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

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

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

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)

A balanced inflammatory response is important for successful fracture healing. The response of osteoporotic fracture healing is deranged and an altered inflammatory response can be one underlying cause. The objectives of this review were to compare the inflammatory responses between normal and osteoporotic fractures and to examine the potential effects on different healing outcomes. A systematic literature search was conducted with relevant keywords in PubMed, Embase, and Web of Science independently. Original preclinical studies and clinical studies involving the investigation of inflammatory response in fracture healing in ovariectomized (OVX) animals or osteoporotic/elderly patients with available full text and written in English were included. In total, 14 articles were selected. Various inflammatory factors were reported; of those tumour necrosis factor-α (TNF-α) and interleukin (IL)-6 are two commonly studied markers. Preclinical studies showed that OVX animals generally demonstrated higher systemic inflammatory response and poorer healing outcomes compared to normal controls (SHAM). However, it is inconclusive if the local inflammatory response is higher or lower in OVX animals. As for clinical studies, they mainly examine the temporal changes of the inflammatory stage or perform comparison between osteoporotic/fragility fracture patients and normal subjects without fracture. Our review of these studies emphasizes the lack of understanding that inflammation plays in the altered fracture healing response of osteoporotic/elderly patients. Taken together, it is clear that additional studies, preclinical and clinical, are required to dissect the regulatory role of inflammatory response in osteoporotic fracture healing.

Cite this article: Bone Joint Res 2020;9(7):368–385.

Skeletal scintigraphy, which has now been established as a useful and accurate method of detecting early skeletal metastases and assessing their response to treatment, has been investigated for its pathological basis. Histological examination of several hundred necropsy specimens, from sixty-eight patients who died from malignant disease, showed a significant increase of osteoid and immature woven bone in the presence of metastatic cancer. Tumour-cell suspensions of the VX2 carcinoma were injected into the medullary cavity or on to the periosteal surface of the ilia or tibiae of New Zealand white rabbits. A combination of bone destruction and new bone formation, similar to the autopsy material, was seen. There were at least two mechanisms for the new bone production. Initially, intramembranous ossification was seen in the fibrous stroma surrounding the tumour. Once the cortex was involved and cortical bone destruction had occurred, large amounts of woven bone resembling fracture callus were laid down. The new bone had a markedly increased avidity for boneseeking isotopes, indicating why skeletal scintigraphy was useful. A further twenty rabbits, in whose ilia the VX2 carcinoma was growing, were treated by local irradiation. When treatment was successful the tumour was destroyed, the production of new bone ceased, and the lesion lost its increased avidity for bone-seeking isotopes, indicating that skeletal scintigraphy could be used to assess the response of skeletal metastases to therapy

Both our own experience with antegrade nailing of the humerus and reports in the literature have made us aware of some of the drawbacks of this technique. Invasion of the intact shoulder is associated with damage to the rotator cuff and possible ectopic calcification, resulting in subacromial impingement. The ‘blind’ percutaneous placement of the top locking screw may endanger the axillary nerve and/or the bicipital tendon. From 1990 to 2000 we performed 144 retrograde nailings, 41 of which were lost to follow-up. For two years we followed up the remaining 103 patients, 71 men and 32 women, who had sustained 83 closed and 20 compound fractures, 14 of which were caused by gunshots. There were 89 recent fractures and 14 cases of nonunion, nine of them the outcome of non-surgical management. Seidel interlocking nails were used in 92 patients and Russell-Taylor in 11. Reaming was invariably done, first to prevent jamming of the nail and fracture propagation, secondly to create endosteal bone transport (equivalent to bone grafting), and thirdly to contribute to bone morphogenetic protein release. The results were encouraging. In fresh fractures callus was present after 5 to 8 weeks and in nonunions after 10 to 14 weeks. In 10 patients, iatrogenic periportal uni-cortical fractures occurred. These healed at the same pace as the original fracture and did not affect the functional recovery. There were no vascular complications. One patient developed transient radial nerve paresis, but there was no permanent neurological damage. No sepsis developed in previously uninfected fractures. Shoulder and elbow function remained normal

Introduction: The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate this to the mechanical fixation stability during the course of healing. Materials and Methods: A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization. Results: Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks) and decreased over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point. Discussion: The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing

