small chondrocytes with labelled nuclei persisted in the cores of new bone trabeculae, but the largest part of the labelled callus was resorbed and replaced by unlabelled new bone.

3. Homografts of labelled callus produced the same results as autografts at twenty-five days, but between twenty-five and forty-five days the donor cells were destroyed by the immune response of the host.

4. Isogenous transplants in host rats treated with ³H-thymidine between nine and thirteen days, when the callus was invaded by new blood vessels, produced many osteogenetic cells with labelled nuclei and made it possible to trace the origin of the new bone. The label appeared in the progenitor cells within twenty-four hours. While remaining thereafter in progenitor cells, it appeared also in osteoclasts (or chondroclasts) and osteoblasts in forty-eight to seventy-two hours, and in osteocytes in ninety-six to 120 hours. Chondrocytes did not proliferate and were not labelled in the eye.

5. Homogenous transplants in host rats treated with ³H-thymidine between five and one days before the operation also produced new bone, but contained no labelled osteoprogenitor or bone cells after twenty-five days in the eye. At forty-five days the donor tissue had been destroyed by the immune response of the host.

6. Devitalised callus was encapsulated in inflammatory connective tissue and scar. When the dead callus was absorbed by the capillaries of the host new bone formation by induction produced a scanty deposit as a delayed event in a few instances.

7. Irrespective of whether it originated in the donor or the host, a connective-tissue cell type that proliferated rapidly and became labelled with ³H-thymidine was identified as a progenitor cell. Differentiation and specialisation as osteoprogenitor cells occurred after the growth of blood vessels into the interior of the callus, and developed inside of excavation chambers in cartilage. Except that the interaction of the donor tissue and host cells leading to new bone formation by induction takes place in the interior of the excavation chamber, the biophysico-chemical mechanism is unknown.

Summary Statement

The Dkk3-derived cells represent a branch of the periosteal mesenchymal lineage that produces fibrocartilage as well as regenerating the periosteal structures.

Secondary fracture healing processes are strongly influenced by interfragmentary motion. Shear movement is assumed to be more critical than axial movement, however experimental results are controversial. Numerical fracture healing models allow to simulate the fracture healing process with variation of single input parameters and under comparable normalized mechanical conditions. Therefore, a direct comparison of different in vivo scenarios is possible. The aim of this study was to simulate fracture healing under several axial and shear movement scenarios and compare their respective time to heal. We hypothesize that shear movement is always more critical than axial loading. For the presented study, we used a corroborated numerical model for fracture healing in sheep. Numerous variations of the movement amplitude, the fracture gap size and the musculoskeletal loads were simulated for comparable axial compressive and shear load cases. In all simulated cases, axial compressive load had less inhibitory influences on the healing process than shear load. Therefore, shear loading is more critical for the fracture healing outcome in general. Thus, our findings suggest osteosynthesis implants to be optimized to limit shear movements under musculoskeletal loading.

Virtual mechanical testing is a method for measuring bone healing using finite element models built from computed tomography (CT) scans. Previously, we validated a dual-zone material model for ovine fracture callus that differentiates between mineralized woven bone and soft tissue based on radiodensity. 1. The objective of this study was to translate the dual-zone material model from sheep to two important clinical scenarios: human tibial fractures in early-stage healing and late-stage nonunions. CT scans for N = 19 tibial shaft fractures were obtained prospectively at 12 weeks post-op. A second group of N = 33 tibial nonunions with CT scans were retrospectively identified. The modeling techniques were based on our published method. 2. The dual-zone material model was implemented for humans by performing a cutoff sweep for both the 12-week and nonunion groups. Virtual torsional rigidity (VTR) was calculated as VTR = ML/φ [N-m. 2. /°], where M is the moment reaction, L is the diaphyseal segment length, and φ is the angle of twist. As the soft tissue cutoff was increased, the rigidity of the clinical fractures decreased and soft tissue located within the fracture gaps produced higher strains that are not predicted without the dual zone approach. The structural integrity of the nonunions varied, ranging from very low rigidities in atrophic cases to very high rigidities in highly calcified hypertrophic cases, even with dual-zone material modeling. Human fracture calluses are heterogeneous, comprising of woven bone and interstitial soft tissue. Use of a dual-zone callus material model may be instrumental in identifying delayed unions during early healing when callus formation is minimal and/or predominantly fibrous with little mineralization. ACKNOWLEDGEMENTS:. This work was supported by the National Science Foundation (NSF) grant CMMI-1943287

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms. Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2. -/-. ). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR. Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones. Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing

Mice are increasingly used for fracture healing research because of the possibility to use transgenic animals to conduct research on the molecular level. Mice from both sexes can be used, however, there is no consensus in the literature if fracture healing differs between female and male mice. Therefore, the aim of the present study was to analyze the similarities and differences in endochondral fracture healing between female and male C57BL/6J mice, since this mouse strain is mainly used in bone research. For that purpose, 12-weeks-old female and male mice received a standardized femur midshaft osteotomy stabilized by an external fixator. Mice were euthanized 10 and 21 days after fracture and bone regeneration was analyzed by biomechanical testing, µCT analysis, histology, immunohistochemistry and gene expression analysis. At day 21, male mice displayed a significantly larger fracture callus than female mice accompanied by higher number of osteoclasts, higher tissue mineral density and absolute values of bone volume, whereas relative bone volume to tissue volume ratio did not differ between the groups. Biomechanical testing revealed significantly increased bending stiffness in both fractured and intact femurs from male vs. female mice, whereas relative bending stiffness of fractured femurs related to the intact femurs did not differ. 10 days after fracture, male mice display significantly more cartilage and less fibrous tissue area in the fracture callus than female mice, whereas bone area did not differ. On the molecular level, male mice displayed increased active β-catenin expression in the fracture callus, whereas estrogen receptor α (ERα) expression was reduced. In conclusion, male mice showed more prominent cartilaginous callus formation, increased mineralization and whole callus tissue formation, whereas functional outcome after fracture did not differ from female mice. This might be due either to the heavier weight of male mice or because of differences in molecular signaling pathways

Objectives. Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown. Methods. We examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-κB on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay. Results. The expression levels of WW Domain Containing E3 Ubiquitin Protein Ligase 1 (Wwp1), SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1), SMAD Specific E3 Ubiquitin Protein Ligase 2 (Smurf2) and Itch were all significantly increased in the fracture callus of WT mice, which was associated with elevated expression of NF-κB members and total ubiquitinated proteins. Callus tissue isolated from Itch KO mice expressed higher levels of osteoblast-associated genes, including Runx2, a positive regulator of osteoblast differentiation, but osteoclast-associated genes were not increased. Both NF-κB RelA and RelB proteins were found to bind to the NF-κB binding site in the mouse Itch promoter. Conclusions. Our findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus. Thus, ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing. Cite this article: J. Liu, X. Li, H. Zhang, R. Gu, Z. Wang, Z. Gao, L. Xing. Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins. Bone Joint Res 2017;6:154–161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1