Background Osteopontin (OPN), also known as bone sialoprotein I or secreted phosphoprotein 1, is a major non-collagenous bone matrix protein. A broad distribution has been detected in embryonic bone, osteoid, and fracture callus [Nomura et al. 2000] pointing out its central role in bone development and healing. It remains unclear weather mechanical conditions influence OPN synthesis and thereby osteoprogenitor cell differentiation. We investigated OPN mRNA-levels of bone marrow derived mesenchymal stem cells (bm-MSC) cultured in a previously described compression bioreactor (CBR) [Matziolis et al. under review] under dynamic compression (DC). Materials Bm-MSCs of 5 different individuals (mean age 61y) were seeded in a fibrin-alginate mix-matrix placed between two slices of lyophyliced cancellous bone. One group of constructs (n=10) underwent DC with 7kPa at 0.05 Hz, resulting in a matrix compression of 1mm at an heigh of 5mm, for 24 hours in the CBR. Constructs cultured under similar conditions but without DC served as control group (n=10). mRNA was extracted out of each construct after ending the DC, following the Trizol®-protocol. After cDNA-synthesis, GEArray Q series (Human Osteogenesis Gene Arrays) were performed and normalized versus GAPDH. Results We found an increase of OPN-expression in all dynamically compressed matrices. In the DC-group we found a mean of 5-fold increase of OPN mRNA compared to the control group (median: 0.43 vs. 0.09, p< 0.001). Discussion and Conclusion The results of this study demonstrate that an in vitro DC of bm-MSCs for 24 hours leads to an increased expression of OPN. We conclude that DC is an important element of early fracture healing by increasing the expression of OPN and thereby modulating progenitor cell differentiation immediately after mechanical instability caused by a fracture

Measurement of the rate of fracture healing is a major problem in fracture research. Bone mineral density (BMD) of fracture callus has been used as a measure of healing in diaphyseal fractures. However, metaphyseal fractures (especially in the elderly) are now the commonest type of fracture and are a significant public health problem. This study investigated whether measurement of BMD at the fracture site in the distal radius can be used as a measure of fracture healing. We recruited 28 patients who had sustained a dorsally displaced distal radial fracture which was deemed to require treatment by intrafocal wire fixation. All patients had acceptable correction of dorsal and radial angle at final x ray (3 months). Wrist function was measured using the Patient Rated Wrist Evaluation (PRWE – a validated outcome measure for use after distal radial fractures), grip strength,and range of motion. All measurements were made at 6, 12 and 26 weeks. BMD was measured at the fracture site (examining the BMD of the medullary bone at the fracture site after removal of wires), in the opposite wrist and the lumbar spine using QCT at 6 weeks after fracture. There was no correlation between fracture site BMD and BMD at the other wrist or the lumbar spine (r < 0.3). The BMD at the fracture site was higher than the BMD at the other wrist (mean 168 vs 70 HU; p< 0.001 paired T test). There was no relationship between fracture site BMD or the ratio of BMDs fracture site / normal wrist, and any of the functional assessments (proportion grip strength recovered, range of motion or PRWE (r < 0.3)). 15 of these patients underwent a second QCT at 12 weeks after fracture. There was no significant change in fracture site BMD between the first and second scan. These data indicate that fracture site BMD is unlikely to be a useful method of measuring metaphyseal bone healing. The increase in BMD at the fracture site was unexpected; possible explanations include impaction of bone or high BMD in woven bone (the relationship of which to bone stiffness is uncertain)

1. Alkaline and acid phosphatase, non-specific esterase and beta-glucuronidase have been estimated and demonstrated histochemically in a series of bone tumours and allied lesions, of which ten were osteogenic sarcomata, ten were giant-cell lesions, eleven were fibroblastic lesions and seven were tumours of cartilage. 2. Osteogenic sarcoma was found to be characterised by high levels of alkaline phosphatase, with rich staining for this enzyme in the tumour cells. Similar high levels of alkaline phosphatase were found in other bone-forming lesions, such as fibrous dysplasia, a giant-cell sarcoma with osteogenic matrix, and fracture callus. 3. Giant-cell lesions were characterised by high levels of acid phosphatase, and intense staining for this enzyme in the osteoclasts. These cells were also found to be rich in non-specific esterase (as shown by the alpha-naphthyl acetate method) and in beta-glucuronidase, but almost or entirely lacking in alkaline phosphatase. High levels of alkaline phosphatase were not found in giant-cell lesions except in relation to osteogenic matrix. 4. Fibroblastic tumours were characterised by moderate levels of all four enzymes, with little or no staining for phosphatases in the tumour cells; non-specific esterase was generally present in a proportion of the cells. 5. In certain lesions intermediate stages in the differentiation of fibroblasts to osteoblasts were found, notably in fibrous dysplasia, in which the biochemical change preceded the histological. In such lesions high total levels of alkaline phosphatase were found. 6. Cartilaginous tumours were characterised by low levels of all four enzymes, and little histochemical staining except in hypertrophied cells in areas of ossification. 7. It was found in general that the enzyme distributions in these neoplasms and other lesions reflected the findings in comparable reactive and growing normal tissues