Secondary bone healing is impacted by the extent of interfragmentary motion at the fracture site. It provides mechanical stimulus that is required for the formation of fracture callus. In clinical settings, interfragmentary motion is induced by physiological loading of the broken bone – for example, by weight-bearing. However, there is no consensus about when mechanical stimuli should be applied to achieve fast and robust healing response. Therefore, this study aims to identify the effect of the immediate and delayed application of mechanical stimuli on secondary bone healing. A partial tibial osteotomy was created in twelve Swiss White Alpine sheep and stabilized using an active external fixator that induced well-controlled interfragmentary motion in form of a strain gradient. Animals were randomly assigned into two groups which mimicked early (immediate group) and late (delayed group) weight-bearing. The immediate group received daily stimulation (1000 cycles/day) from the first day post-op and the delayed group from the 22nd day post-op. Healing progression was evaluated by measurements of the stiffness of the repair tissue during mechanical stimulation and by quantifying callus area on weekly radiographs. At the end of the five weeks period, callus volume was measured on the post-mortem high-resolution computer tomography (HRCT) scan. Stiffness of the repair tissue (p<0.05) and callus progression (p<0.01) on weekly radiographs were significantly larger for the immediate group compared to the delayed group. The callus volume measured on the HRCT was nearly 3.2 times larger for the immediate group than for the delayed group (p<0.01). This study demonstrates that the absence of immediate mechanical stimuli delays callus formation, and that mechanical stimulation already applied in the early post-op phase promotes bone healing

Cyclooxygenase-2 (COX-2) activity is necessary for fracture healing to proceed normally. In most cell types, COX-2 is inductively expressed and acts in a coordinated pathway to produce prostaglandins, which affect many physiological processes including inflammation. In the fracture callus, however, COX-2 expression and the molecular and cellular processes affected by COX-2 activity remain poorly understood. Using LC-MS/MS and xMAP, we measured fracture callus prostaglandin and inflammatory cytokine levels. We found that inflammatory cytokines rapidly peaked after fracture before declining to normal levels by day 4 after fracture. However, callus prostaglandin levels did not peak until 4 days after fracture before returning to normal levels by day 10. We used immunohistochemistry to detected COX-2 expression in callus cells and found that COX-2 was expressed in callus chondrocytes and osteoclasts during endochondral ossification, including those osteoclasts at the callus chondro-osseous junction. Targeted deletion of the COX-2 gene (Ptgs2) in osteoclasts or in chondrocytes was found to delay fracture healing. Using cell-based experiments, we found that COX-2 expression could be induced in osteoclasts by osteopontin treatment, suggesting an integrin-dependent induction of COX-2 expression in osteoclasts. This was confirmed in vivo using mice lacking osteopontin or integrin ß3. Immunohistochemistry also showed abundant osteopontin expression at the callus chondro-osseous junction. The results indicate that COX-2 expression in osteoclasts is controlled by integrin-dependent signalling, that COX-2 expression in osteoclasts and chondrocytes is necessary for fracture healing to proceed normally, and that COX-2 expression in chondro-osseous junction osteoclasts may be induced by osteopontin-dependent signalling by chondrocytes

Summary Statement. This study demonstrated that Sclerostin monoclonal antibody (Scl-Ab) enhanced bone healing in the rat osteotomy model. Scl-Ab increased callus size, callus bone volume fraction, rate of callus bone formation and fracture callus strength. Introduction. Sclerostin is a protein secreted by osteocytes and is characterized as a key inhibitor of osteoblast-mediated bone formation. Previous studies demonstrated that treatment with a sclerostin monoclonal antibody (Scl-Ab) results in significantly increased bone formation, bone mass and strength in rat closed fracture model (1–2). However, the effects of Scl-Ab on healing of open fracture model have not yet been reported in rats. Previously in ORS and ASBMR Annual Meeting, we have reported that Scl-Ab promoted the open fracture healing at week 3 and week 6 post-fracture. Here we extended our investigation for up to week 9 with additional histological assessments and dynamic histomorphometric analysis to investigate the effects of systemic administration of Scl-Ab on a later phase of fracture repair. Patients & Methods. Animal research ethics approval was obtained from our institute (reference No. 09/042/MIS), and the institute's guidelines for the care and use of laboratory animals were followed. In total, 120 six-month-old male SD rats were randomly divided into Scl-Ab group and vehicle group after a transverse osteotomy performed at the mid-shaft of right femur with internal fixation. One day post-surgery, rats were treated with a rodent Scl-Ab (Scl-Ab IV, s.c. injection, 25 mg/kg, 2 times per week) or vehicle for 3, 6 or 9 weeks. The progress of fracture healing for each animal was monitored weekly by digital radiography. Images acquired 3, 6 and 9 weeks post-operation were analyzed by ImageJ to quantify the total area of the fracture calluses. After euthanasia, femora were collected and subjected to the following analyses: micro-CT for bone mineral density (BMD) and callus volume fraction (BV/TV), micro-CT-based angiography for angiogenesis, histological evaluation and dynamic histomorphometry, and four-point mechanical testing for ultimate load, energy to failure and stiffness (3–6). Two-way ANOVA with Bonferroni post-hoc test was used to analyze the data. Significance level was set at P<0.05. Results. Radiographically, Scl-Ab treatment groups had significantly larger fracture calluses compared with respective vehicle group starting from week 3 post-fracture by quantitative analysis. Micro-CT analysis showed that Scl-Ab treatment groups had significantly higher callus bone volume fraction (+16–23%, P<0.01) and BMD (+15–16%, P<0.01) compared with respective vehicle groups at all time points post-fracture. Histological analysis also revealed more bone and less cartilage tissue in calluses in Scl-Ab group starting at week 3, which is explained by faster in the rate of new bone formation in fluorescence microscopy. Micro-CT based angiography demonstrated that Scl-Ab significantly enhanced neovasculation at the fracture calluses at week 3. Four-point bending test showed significantly higher ultimate load in Scl-Ab group than vehicle group at week 6 (+98%, P<0.01) and week 9 (+45%, P<0.05) post-fracture. In addition, ultimate load at week 6 of Scl-Ab group was at the similar level as seen at week 9 of the vehicle group, indicating the increased healing by Scl-Ab in this model. Stiffness (week 6 and 9) and energy to failure (week 6) were also tended higher in Scl-Ab group. Discussion/Conclusion. This study demonstrated that Scl-Ab enhanced bone healing in the rat osteotomy model. Scl-Ab increased callus size, callus bone volume fraction, rate of callus bone formation and fracture callus strength. Neovasculation was enhanced in the Scl-Ab group at week 3, implying Scl-Ab may enhance coupling of osteogenesis and angiogenesis. Scl-Ab treatment also resulted in more bone and less cartilage tissue in fracture calluses. Our results indicated that the systemic administration of Scl-Ab enhanced open fracture healing in rat femoral osteotomy model

Introduction. The fracture healing outcome is often evaluated via ex vivo testing of the fracture callus. However, there is only a small time window, where the callus stiffness is significantly different, i.e. a delayed fracture healing might be undetected if the time point of sacrifice is improper. The aim of this study was to develop an in vivo monitoring concept, which allows determining the fracture callus stiffness in vivo over the whole healing time in rats. Hypothesis. The fracture callus stiffness can be monitored by measuring the deformation of the external fixation device during gait analysis at several healing time points. Materials & Methods. The right femurs of sixteen wistar rats were osteotomized and stabilized with an external fixation device (stiffness 119 N/mm or 32 N/mm). The fixator body was instrumented with a stain gauge to measure the deformation. Gait analysis was performed once per week in a gait wheel equipped with a ground reaction force measuring device. Results. The deformation of the fixation devices decreased over the healing time indicating an increase of the callus stiffness. The flexible fixated group showed a later increase of the callus stiffness indicating a delay in fracture healing. Discussion & Conclusion. Measuring the deformation of the fixator and gait analysis provides a powerful tool to monitor the fracture healing process in rats. With this, it is possible to detect a delayed fracture healing process more reliable than with ex vivo analyses