Mast cells (MC), the tissue-based effector cells in allergic diseases, have many functions. Within bone tissue, they have been linked with new blood vessel formation and marrow fibrosis and it has been proposed that they are capable of promoting osteoclastic bone resorption. MC numbers are known to increase in a number of osteolytic conditions e.g. osteoporosis, hyperparathyroidism and periodontitis. In fracture callus, too, large numbers of MC are present, especially during the onset of remodelling where it is believed they may be responsible for osteoclast recruitment and/or differentiation. The aim of this study was to look for further evidence of mast cell (MC) involvement in pathological bone resorption. MC activity was assessed in tissue sections of osteolytic conditions including Paget’s disease of bone, rheumatoid arthritis and fibrous dysplasia together with several benign and malignant bone tumours. MCs were identified by toluidine blue staining and by immunostaining with a commercial antibody against MC tryptase. Extensive infiltration of mast cells was observed in fibrous dysplasia, rheumatoid arthritis and Paget’s disease of bone and mast cell accumulation was seen at the bone resorbing margin of a number of enlarging bone tumours including osteosarcoma, giant cell tumour of bone, osteoma and osteoid osteoma. MCs, along with other inflammatory cells, are known to accumulate at the margins of soft tissue tumours where they are thought to promote tumour growth. The current findings are consistent with a similar role for mast cells in the primary bone tumours examined. In each of the conditions studied, an additional role for MC may be that of promoting bone lysis. MC are known to contain numerous factors including TNF-alpha and IL-1, which are potent stimulators of osteoclast formation and activity. It is concluded that MCs may contribute to the fibrosis, angiogenesis and increased bone resorption seen in certain metabolic bone diseases. MC activity may also be an important factor contributing to the lysis that occurs in numerous other pathological situations including at the margins of aggressive primary bone tumours and skeletal metastases, leading to the expansion of these lesions

During fracture repair, a number of growth factors and cytokines are present at elevated levels at the fracture site such as Transforming Growth Factor Beta (TGF-), Fibroblast Growth Factor (FGF) and Platelet Derived Growth Factor (PDGF). The aim of the study was to investigate the presence of these growth factors in healing fractures and fracture non-unions, in order to test the hypothesis that atrophic non-unions express a lower level of growth factors than hypertrophic non-unions and healing fractures. Biopsies were taken from the fracture site of 23 patients (mean age 46) with uninfected non-unions, 12 patients with hypertrophic (mean 13.8 months after fracture) and 11 patients with atrophic (mean 16.5 months after fracture). A comparison group of biopsies from early fracture callus (one to four weeks after fracture) in five patients with healing fractures was also included. Five-micron paraffin sections were immunohistochemically stained for TGF-, FGF-II and PDGF. Growth factors were then assessed in six different cell types. Fibroblasts, endothelial cells and macrophages were found to express TGF-, FGF-II and PDGF in all three-fracture groups. Osteoblasts, osteoclasts and chondrocytes were not present in the healing fracture group. The growth factor expression in osteoblasts, osteoclasts and chondrocytes in the non-union groups were found to be variable, however, the expression of these growth factors appeared to be less in the atrophic non-unions than hypertrophic non-unions. The expression of these growth factors was found to be less in the atrophic non-union group than the hypertrophic non-union group in osteoblasts, osteoclasts and chondrocytes. These results may have relevance for new therapies that can be aimed at delivering growth factors to treat fracture non-unions. By further investigation of the differential expression of these growth factors it may be possible to determine which factors are likely to stimulate fracture healing

Aims

To fully verify the reliability and reproducibility of an experimental method in generating standardized micromotion for the rat femur fracture model.

Aims

The incidence of atypical femoral fractures (AFFs) continues to increase. However, there are currently few long-term studies on the complications of AFFs and factors affecting them. Therefore, we attempted to investigate the outcomes, complications, and risk factors for complication through mid-term follow-up of more than three years.