Background and Hypothesis: High-energy fractures associated with severe soft tissue injury have a significant incidence of delayed or non-union. The soft tissue envelope may adversely contribute to the healing of a fracture, not only in stripping of the periosteal blood supply, development of compartment syndrome or tissue interposition between the bone ends but also in its ability to generate an intense acute inflammatory response. Inflammation is the initiator of healing; in clinical scenarios of impaired inflammation (immune deficiency, NSAIDs, corticosteroids) healing is delayed; interestingly, in injury with excess inflammation (CVA, MI) healing is also delayed. Would the inflammatory response following high-energy fractures contribute beneficially or adversely to the healing of the underlying fracture? Using an in-house murine femoral fracture model which reliably demonstrated features of delayed fracture healing when associated with a severe overlying muscle crush injury we proposed these hypotheses:. That fracture callus with overlying muscle crush would contain raised expression of acute inflammatory cytokines (IL-1β, IL-6 and TNF-α). That application of locally applied blocking antibodies to these inflammatory cytokines might negate excessive cytokine release and modulate fracture healing in this model. Methods: Total RNA was extracted from normal fracture callus (FO) and muscle crush fracture callus (MC) at day 2, day 4 and day 8. Semi-quantitative RT-PCR was used to compare IL-1β, IL-6 and TNF-α mRNA expression. Histomorpometric analysis of ICC stained sections of the FO and the MC groups was used to estimate IL-1β, IL-6 and TNF-α protein expression within the callus. Positively staining areas for the cytokine within the callus were a semi-quantified and compared between groups. Finally, blocking antibodies to IL-1β and TNF-α were injected into MC fracture callus at day 0, 4 and 8. Control MC group had vehicle only injected. Fracture healing was measured using radiological, histomorphological and biomechanical outcome measures. Following a pilot dosing experiment, the effect of blocking antibodies on fracture healing was compared between MC and MC with antibody groups. Results: The MC group IL-1β mRNA expression was significantly higher than FO at day 4 and day 8 (p=0.05). ICC for IL-1β protein expression was higher on day 4 and on day 8 in the MC group, significant at day 8 (p=0.03). TNF-α mRNA expression in the MC group at day 8 was significantly higher than the FO group (p=0.05). ICC for TNF-α protein in the MC group peaked at day 8 and was significantly higher than the FO group (p< 0.03). IL-6 mRNA expression was significantly raised in the MC group at day 4 and 8 compared with the FO group (p=0.05). ICC for IL-6 protein showed significantly increased expression at day 8 in the MC group (p=0.05). The patterns of expression of the mRNA and proteins were similar. Injection of anti-TNF-α antibodies into MC mice caused more new bone formation on day 16 (p=0.03) and day 24 (p=0.06), stiffer calluses at day 24 (p=0.01) and faster fracture gap obliteration at day 16 (p=0.05) and day 24 (p=0.001). IL-1β blockade had slightly less effect, more new bone formationd ay 16 (p=0.01) and day 24 (p=0.03), slightly stiffer (p=0.08), but no significant difference in fracture gap obliteration from controls. Conclusion: The effect of muscle crush around the fracture callus was to increase and prolong the expression of inflammatory cytokines with the callus. The effect of blocking these excessive inflammatory cytokines in our model was to improve fracture healing. Excessive inflammatory cytokines (IL-1β, IL-6, TNF-α) in bone impair new bone production by osteoblasts, inhibit the recruitment and differentiation of mesenchymal precursors and promote osteoclastogenesis. The mechanism of action of blocking antibodies may be due to inhibition of the antiosteogenic effects of these cytokines

Fracture healing is an issue that has not yet been fully elucidated. It is generally accepted in the literature that head trauma accelerates fracture healing and causes higher volume callus tissue. Recent studies have examined the relationship between head trauma and fracture healing more molecularly. Based on this research; the aim of this study is to show the effect of head trauma on fracture healing radiologically and histologically and to investigate the relationship between serum β-Catenin level and fracture healing with the experiment we performed on rats. A total of 36 Wistar Albino female rats with a mean age of 24 weeks were included in the study with the permission of Mersin University Animal Experiments Local Ethics Committee. Six rats in the first group were not traumatized and their blood samples were collected on the day of the experiment started, end of the third week and end of the sixth week. In the second group, only head trauma was performed and blood samples were collected at the end of the third and sixth weeks. In the third group, only open femoral fracture model was applied, blood samples were collected at the third and sixth weeks and AP and Lateral radiographs of the fractured femurs were taken. After sacrification, femurs were dissected from the surrounding soft tissues and subjected to histological examination. In the fourth group, both head trauma and open femur fracture model were applied, blood samples were collected at the end of third and sixth weeks and AP and Lateral radiographs of the fractured femurs were taken. After sacrification, femurs were dissected from the surrounding soft tissues and subjected to histological examination. The expression level of β-Catenin was measured by PCR from all blood samples. Direct radiographs of the third and fourth groups at 3 and 6 weeks were evaluated by two orthopedists according to Rust and Lane & Sandhu scoring system. The histomorphometric examination was performed by evaluating the Huo scoring and the ratio of fracture callus components (cartilage callus, bone callus, fibrous callus) to areas. According to PCR analysis, the change of expression of β-Catenin by weeks was not statistically significant in the first and second groups. However, a statistically significant decrease was observed in the 0–6 week interval in the third and fourth groups (p = 0.002, p <0.0001, respectively). In the radiological examination, the union scores of the rats with head trauma + femoral fracture were higher than the isolated femoral fractures at 3 weeks and 6 weeks. In histomorphometric examination, no statistically significant difference was found between head trauma + femur fracture group and isolated femur fracture group. In addition, there was no correlation between the groups in the correlation studies between radiological findings, histomorphmetric findings and PCR findings. Considering that each molecule involved in fracture healing processes has a time interval and concentration; We concluded that the expression levels of β-catenin can be repeated in smaller time periods including the early stages of fracture healing

Aims: Cell enlargement or hypertrophy is an intermediate transitional process in the transformation of Òsoft callusÒ into bone. The purpose of this study was to determine whether it is caused by an osmotic phenomenon. Following bone fracture, there is a local increase in tissue ßuid due to inßammation and neovascularisation. According to the osmosis principle, cells bathed in excess tissue ßuid swell. Methods: The specimens examined were 1 and 2-week old closed fractures of the right tibia of 12 NZW rabbits created by a drop tower technique. The specimens were prepared for routine histology. Thin sections were stained for haematoxylin and eosin and examined with the light microscope. Results: Cell enlargement and cell rupture were observed principally in the vicinity of the blood vessels. There was a hierarchy of cell sizes with the larger cells close to the blood vessels and the smaller ones further away from the vessels. Conclusions: According to these þndings, the fracture callus exhibits features which raise the possibility that an osmotic phenomenon is responsible for cell enlargement. The resulting increase in cell turgidity makes the fracture callus progressively stiffer and increases tissue strain. Cell enlargement also causes the tissue to expand. This may be the mechanism by which fracture callus migrates and bridges the fracture cleft