Introduction: Our previous work has shown that angiogenesis occurs within the cartilaginous callus during long bone fracture healing. 1. Our aim in this study was to investigate the mechanisms involved in endochondral ossification within callus tissue during the secondary stages of fracture healing. Methods: In this study, immunohistochemical techniques were used to localise the following proteins within the fracture callus at different times following injury. The angiogenic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were localised; bFGF is also involved in matrix remodelling and cell proliferation. In addition, urokinase plasminogen activator and its receptor (uPA and uPAr respectively), a proteolytic enzyme involved in matrix remodelling, was immunolocalised. The model used for the study was a standardised midtibial osteotomy performed on New Zealand white rabbits. Results: Results showed that from early time points, VEGF and bFGF were detected in pericellular locations around the chondrocytes. However, VEGF was only detected around the chondrocytes in close proximity to infiltrating vessels whereas homogenous localisation of bFGF was seen throughout the cartilage. Urokinase was localised throughout the cartilage callus as well as within vascular cavities and its receptor was detected on the chondrocyte cell surface from early time points. Discussion: The immunolocalisation of VEGF around large hypertrophic chondrocytes in close proximity to infiltrating vessels suggests that this growth factor plays a role in chondrocyte hypertrophy and death, similar to its role in the growth plate. In contrast, bFGF was strongly detected throughout cartilage from early time points, suggesting that it may also be involved with cartilage proliferation during callus formation and subsequent matrix remodelling. The localisation of urokinase around chondrocytes and within vascular cavities suggests that this enzyme plays an important role in matrix remodelling and the vascularisation of the cartilage. In conclusion, our work suggests that the following sequence of events takes place within the cartilaginous callus. Firstly, bFGF is involved in the rapid proliferation of chondrocytes in the early stages of cartilage formation following fracture. Chondrocytes then express high levels of urokinase and its receptor which is actively involved in the degradation of cartilage matrix and formation of vascular spaces which leads to angiogenesis, VEGF expression, chondrocyte hypertrophy and eventual death

Aims

Cigarette smoking has a negative impact on the skeletal system, causes a decrease in bone mass in both young and old patients, and is considered a risk factor for the development of osteoporosis. In addition, it disturbs the bone healing process and prolongs the healing time after fractures. The mechanisms by which cigarette smoking impairs fracture healing are not fully understood. There are few studies reporting the effects of cigarette smoking on new blood vessel formation during the early stage of fracture healing. We tested the hypothesis that cigarette smoke inhalation may suppress angiogenesis and delay fracture healing.

Aims

The present study investigates the effectiveness of platelet-rich plasma (PRP) gel without adjunct to induce cartilage regeneration in large osteochondral defects in a rabbit model.

Despite biomechanical well established implants and improved operation techniques we still have a too high rate of complications in orthopaedic and trauma surgery like non-union, implant loosening or implant associated infections. The development of bioactive implants could improve the clinical outcome. Growth factors are important regulators of bone metabolism. During fracture healing many growth factors or cytokines were locally released at the facture site. In several studies, different growth factors demonstrated osteoinductive and fracture stimulating properties. In vitro and in vivo studies showed a stimulating effect of Insulin-like growth factor-I (IGF-I), Transforming growth factor-A71 (TGF-A71) and Bone morphogenetic protein-2 (BMP-2) on osteo- and chondrogenetic cells. The exact effectiveness and the interaction of these growth factors during fracture healing is not known so far. Further, the local application of these factors for therapeutically use in fracture treatment is still a problem. A biodegradable poly(D,L-lactide)-coating of implants allows the local and controlled release of incorporated growth factors directly at the fracture site. The coated implant serves on the one hand for fracture stabilization and on the other hand as a drug delivery system. The coating has a high mechanical stability. The incorporated growths factors remain biologically active in the coating and were released in a sustained and controlled manner. To investigate the effect of locally released growth factors IGF-I, TGF-A71 and BMP-2 and the carrier PDLLA on fracture healing, standardised closed fracture models were developed with a close relationship to clinical situation. Further, possible local and systemic side effects were analysed. The results demonstrated a significantly higher stimulating effect of IGF-I on fracture healing compared to TGF-A71. The combined application of both growth factors showed a synergistic effect on the mechanical stability and callus remodeling compared to single treatment. The local release of BMP-2 also enhanced fracture healing significantly – comparable to combination of IGF-I and TGF-A71. However, a higher rate of mineralisation was measurable outside the fracture region using BMP-2 in a rat fracture model. Using a large animal model on pigs with a 1 mm osteotomy gap, the effectiveness of locally released growths factors could be confirmed. Further, the PDLLA-coating without any incorporated growth factors demonstrated a significantly effect on healing processes in both models. These investigations showed, that the local release of growth factors from PDLLA coated implants significantly stimulate fracture healing without any local or systemic side effects. Comparing systemic with local stimulation techniques, we found an improvement of fracture healing by systemic administration of growth hormone and local application of IGF-I and TGF-A71. However, the combined use of both simulation techniques did not lead to a further increase of healing processes. Investigations on the effectiveness and the interaction of growth factors during fracture healing demonstrated an dramatic effect in the early phases of healing processes. The growth factors stimulate the differentiation of osteoblasts with a higher production of collagen I in vitro and increase osteogenesis and vascularisation of the fracture callus in vivo. Further applications of the coating technology are the use of PDLLA and growth factor coated cages for the stimulation of intervertebral fusion and the use of PDLLA and Gentamicin coated implants in order to prevent implant associated infections. The first patients with open tibia fractures were treated with PDLLA and Gentamicin coated IM nails

Introduction: A number of clinical and experimental studies suggest that an intact nervous system is essential for normal fracture healing. In the present study, we analysed the occurrence of regenerating and mature nerve fibres over time in fracture callus. Using antibodies against neuronal proteins specific for nerve regeneration (growth associated protein – GAP-43) and nerve maturity (protein gene product – PGP 9.5) it is possible to demonstrate regeneration and end differentiation of nerves by immunohistochemistry. Methods: Twelve male Sprague Dawley rats, weighing 230–290 g were used. The right tibias were fractured under HypnormÒ anaesthesia and fixed with a 17-G cannula needle in the medullary canal. The left un-fractured tibia served as an internal control. X-rays was used to monitor progress of fracture healing. Three rats were killed at 3 days, 1, 2 and 3 weeks post-fracture and right and left tibia were prepared for immunohistochemistry. The tissue sections (15 mm thick) were incubated with antiserum to GAP-43 and then with biotinylated antibodies. Cy2-conjugated avidin was used for the fluorescent staining. For double staining, after the staining with first antibody, the sections were incubated with avidin blocking solution followed by biotin blocking solution. Incubation with the second antiserum to PGP 9.5 was performed in the same manner as for the first peptide. For fluorescent staining of PGP 9.5, the sections were incubated with Cy3-conjugated avidin. A Nikon epifluorescence microscope was used for photog. Results: In the un-fractured tibia. PGP 9.5-positive nerve fibres were consistently identified in periosteum, muscles and connective tissues. A number of nerve fibres also expressed GAP-43, although there were no signs of nerve sprouting, i.e. regeneration. In the fractured tibia, many GAP-43-positive nerves were identified already at 3 days post-fracture in the hematoma and periosteum. At 1 week, abundant sprouting of these nerves was seen in cartilaginous callus and hyperplastic periosteum. A number of nerve terminals were observed very close to the chondroid cells in the fibrocartilage of the fracture gap. At 2 and 3 weeks, GAP 43-positive fibres gradually shifted from the fibrocartilage area towards the outlying hyperplastic periosteum. Double staining studies showed that an increased expression of GAP-43 as compared to PGP 9.5 occurred in the early period of fracture healing. This relationship changed at 3 weeks when enhanced PGP 9.5 and less GAP 43 expression was found. Discussion: Our study suggests that there was an intense nerve regeneration in the early phase of fracture healing. Thus, a prominent expression of GAP-43 was seen in sprouting nerves in the hyperplastic periosteum and the callus fibrocartilage as early as 1 week post-fracture. This expression remained high in the fractures up to 3 weeks, when healing was essentially completed. Possibly, this persistent occurrence of GAP-43 is necessary for the ensuing ossification and bone remodeling. PGP 9.5 expression was markedly low at one week, but became pronounced at 3 weeks, probably reflecting functional maturation of the regenerated nerve in the healing fracture. It may prove that strong regenerative capability of nerves seen in the fractures is a prerequisite for normal fracture healing. Our results point to the possibility that regenerating nerves provide the delivery system for GAP-43 and other neuronal mediators required for normal callus formation and/or neovascularization

Objectives

Experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) may have negative effects on fracture healing. This study aimed to assess the effect of immediate and delayed short-term administration of clinically relevant parecoxib doses and timing on fracture healing using an established animal fracture model.