Objectives. Bone fracture healing is regulated by a series of complex physicochemical and biochemical processes. One of these processes is bone mineralization, which is vital for normal bone development. Phosphatase, orphan 1 (PHOSPHO1), a skeletal tissue-specific phosphatase, has been shown to be involved in the mineralization of the extracellular matrix and to maintain the structural integrity of bone. In this study, we examined how PHOSPHO1 deficiency might affect the healing and quality of fracture callus in mice. Methods. Tibial fractures were created and then stabilized in control wild-type (WT) and Phospho1. -/-. mice (n = 16 for each group; mixed gender, each group carrying equal number of male and female mice) at eight weeks of age. Fractures were allowed to heal for four weeks and then the mice were euthanized and their tibias analyzed using radiographs, micro-CT (μCT), histology, histomorphometry and three-point bending tests. Results. The μCT and radiographic analyses revealed a mild reduction of bone volume in Phospho1. -/-. callus, although it was not statistically significant. An increase in trabecular number and a decrease in trabecular thickness and separation were observed in Phospho1. -/-. callus in comparison with the WT callus. Histomorphometric analyses showed that there was a marked increase of osteoid volume over bone volume in the Phospho1. -/-. callus. The three-point bending test showed that Phospho1. -/-. fractured bone had more of an elastic characteristic than the WT bone. Conclusion. Our work suggests that PHOSPHO1 plays an integral role during bone fracture repair and may be a therapeutic target to improve the fracture healing process. Cite this article: M. W. Morcos, H. Al-Jallad, J. Li, C. Farquharson, J. L. Millán, R. C. Hamdy, M. Murshed. PHOSPHO1 is essential for normal bone fracture healing: An Animal Study. Bone Joint Res 2018;7:397–405. DOI: 10.1302/2046-3758.76.BJR-2017-0140.R2

Previously, we reported impaired biomechanical bone properties and inferior bone matrix quality in tachykinin1 (Tac1)-deficient mice lacking the sensory neuropeptide substance P (SP). Additionally, fracture callus development is affected by the absence of SP indicating a critical effect of sensory nerve fibers on bone health and regeneration. For α-calcitonin gene-related peptide (α-CGRP)-deficient mice, a profound distortion of bone microarchitecture has also been described. We hypothesize that SP and α-CGRP modulate inflammatory as well as pain-related processes and positively affect bone regeneration during impaired fracture healing under osteoporotic conditions. Therefore, this study investigates the effects of SP and α-CGRP on fracture healing and fracture-related pain processes under conditions of experimental osteoporosis using SP- and α-CGRP-deficient mice and WT controls. We ovariectomized female WT, Tac1−/− and α-CGRP−/− mice (age 10 weeks, all strains on C57Bl/6J background) and set intramedullary fixed femoral fractures in the left femora 28 days later. We analyzed pain threshold (Dynamic Plantar Aesthesiometer Test) and locomotion (recorded at day and night, each for 1 hour, EthoVision®XT, Noldus) at 5, 9, 13, 16 and 21 days after fracture. At each time point, fractured femora were prepared for histochemical analysis of callus tissue composition (alcian blue/sirius red staining). Pain threshold is significantly higher in Tac1−/− mice 13 days after fracture and tends to be higher after 21 days compared to WT controls. In contrast, touch sensibility was similar in α-CGRP−/− mice and WT controls but compared to Tac1−/− mice pain threshold was significantly lower in α-CGRP−/− mice 13 and 16 days and tends to be lower 21 days after fracture. Locomotion of Tac1−/− mice during daylight was by trend higher 9 days after fracture and significantly higher 16 days after fracture whereas nightly locomotion is reduced compared to WT mice. Analysis of locomotion during daylight or night revealed no differences between α-CGRP−/− and WT mice. During early fracture healing phase, 5 and 9 days after fracture, transition of mesenchymal to cartilaginous callus tissue tends to be faster in Tac1−/− mice compared to WT controls whereas no difference was observed during late stage of fracture healing, 13, 16 and 21 days after fracture. In contrast, callus tissue maturation seems to be similar in α-CGRP−/− and WT mice. Our data indicate different effects of SP and α-CGRP on fracture healing under conditions of experimental osteoporosis as a model for impaired bone tissue. Lack of α-CGRP seems to have no effects, but loss of SP affects locomotion throughout osteoporotic fracture healing and fracture-related pain processes during late phases of osteoporotic fracture healing. This indicates a modified role of SP during fracture healing under impaired versus healthy conditions, where SP changed early fracture-related pain processes and had no influence on callus tissue composition

Myostatin (GDF-8) is known to play an important role in muscle regeneration, and myostatin is also expressed during the early phases of fracture healing. In this study we used fluorescent immunohistochemistry to define the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. We then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results show that while myostatin was constitutively expressed in the cytoplasm of intact skeletal muscle fibers, a pool of intense myostatin staining was observed amongst injured skeletal muscle fibers 12-24 hours post-surgery. Myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 ug/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also dose-dependently decreased fracture callus total bone volume by 23% and 47% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant, dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that myostatin may inhibit bone repair after traumatic musculoskeletal injury through both autocrine (soft-callus chondrocytes) and paracrine (surrounding injured muscle fibers) mechanisms. Thus, early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury

We studied the effect of vitamin C on fracture healing in the elderly. A total of 80 elderly Osteogenic Disorder Shionogi rats were divided into four groups with different rates of vitamin C intake. A closed bilateral fracture was made in the middle third of the femur of each rat. Five weeks after fracture the femora were analysed by mechanical and histological testing. The groups with the lower vitamin C intake demonstrated a lower mechanical resistance of the healing callus and a lower histological grade. The vitamin C levels in blood during healing correlated with the torque resistance of the callus formed (r = 0.525). Therefore, the supplementary vitamin C improved the mechanical resistance of the fracture callus in elderly rats. If these results are similar in humans, vitamin C supplementation should be recommended during fracture healing in the elderly

Endochondral ossification involves a well ordered sequence of cellular events. Chondrocytes change their morphology and functions and are ultimately removed by the process of apoptosis. A variety of apoptotic-related signals have been characterised. These include Fas receptor (FasR)/Fas ligand (FasL), p53 and Bcl family. However, there is little known regarding the activity of these signals in the process of fracture healing. The purpose of this study was to investigate mRNA expression of apoptotic signals using RNase protection assay (RPA) and immunohistochemistry in endochondral bone formation. BALB/C mice aged 8 to 10 weeks were used for this study. First, a transverse fracture was made in the right tibia. Mice were euthanised at 1, 2 and 3 weeks postfracture. The calluses were harvested and studied for the expression of caspase-8, a key enzyme of apoptosis, and apoptosis inducers: tumour necrosis factor-alpha (TNF-α) and its receptor p55, FasL and Fas receptor (FasR), and TNF-related apoptosis-inducing ligand (TRAIL). Four mice at each timepoint were used for immunostaining of fracture callus. Sections were incubated with primary antibody then labelled by avi-din-biotin complex method. Another four to ten tibiae were used for RPA. Fracture callus were harvested and snap frozen in liquid nitrogen. RNA was isolated by TRI reagent and BCP, and mRNAs expression of apoptotic signals were detected. At each timepoint, mRNA of caspase-8, TNF-α, p55, FasL,FasR and TRAIL were detected by RPA. Immunostainings clearly showed that those apoptotic-related proteins were expressed by callus chondrocytes. Cartilaginous callus is replaced by woven bone in endochondral ossification. In this process, chondrocytes should be removed by the process of apoptosis in which death factors are elaborated directly in both an autocrine and paracrine manner

Objectives. As one of the heat-stable enterotoxins, Staphylococcal enterotoxin C2 (SEC2) is synthesized by Staphylococcus aureus, which has been proved to inhibit the growth of tumour cells, and is used as an antitumour agent in cancer immunotherapy. Although SEC2 has been reported to promote osteogenic differentiation of human mesenchymal stem cells (MSCs), the in vivo function of SCE2 in animal model remains elusive. The aim of this study was to further elucidate the in vivo effect of SCE2 on fracture healing. Materials and Methods. Rat MSCs were used to test the effects of SEC2 on their proliferation and osteogenic differentiation potentials. A rat femoral fracture model was used to examine the effect of local administration of SEC2 on fracture healing using radiographic analyses, micro-CT analyses, biomechanical testing, and histological analyses. Results. While SEC2 was found to have no effect on rat MSCs proliferation, it promoted the osteoblast differentiation of rat MSCs. In the rat femoral fracture model, the local administration of SEC2 accelerated fracture healing by increasing fracture callus volumes, bone volume over total volume (BV/TV), and biomechanical recovery. The SEC2 treatment group has superior histological appearance compared with the control group. Conclusion. These data suggest that local administration of SEC2 may be a novel therapeutic approach to enhancing bone repair such as fracture healing. Cite this article: T. Wu, J. Zhang, B. Wang, Y. Sun, Y. Liu, G. Li. Staphylococcal enterotoxin C2 promotes osteogenesis of mesenchymal stem cells and accelerates fracture healing. Bone Joint Res 2018;7:179–186. DOI: 10.1302/2046-3758.72.BJR-2017-0229.R1