Introduction. Septic arthritis of the hip in the newborn baby can result in severe long term sequelae in the form of dislocation of the hip, loss of movement and growth disturbance with leg length discrepancy. Reconstructive procedures for this difficult problem have not been satisfactory. Patient and methods. This series comprises eight patients with septic hip sequelae . All of them had old septic hip and leg length discrepancy ranging from 4 cm to 15 cm. all underwent hip stabilization by pelvic support osteotomy and simultaneous limb lengthening and angular deformities correction using the Ilizarov frame. The age at the operation range from 8 years to 19 years, with an average follow up 4 years. Results. The average of limb lengthening gained was 9cm. All hips remain stable and loss of the range of hip and knee motion was not significant. Other complications include pin tract infection one delayed consolidation of callus and one fracture of callus. Conclusion. The Ilizarov hip reconstruction is a good option for the treatment of the late sequelae of infantile hip infection in adolescents. It should be considered a promising choice for the management of late sequelae of infantile hip infection

Objectives

Opening wedge high tibial osteotomy (HTO) is an established surgical procedure for the treatment of early-stage knee arthritis. Other than infection, the majority of complications are related to mechanical factors – in particular, stimulation of healing at the osteotomy site. This study used finite element (FE) analysis to investigate the effect of plate design and bridging span on interfragmentary movement (IFM) and the influence of fracture healing on plate stress and potential failure.

Aims

In this randomized study, we aimed to compare quality of regenerate in monolateral versus circular frame fixation in 30 patients with infected nonunion of tibia.

Aims

The primary aim of this study was to develop a reliable, effective radiological score to assess the healing of humeral shaft fractures, the Radiographic Union Score for HUmeral fractures (RUSHU). The secondary aim was to assess whether the six-week RUSHU was predictive of nonunion at six months after the injury.

Large bone defects remain a tremendous clinical challenge. There is growing evidence in support of treatment strategies that direct defect repair through an endochondral route, involving a cartilage intermediate. While culture-expanded stem/progenitor cells are being evaluated for this purpose, these cells would compete with endogenous repair cells for limited oxygen and nutrients within ischaemic defects. Alternatively, it may be possible to employ extracellular vesicles (EVs) secreted by culture-expanded cells for overcoming key bottlenecks to endochondral repair, such as defect vascularization, chondrogenesis, and osseous remodelling. While mesenchymal stromal/stem cells are a promising source of therapeutic EVs, other donor cells should also be considered. The efficacy of an EV-based therapeutic will likely depend on the design of companion scaffolds for controlled delivery to specific target cells. Ultimately, the knowledge gained from studies of EVs could one day inform the long-term development of synthetic, engineered nanovesicles. In the meantime, EVs harnessed from in vitro cell culture have near-term promise for use in bone regenerative medicine. This narrative review presents a rationale for using EVs to improve the repair of large bone defects, highlights promising cell sources and likely therapeutic targets for directing repair through an endochondral pathway, and discusses current barriers to clinical translation.

Cite this article: E. Ferreira, R. M. Porter. Harnessing extracellular vesicles to direct endochondral repair of large bone defects. Bone Joint Res 2018;7:263–273. DOI: 10.1302/2046-3758.74.BJR-2018-0006.

Objectives

Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM.