Aim: Antibiotics are currently used during fracture healing for prevention or treatment of infection. Quinolones are well known to delay fracture healing, but little is known about other antibiotics. Cefazolin is the most commonly used drug for antibiotic prophylaxis, but many centres use cefuroxime. When allergy to cephalosporins is present, current recommendations include clindamicin or vancomicin. The purpose of this study is to know if other commonly used antibiotics can delay fracture healing. Methods: 100 male 3-months-old Wistar rats were used. After anaesthesia with ketolar, a closed fracture in the middle third of the femur was carried out. Rats were divided in five groups (20 rats each): one receiving cefazolin (a first generation cephalosporin, CZ), other receiving cefuroxime (a second generation cephalosporine, CF), other vancomicin (group V), other clindamicin (group CL) and the other receiving placebo (P) for 4 weeks. Group CZ received a subcutaneous dose of 50mg/kg/daily, Group CF received a dose of 100 mg/Kg/daily, Group V received a dose of 20 mg/Kg/daily, Group CL received a dose of 25 mg/Kg/daily and group P received water. 4 weeks later rats were killed and femora extracted. A mechanical test (low speed torsion) was performed to evaluate healing. All four groups (CZ, CF, V, CL) were compared to placebo through ANOVA. Results: Six bones were discarded because of technical errors, no infections were found. The maximum torque achieved by the calluses before breaking were 240 mNm in group P (n=18), 238 in group CZ (n=20), 178 in group CF (n=19), 167 in group V (n=19), and 205 in group CL (n=18). When compared to placebo, cefazolin and clindamicin showed no statistical differences (N.S, p> 0,10), vancomicin had lower callus strength (p=0,015), and cefuroxime had also lower callus strength near the significance level (p=0,084). Conclusion: The mechanical strength of fracture callus is similar when rats are given cefazolin or clindamicin during fracture healing. The mechanical strength of fracture callus is lower when vancomicin (and probably cefuroxime) is given. If these results are similar to human, cefazolin and clindamicin are safe drugs to use during fracture healing. If possible, vancomicin (and perhaps cefiuroxime) use during fracture healing should be restricted

The quantitative measurement is required for fracture healing evaluation in clinical studies approaching toward Evidence Based Medicine. Early detection of fracture healing impairment is demanded to introduce as early as possible the optimal treatment leading to recovery. Clinically validated and tested methods usually utilize X-ray and CT data. The aim of this study was to improve assessment of fracture healing progress or impairment examined with X-ray and computed tomography (CT) and to determine the usefulness of quantitative methods based on newly developed 3D image analyzing software. An original software “3D Reconstructor” was developed. The application operates on medical image data stored in DICOM format. 3D Reconstructor v.2.0 allows 2D reconstruction in custom surface directs through data block and 3D reconstruction as well. The usefulness of the application was evaluated for long bone fracture healing with three-dimensional presentation of fracture callus. Twenty otherwise healthy fracture patients (average age 43 years) with fractures of the tibial shaft, humerus, forearm, and clavicle were enrolled and underwent chronological series X-ray imaging and spiral CT scanning when it was clinically justified and necessary. Patients were preselected for CT study because of healing impairment suspicion. Computer Tomography examination was performed 14 to 56 weeks after injury (GE Pro Speed SX, slice 2–3 mm, image reconstruction 1 – 1,5 mm, 120 kV, 100 mAs). Qualitative assessment included fracture line/margins, fracture gap, external callus appearance, callus-to-cortex ratio, bridging, and radiologic union. Quantitative assessment of CT density changes (Hounsfield units [HU]) in the fracture gap was performed in patients using 3D Reconstructor multimodality image analysis software. Regions of interest (ROI) were analyzed in details by quantitative and qualitative assessment. Complete bone union was confirmed in 2 cases, delayed union in 8 cases and nonunions in 10 cases. The use of 3D Reconstructor enhanced quantitative evaluation of fracture callus in all evaluated cases. Low union rate observed in computed tomography derives from patient’s preselection based on clinical observations. Reliable fracture healing assessment can be enhanced with computed tomography and Computer Aided Diagnostic methods that supplement quantitative analysis of CT images. CAD quantitative assessment of the fracture gap and callus was successfully performed using 3D Reconstructor software in clinical cases. Higher accuracy of pseudoarthrosis and delayed union diagnosis was achieved. The reliable diagnosis in fracture cases may be supported with Computer Aided Diagnostic Methods. Still ongoing project, “3D Reconstructor” seems to be more accurate over not “armed” evaluation of original CT DICOM files of fracture healing in selected cases

Fracture repair is a complex physiological process during which bone shows the remarkable ability to mount a repair process, restoring its mechanical integrity and anatomical configuration by original osseous tissue. Programmed cell death, or apoptosis, is a naturally occurring cell suicide pathway with a homeostatic function in the maintenance of continuously renewing tissues. The present study investigated the relation between cell proliferation and cell death (apoptosis) during fracture healing in a mouse femoral model. Left femoral osteotomies were performed in 20 male CFLP mice (35–45g), immobilised with uniplanar external fixators. 4 animals were sacrificed on days 2, 4, 8, 16 and 24 post-fracture and fracture callus collected for paraffin embedding. Localisation of cell proliferation was examined using immunohistochemistry with proliferating cell nuclear antigen (PCNA) monoclonal antibody. Apoptotic cells were visualised with the terminal deoxynucleotidyl transferase (TdT)–mediated dUTP-biotin nick end-labelling (TUNEL) method. Random images of each time specific specimen were captured via a digital camera and the positive labelling indices of PCNA and TUNEL labelling were calculated and statically compared. Cell proliferation and apoptosis were found co-existing during the entire period of fracture healing studied. Cell proliferation was predominant in the early phases of fracture healing (days 2–8). PCNA positive labelling index peaked at day 8 (p< 0.01, t-test) and PCNA-positive cells were not limited to the fracture gap mesenchymal tissues but extended in the periosteum along most of the fractured femur. TUNEL positive labelling was minimal in the early stages (days 2–8). In later stages of fracture healing (days 16–24), PCNA expression declined as intramembranous and endochondral ossification spread within the fracture site and apoptosis was the dominant cell activity with the TUNEL positive labelling index peaked at day 16 (p< 0.05, t-test) and then declined sharply at day 24. The current study indicated that apoptosis was a normal concomitant during fracture repair, confirming programmed cell death in chondrocytes and bone cells, and that cell proliferation and apoptosis were tempero-spatially dependent. These findings support the view that apoptosis is a natural process, genetically programmed and active during fracture repair. The demonstration of a mixture of proliferative and apoptotic cell populations in the regenerating tissues of fracture callus, suggests that apoptosis and cell proliferation may be regulated by local factors during fracture healing

Introduction: Collagen type X is secreted by hypertrophic chondrocytes during fracture repair. Its precise role is uncertain. This study uses a knockout mouse model in which the collagen X gene is removed to examine its function. Method: Bilateral femoral fractures were created in type X collagen knockout mice (mutant) and normal mice (wild type), and were stabilized using an external fixator. The mice were sacrificed 7, 10, 14, 21, 28 and 60 days after fracture. Fracture healing was followed by x-rays, histology, gene expression studies, immuno-histochemistry and mechanical testing. Results: In the mutant mice, bony union was delayed, there was abnormal persistence of aggrecan up to 60 days after fracture. Histology reviewed amorphous acellular areas surrounded by osteoclasts at 21 and 28 days, while mechanical testing revealed that at 14 days after fracture, mutant callus was stiffer than the wild type, but the trend is reversed at 28 and 60 days. Discussion: This study contributes to the understanding of the basic mechanisms involved in fracture repair. The data suggest that collagen type X plays a significant role in bone remodeling during fracture healing. Its absence results in delayed union and abnormalities within the fracture callus

The February 2024 Wrist & Hand Roundup³⁶⁰ looks at: Occupational therapy for thumb carpometacarpal osteoarthritis?; Age and patient-reported benefits from operative management of intra-articular distal radius fractures: a meta-regression analysis; Long-term outcomes of nonsurgical treatment of thumb carpometacarpal osteoarthritis: a cohort study; Semi-occlusive dressing versus surgery in fingertip injuries: a randomized controlled trial; Re-fracture in partial union of the scaphoid waist?; The WALANT distal radius fracture: a systematic review; Endoscopic carpal tunnel release with or without hand therapy?; Ten-year trends in the level of evidence in hand surgery.

Aims

Tobacco, in addition to being one of the greatest public health threats facing our world, is believed to have deleterious effects on bone metabolism and especially on bone healing. It has been described in the literature that patients who smoke are approximately twice as likely to develop a nonunion following a non-specific bone fracture. For clavicle fractures, this risk is unclear, as is the impact that such a complication might have on the initial management of these fractures.

Aims

With the ageing population, fragility fractures have become one of the most common conditions. The objective of this study was to investigate whether microbiological outcomes and fracture-healing in osteoporotic bone is worse than normal bone with fracture-related infection (FRI).

Aims

The optimal management of posterior malleolar ankle fractures, a prevalent type of ankle trauma, is essential for improved prognosis. However, there remains a debate over the most effective surgical approach, particularly between screw and plate fixation methods. This study aims to investigate the differences in outcomes associated with these fixation techniques.

Aims

Alcoholism is a well-known detrimental factor in fracture healing. However, the underlying mechanism of alcohol-inhibited fracture healing remains poorly understood.

Objectives. Recent studies have shown that modulating inflammation-related lipid signalling after a bone fracture can accelerate healing in animal models. Specifically, decreasing 5-lipoxygenase (5-LO) activity during fracture healing increases cyclooxygenase-2 (COX-2) expression in the fracture callus, accelerates chondrogenesis and decreases healing time. In this study, we test the hypothesis that 5-LO inhibition will increase direct osteogenesis. Methods. Bilateral, unicortical femoral defects were used in rats to measure the effects of local 5-LO inhibition on direct osteogenesis. The defect sites were filled with a polycaprolactone (PCL) scaffold containing 5-LO inhibitor (A-79175) at three dose levels, scaffold with drug carrier, or scaffold only. Drug release was assessed in vitro. Osteogenesis was assessed by micro-CT and histology at two endpoints of ten and 30 days. Results. Using micro-CT, we found that A-79175, a 5-LO inhibitor, increased bone formation in an apparent dose-related manner. Conclusions. These results indicate that 5-LO inhibition could be used therapeutically to enhance treatments that require the direct formation of bone

Objective. The optimal positioning of the acetabular component is a relevant prognostic factor in total hip arthroplasty (THA). Because of substantial errors of manual technique in cup placement even with experienced surgeon, computer aided navigation system has been developed in recent years. However, existence of the hardware around acetabulum likely deteriorates the accuracy of the navigation system, namely in revision THA case and postoperative status of pelvic fracture. Here we report a case who we successfully performed THA using CT based navigation system although there were multiple hardware around acetabulum due to osteosynthesis for the previous pelvic fracture. Case presentation. A forty-one years old man presented with intolerable hip pain with severe radiographic osteoarthritic findings in left hip joint. He had sustained left pelvic fracture and posterior hip dislocation due to traffic accident and undergone osteosynthesis using multiple plates and screws when he was forty years old. However, progressive collapsing of femoral head and acetabulum occurred. Then, we indicated THA for his situation and planned to apply the CT based navigation system (Stryker CT based hip Ver.1.1 softwear and Cart II system). Preoperative workup revealed incomplete union of posterior and superior acetabular wall and we had to retain plates and screws for the stable fixation of acetabular cup. The existence of the hardware made it complicated to perform three dimensional planning and templating. Meticulous surface editing of pelvis to exclude the metal artifact and fibrocartilagenous tissue was needed to achieve accurate surface registration. In the operation room, we had to use unusual way of registration to complete two steps of registration. In the first step (roughly matching between patient's physical pelvic surface and edited pelvic surface in work station using corresponding 5 points), we utilized head of screw and hole of the plate which we could easily identify intraoperatively, in addition to ASIS and innominate groove. In the second step (strict matching using more than 30 points of pelvic surface), we had to identify the pelvic bony surface, as excluding the metal surface and fibrocartilagenous tissue such as fracture callus. These efforts enabled us to accomplish substantial accuracy of registration with RMS of 0.5 mm. Final cup orientation at the end of surgery was 41° of inclination and 25° of anteversion. Postoperative CT scan revealed that cup placement angle was 40° of inclination and 25° of anteversion, almost identical with intraoperative value. Conclusion. Our experience showed that CT based navigation system provided accurate placement of the acetabular component in a case having multiple hardware as well as in normal primary THA. Although we need additional efforts such as meticulous preoperative planning, extra operation time, CT based navigation system has great advantages to minimize the mal-placement of the cup in complicated case

Clinical evidence that patients with type 2 diabetes mellitus (T2DM) have increased risk of fractures is reported. Furthermore, thiazolidinediones, used to treat T2DM increases the risk of secondary osteoporosis & subsequent fractures. The osteogenic potency of metformin is reported in vitro, few studies have investigated the effects of metformin on bone mass and fracture healing in vivo. We aimed to investigate the effects of metformin on fracture healing in vivo. Method. 20 female Wistar rats aged 3 months were randomly divided in two groups, one group receiving saline, the other group receiving metformin administered orally via the drinking water at a concentration of 2mg/ml. After 4 weeks of metformin treatment, a mid-diaphyseal, open External fixation fracture was performed. Rats were sacrified 4 weeks later. Right contralateral tibia and left osteotomised femora were excised, bone architecture analysed by micro-CT in the right tibia. Results. No significant differences were noted between the two groups. Fracture callus volume and mineral content after 4 weeks were similar in metformin and saline groups. Discussion Our results indicate that while metformin has no adverse effects on bone, it does not promote bone mass, as suggested by in vitro studies. This confirms clinical data which have not shown direct links between metformin and decreased fracture risk