Thrombin related peptide (TP 508) is a 23 amino-acid synthetic peptide that mimics a portion of the receptor-binding domain of the human thrombin molecule. Thrombin triggers both proteolytic activated receptors and non proteolytic activated receptors to bring about a mixture of responses ranging from tissue breakdown and clot formation, to new vessel formation and tissue repair. TP 508 stimulates only the non proteolytic activated receptors, and this initiates repair and angiogenesis but not clot formation or tissue breakdown Previous studies have shown that TP508 can stimulate repair in the dermal and musculoskeletal tissues by promoting angiogenesis and enhancing the proliferation and migration of cells. High energy fractures are associated with a delay in healing. We hypothesized that high energy fracture healing would be improved with the use of TP508, and that the dose and site of application would have importance. Methods: 80 CD 1 Mice were randomised into four groups; all underwent a high energy quadriceps muscle crush and a femoral fracture on the left hind limb. In each case the fracture was reduced and held with an external fixator. At the time of operation Group I received a dose of 100ìg TP 508 into the fracture, Group II 100ìg into the surrounding damaged soft tissue, Group III a dose of 10ìg into the fracture, and group IV (the control group) received PBS carrier into the fracture. 24 animals were sacrificed on day 21 and the remaining 56 mice on day 35. Of the 35 day old animals 8 in each group had both femora harvested and the biomechanical properties were tested using the 3-point bending technique. Specimens from the 21 day old animals and remaining 35 day old animals were used for histological analysis. All 80 animals had digital radiographs taken each week. Using image analysis software five pixel density graphs were generated across each fracture gap. A validated semi quantitative analysis was used to score each graph and the total accumulated for each radiograph. The width of the fracture calus was measured and expressed as a ratio of the femur diameter. Results: Mechanical testing showed significantly greater stiffness in group I when compared to control (p < 0.05), and a dose dependent trend of increasing strength. Radiographic analysis showed greater healing of fracture and callus formation in Group I compared to Groups II, III, and IV, at both three and five weeks post-fracture (P< 0.05). Histological analysis showed an increase in bone formation in group I compared to the other groups. Conclusion: This data from this model, suggests that TP508 enhances healing in high energy fractures. The results also suggest that the effects of TP508 are dose dependant, and are greater when delivered into the fracture site

Aims

Recent studies of nonoperatively treated displaced midshaft clavicular fractures have shown a high incidence of nonunion and unsatisfactory functional outcome. Some studies have shown superior functional results and higher rates of healing following operative treatment. The aim of this study was to compare the outcome in these patients after nonoperative management with those treated with fixation.

Objectives

Secondary fracture healing is strongly influenced by the stiffness of the bone-fixator system. Biomechanical tests are extensively used to investigate stiffness and strength of fixation devices. The stiffness values reported in the literature for locked plating, however, vary by three orders of magnitude. The aim of this study was to examine the influence that the method of restraint and load application has on the stiffness produced, the strain distribution within the bone, and the stresses in the implant for locking plate constructs.

Objectives

We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days.

This study was designed to test the hypothesis that the sensory innervation of bone might play an important role in sensing and responding to low-intensity pulsed ultrasound and explain its effect in promoting fracture healing. In 112 rats a standardised mid-shaft tibial fracture was created, supported with an intramedullary needle and divided into four groups of 28. These either had a sciatic neurectomy or a patellar tendon resection as control, and received the ultrasound or not as a sham treatment. Fracture union, callus mineralisation and remodelling were assessed using plain radiography, peripheral quantitative computed tomography and histomorphology.

Daily ultrasound treatment significantly increased the rate of union and the volumetric bone mineral density in the fracture callus in the neurally intact rats (p = 0.025), but this stimulating effect was absent in the rats with sciatic neurectomy. Histomorphology demonstrated faster maturation of the callus in the group treated with ultrasound when compared with the control group. The results supported the hypothesis that intact innervation plays an important role in allowing low-intensity pulsed ultrasound to promote fracture healing.

Objectives

The radiographic union score for tibial (RUST) fractures was developed by Whelan et al to assess the healing of tibial fractures following intramedullary nailing. In the current study, the repeatability and reliability of the RUST score was evaluated in an independent centre (a) using the original description, (b) after further interpretation of the description of the score, and (c) with the immediate post-operative radiograph available for comparison.

Congenital pseudarthrosis of the tibia is an uncommon manifestation of neurofibromatosis type 1 (NF1), but one that remains difficult to treat due to anabolic deficiency and catabolic excess. Bone grafting and more recently recombinant human bone morphogenetic proteins (rhBMPs) have been identified as pro-anabolic stimuli with the potential to improve the outcome after surgery. As an additional pharmaceutical intervention, we describe the combined use of rhBMP-2 and the bisphosphonate zoledronic acid in a mouse model of NF1-deficient fracture repair.

Fractures were generated in the distal tibiae of neurofibromatosis type 1-deficient (Nf1^+/−) mice and control mice. Fractures were open and featured periosteal stripping. All mice received 10 μg rhBMP-2 delivered in a carboxymethylcellulose carrier around the fracture as an anabolic stimulus. Bisphosphonate-treated mice also received five doses of 0.02 mg/kg zoledronic acid given by intraperitoneal injection.