Introduction. The delay looks radiographically as a fracture callus not very evident or absent 6 months after osteosynthesis. Patients undergo a long period of immobilization and this fact causes the increase the social cost of the disease. The technique we suggest aims to the reduce the period of immobilization and as a consequence the management costs of the disease. Materials and methods. Our technique includes the infiltration of the delay focus with platelet rich fibrin, bone marrow concentrated and demineralized bone matrix. Outpatients and radiographic checks were carried out 3, 6 and 12 months after surgery. The treatment was considered fail in case of absence of bone callus at 3 out of 4 corticals at the rx after 6 months from surgery. Results. From November 2008 we treated 14 patients (average age 35 years, range 18 to 53). The treatment was performed after an average period of 14 months (range 5 to 38) from the fracture. The average follow up was 12 months (range 3–30). After 3 months from surgery, seven patients bear full load, did not feel pain and X-ray showed an increase of osteogenesis. Two cases failed; in one of them the patient underwent another infiltration and in the other one we replaced the fixation. Conclusion. Our tecnique lets a reduction of healing times in patients with delay and can be performed through the One Day Surgery programme

Introduction. Bisphosphonates are among the most commonly prescribed drugs in Osteoporotic Patients. Their mode of action is anti-resorptive. Since remodeling is a key step in fracture healing, there has been concern regarding the effect of bisphosphonates on fracture healing. Objectives. To assess the effect of alendronate on fracture healing in the rabbit ulna osteotomy model. Materials and methods. 16 New Zealand white rabbits were divided into 2 equal groups. Bilateral ulnar osteotomies were performed in the first week. Group 1 was the control group and group 2 was gavaged with alendronate solution (human equivalent dose). 2 rabbits were euthanised at 3 and 6 weeks and the remaining 4 rabbits were euthanised at 8 weeks. Fracture healing was assessed radiologically, with mechanical testing using the Instron 4302 materials testing machine and histologically, in that order. Results. The fractures healed satisfactorily in all the control group animals. However, in the alendronate treated group, there was an abundance of woven bone and little lamellar bone in the callus. However there was no significant difference in mechanical testing. In addition we did not find any evidence of Osteonecrosis in the Bisphosphonate treated group. Conclusion. Bone remodelling in the alendronate treated group is slower but a larger amount of bone callus is formed around the fracture, thus giving the fracture callus a higher ultimate load to failure at an earlier stage

Background:. In recent times there has been an increasing trend towards surgical intervention in paediatric femoral shaft fractures with widening indications. Titanium elastic nails and external fixation are two widely practiced procedures for such fractures. Materials & Methods:. We report a series of 48 children with 52 fractured femurs, 18 being managed by TENS and 34 in a linear external fixator. Children were aged between 3.5 to 12 years and the fractures were stabilised after an optimal closed reduction on a normal theatre table under image intensifier control. Fracture site distribution was nearly uniform in both the groups. Though most children were assigned to any of the groups at random, external fixators were applied on many younger children and those having financial constraints. Results:. The average age of children in the TENS group was 7.4 years and the average fracture healing time was 9.4 weeks. In the ex-fix group the figures were 5.6 years and 8.6 weeks respectively. Fixators were removed when good callus formation was seen on at least three cortices; average fixator time was 7.5 weeks. Fracture callus formation was slower in TENS group. Soft-tissue irritation at the nail entry points was the commonest complication for the TENS whereas pin-track infection was problem in the ex-fix group. Conclusion:. Management of paediatric femoral shaft fractures has changed to include more interventions. Flexible or elastic nailing like the TENS is a versatile and popular technique, however stabilisation in an external fixator also produce comparable results. External-fixation is an easier, cheaper and shorter procedure, and a mini ex-fix becomes a convenient external splint for smaller children who can be more conveniently nursed. Moreover ex-fix removal is an office procedure without anaesthesia

Introduction. Subchondral insufficiency fracture (SIF) needs to be differentiated from osteonecrosis. The purpose of this study was to evaluate the imaging and histopathological findings of patients 60 years or older with radiological evidence of subchondral collapse of the femoral head. Methods. We reviewed 77 consecutive hips in 56 patients aged 60 years or older, which showed subchondral collapse on radiographs. According to the shapes of low-intensity bands on T1-weighted images of magnetic resonance imaging (MRI), the patients were divided into 2 groups. Group A showed a concavity of the articular surface: characteristic appearances of osteonecrosis, and Group B showed an irregular convexity of the articular surface: characteristic appearances of SIF. Results. Thirty-four patients (60.7%) were categorized as Group A and 22 patients (39.3%) as Group B. All the cases in Group A had a history of corticosteroid intake or alcohol abuse. In Group B, the proportion of women as well as unilateral involvement was significantly higher than those in Group A. In 30 cases with histopathological examination, 16 were in Group A and 14 were in Group B. All the cases in Group A demonstrated characteristic features of osteonecrosis. In Group B, the band on T1-weighted images corresponded to viable reparative tissue including granulation tissue and fracture callus, which are characteristic histological findings in SIF. Conclusion. The results of this study suggest that the patient's clinical features as well as the shapes of the low-intensity bands on T1-weighted images provide a clinically concise tool for the differentiation of SIF from osteonecrosis

1. Experimental fracture callus in rats contains mast cells as a normal morphological element. 2. The mast cell count undergoes peculiar variations in the normal course of events in experimentally delayed or accelerated bone repair. 3. A hypothesis is presented in which the tissue mast cell granules are regarded as calcium transporters in the mineral phase of callus formation, a process probably corresponding to Selye's concept of "mastocalciphylaxis" and "mastocalcergy."

1. The bone changes in four autopsied cases of Cushing's syndrome are described. The changes take the form of osteoporosis, which is most marked in the spine and the ribs. 2. The osteoporosis results from impaired osteoblastic bone formation in the presence of a normal degree of osteoclastic bone resorption. 3. Histological abnormalities of fracture callus in Cushing's syndrome indicate interference with the proliferation of osteoblasts and cartilage cells and with the formation of new tissue by these cells. 4. The bone changes in Cushing's syndrome are comparable with those produced in experimental animals by the administration of A.C.T.H. or cortisone

To elucidate the molecular biology of fracture healing, murine models are preferred. We performed a study with the first internal fixation system that allows studying murine fracture healing in a controlled mechanical environment, to characterise the timing of the fracture healing cascade with this model, based on a histological evaluation. Femoral osteotomies were performed in 68 male C57BL/six mice and stabilised with locking internal fixation plates in either stiff, or defined, flexible configurations. Healing progression was studied at 10 time points between 3 and 42 days post- surgery. After surgery, mice were radiographed to confirm the correct implant positioning. After sacrifice, the extracted femora were processed for decalcified histology. Thin sections were taken as serial transverse sections and stained for subsequent histomorphometric analysis and three-dimensional reconstruction of the different fracture callus tissues. The surgery was successful in 62 animals. Only six6 (8.8%) animals had to be sacrificed due to complications during surgery. The post-operative radiographs demonstrated a high reproducibility of implant positioning and no implant failure or screw loosening occurred during the experimental period. The improved consistency in surgical technique leading to more uniform results represents a key advantage of this system over other mouse fracture healing models. As such, it may allow a reduction in the sample size needed in future murine fracture healing studies. The histological evaluation confirmed the lack of a periosteal callus, and exclusively endosteal, intramembraneous bone formation in the bones stabilised with the stiff implants. The bones that were stabilised with the more flexible internal fixation plates showed additional endochondral ossification with extensive, highly asymmetrical, periosteal callus formation. Our results demonstrate that this murine fracture model leads to different healing patterns depending on the flexibility of the chosen plate system. This allows researchers to investigate the molecular biology of fracture healing in different ossification modes by selection of the appropriate fixation

Introduction. Amino acids like arginine and lysine have been suggested to hasten the process of fracture healing by improving the local blood supply, supplementing growth factors, and improving collagen synthesis. We studied the role of lysine and arginine in the fracture repair process with regard to the rate of healing, probable mechanisms involved in the process, and mutual synergism between these agents. Materials and methods. In an experimental study, 40 rabbits were subjected to ulnar osteotomy. They were distributed in control (14) and test groups (26). Twenty-six animals in the test group were fed with a diet rich in lysine and arginine. Both the groups were followed radiologically and histologically till union. Results. Ten weeks postoperatively, there was difference evident radiologically between those supplemented with lysine and arginine, indicating that these components enhance the healing in the later part of bone remodeling, canal restoration, and medullary as well as cortical continuity and repair. X-rays obtained at weeks 9, 10, and 12 in both the groups showed statistical significance. These findings showed that healing is better in the test group in terms of increased vascularity in the early part of healing, i.e., at approx. 2–3 weeks and in terms of bone matrix, Haversian system formation, and cortical repair in the later part of healing, i.e., at approx. 9–12 weeks between the two groups. There was better healing of osteotomy in terms of better vascularization, callus formation, and mineralization in the test group. The time of healing in the test group was reduced by a period of 2 weeks. Discussion. NO is expressed during fracture healing in rats and humans, as after fracture, mRNA, protein, and enzymatic activity iNOS have been identified at the fracture callus with maximum activity at day 15. Thus, the initial better healing, by 3 weeks, in the test group rabbits can be explained by the fact that the iNOS activity mediates an increased vascularity at the fracture site. The mRNA activity for eNOS and bNOS was induced slightly later than that for iNOS, which was consistent with a temporal increase in the calcium-dependent NOS activity that gradually increased up to day 30. All calcium-dependent processes like collagen recruitment for Haversian system formation, better bone matrix, and cortical repair were significantly better at any point of time, in the rabbits that were supplemented with arginine; however, lysine has also an important role in these processes. Arginine may influence bone formation by enhancing local IGF-I production. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Arginine being nitric oxide donor increases angiogenesis. Summary. Amino acids like arginine and lysine may hasten fracture healing. Adjuvant amino acid treatment is having inherent advantage in being nontoxic, inexpensive, and a simple oral therapy

Aims

Fibrinolysis plays a key transition step from haematoma formation to angiogenesis and fracture healing. Low-magnitude high-frequency vibration (LMHFV) is a non-invasive biophysical modality proven to enhance fibrinolytic factors. This study investigates the effect of LMHFV on fibrinolysis in a clinically relevant animal model to accelerate osteoporotic fracture healing.

This is a clinically based study to assess the reliability of fracture stiffness as a measurement of clinical union and investigate other indicators which may aid the clinician to accurately determine when fracture fixation may be removed. A fracture bending stiffness in the sagittal plane of 15Nm/deg. has been stated as a satisfactory endpoint at which an external fixator may be removed from diaphyseal fractures of the tibia. However using this as a measure to determine when to remove support in a study of 76 patients 4 continued to a malunion. Fracture callus properties were measured in clinic. The fixator was removed for the tests and a specially designed system was used to measure displacement and load. Fracture stiffness was measured in different planes and at various loading rates. Passive stressing of the leg was performed whilst fracture displacement was recorded. A constant load was applied for a longer period to assess creep properties. Fracture stiffness was found to vary between different planes of measurement and on load rate. The visco-elastic characteristics of the callus changed with time. In early measurements, the callus absorbed a large proportion of energy when a load was applied. Later tests showed a progressive change with the callus absorbing less energy. This demonstrates that the properties of the callus changed with time, with the viscous element diminishing and the elastic element increasing. This sometimes occurred with no change in the measured fracture stiffness. Further investigation is needed, focusing on the visco-elastic properties of callus, to develop a more reliable method of determining clinical union

We describe a new method of biological repair of osteochondral defects. In rabbit knees an osteochondral defect was reconstructed with a callo-osseous graft made of a superficial sheet of medullary fracture callus attached to a base of cancellous bone. This was taken from the iliac bone of the same animal which had been osteotomised ten days earlier. The reparative tissues were evaluated for 24 weeks by quantitative histology, biochemical analysis of the uronic acid content, and immunohistochemical staining of collagen constituents. The callo-osseous graft provided significantly faster and better repair of the articular surface than an untreated defect or a callo-osseous graft in which the cells had been devitalised by irradiation before transplantation. Our findings indicate that the callo-osseous graft contributes to the repair process by providing both favourable extracellular matrices and pluripotential mesenchymal cells. Our study tested the hypothesis that early medullary callus generates hyaline cartilage instead of bone after transfer to an articular surface

Summary Statement. Parathytorid hormone-related protein (107–111) loaded onto biopolymer-coated nanocrystalline hydroxyapatite (HA. Glu. ) improves the bone repair in a cavitary defect in rat tibiae. Introduction. Biopolymer-coated nanocrystalline hydroxyapatite (HA. Glu. ) made as macroporous foams are promising candidates as scaffolds for bone tissue engineering applications. They exhibit optimal features, promoting internalization, proliferation and differentiation of osteoprogenitors, with an adequate cell colonization over the entire scaffold surface. Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Its 107–111 epitope (osteostatin) exhibits osteogenic properties at least in part by directly acting on osteoblasts. The main aim of this study was to evaluate whether osteostatin loading into HA. Glu. scaffolds might improve their bone regeneration capacity. Materials and Methods. HA. Glu. scaffolds were prepared as previously described (Sánchez-Salcedo S et al. J. Mater. Chem. 2010; 20:6956-61). Osteostatin was adsorbed onto HA. Glu. material by dipping into a solution containing this peptide at 100 nM (in phosphate-buffered saline, pH 7.4), following a standard protocol. We performed a cavitary defect (2 mm in diameter and 3 mm in depth) in both distal tibial metaphysis using a drill under general anesthesia in male Wistar rats (n=8) of 6 months of age. Unloaded HA. Glu. material (7 mg) was implanted into left tibial defects, whereas rigth tibial defects received the osteostatin-loaded material. Animals were sacrificed after 4 weeks for histological, μ-computerised tomography and gene expression analysis of the callus. Our protocol was approved by the Institutional Animal Care and Use Committee at the IIS-FJD. Mouse osteoblastic MC3T3-E1 cells were grown in differentiation medium (α-MEM with 10% fetal bovine serum, 50 µg/ml ascorbic acid, and 10 mM β-glycerolphosphate), in the presence or absence of HA. Glu. material with or without osteostatin. Cell viability (assessed by trypan blue staining), alkaline phosphatase (ALP) activity and mineralization (alizarin red) were analyzed at different culture times. Results. The mean uptake of osteostatin by HA. Glu. scaffolds was about 60 % (representing 0.7 μg/implanted scaffold) after 24 h of loading, and they released a mean of 80 % of loaded peptide to the surrounding medium within 1–24 h. At 4 weeks, this osteostatin-containing HA. Glu. material significantly increased the bone volumen fraction and trabecular thickness of regenerating bone in the tibial methaphysis, compared to those observed with unloaded HAGlu scaffolds. In addition, osteostatin-coated HA. Glu. scaffolds increased (2-fold) the gene expression of osteocalcin and vascular cell adhesion molecule 1, but decreased (2-fold) that of the Wnt inhibitors, SOST and Dickkopf homolog 1 (DKK-1) in the fracture callus. In MC3T3-E1 cell cultures, osteostatin-loaded HA. Glu. material increased cell viability and ALP activity (each by 30%), and matrix mineralization (by 50%) at days 4 and 10, respectively. Conclusions. These results indicate that osteostatin loading improves the bone regeneration capacity of HA. Glu. scaffolds. Our findings suggest that these scaffolds might be promising implants in orthopaedic applications. This work has been supported by a grant from Comunidad Autónoma de Madrid (S-2009/MAT/1472)