When only rhBMP but no zoledronic acid was used to promote repair, 75% of fractures in Nf1^+/− mice remained ununited at three weeks compared with 7% of controls (p < 0.001). Systemic post-operative administration of zoledronic acid halved the rate of ununited fractures to 37.5% (p < 0.07).

These data support the concept that preventing bone loss in combination with anabolic stimulation may improve the outcome following surgical treatment for children with congenital pseudarthoris of the tibia and NF1.

Fracture repair occurs by two broad mechanisms: direct healing, and indirect healing with callus formation. The effects of bisphosphonates on fracture repair have been assessed only in models of indirect fracture healing.

A rodent model of rigid compression plate fixation of a standardised tibial osteotomy was used. Ten skeletally mature Sprague–Dawley rats received daily subcutaneous injections of 1 µg/kg ibandronate (IBAN) and ten control rats received saline (control). Three weeks later a tibial osteotomy was rigidly fixed with compression plating. Six weeks later the animals were killed. Fracture repair was assessed with mechanical testing, radiographs and histology.

The mean stress at failure in a four-point bending test was significantly lower in the IBAN group compared with controls (8.69 Nmm^-2 (sd 7.63) vs 24.65 Nmm^-2 (sd 6.15); p = 0.017). On contact radiographs of the extricated tibiae the mean bone density assessment at the osteotomy site was lower in the IBAN group than in controls (3.7 mmAl (sd 0.75) vs 4.6 mmAl (sd 0.57); p = 0.01). In addition, histological analysis revealed progression to fracture union in the controls but impaired fracture healing in the IBAN group, with predominantly cartilage-like and undifferentiated mesenchymal tissue (p = 0.007).

Bisphosphonate treatment in a therapeutic dose, as used for risk reduction in fragility fractures, had an inhibitory effect on direct fracture healing. We propose that bisphosphonate therapy not be commenced until after the fracture has united if the fracture has been rigidly fixed and is undergoing direct osteonal healing.

Cite this article: Bone Joint J 2013;95-B:1263–8.

The August 2014 Research Roundup³⁶⁰ looks at: Antibiotic loaded ceramic of use in osteomyelitis; fibronectin implicated in cartilage degeneration; Zinc Chloride accelerates fracture healing in rats; advertisements and false claims; Net Promoter Score: substance or rhetoric?; aspirin for venous thromboembolism prophylaxis and dissection, stress and the soul.

The widespread use of MRI has revolutionised the diagnostic process for spinal disorders. A typical protocol for spinal MRI includes T1 and T2 weighted sequences in both axial and sagittal planes. While such an imaging protocol is appropriate to detect pathological processes in the vast majority of patients, a number of additional sequences and advanced techniques are emerging. The purpose of the article is to discuss both established techniques that are gaining popularity in the field of spinal imaging and to introduce some of the more novel ‘advanced’ MRI sequences with examples to highlight their potential uses.

Cite this article: Bone Joint J 2015;97-B:1683–92.

The August 2013 Trauma Roundup³⁶⁰ looks at: reverse oblique fractures do better with a cephalomedullary device; locking screws confer no advantage in tibial plateau fractures; it’s all about the radius of curvature; radius of curvature revisited; radial head replacement in complex elbow reconstruction; stem cells in early fracture haematoma; heterotrophic ossification in forearms; and Boston in perspective.

This prospective multicentre study was undertaken to determine whether the timing of the post-operative administration of bisphosphonate affects fracture healing and the rate of complication following an intertrochanteric fracture. Between August 2008 and December 2009, 90 patients with an intertrochanteric fracture who underwent internal fixation were randomised to three groups according to the timing of the commencement of risedronate treatment after surgery: Group A (from one week after surgery), Group B (from one month after surgery), and Group C (from three months after surgery). The radiological time to fracture healing was assessed as the primary endpoint, and the incidence of complications, including excessive displacement or any complication requiring revision surgery, as the secondary endpoint. The mean time to fracture healing post-operatively in groups A, B and C was 10.7 weeks (sd 4.4), 12.9 weeks (sd 6.2) and 12.3 weeks (sd 7.1), respectively (p = 0.420). At 24 weeks after surgery, all fractures had united, except six that had a loss of fixation. Functional outcomes at one year after surgery according to the Koval classification (p = 0.948) and the incidence of complications (p = 0.386) were similar in the three groups.

This study demonstrates that the timing of the post-operative administration of bisphosphonates does not appear to affect the rate of healing of an intertrochanteric fracture or the incidence of complications.

Although mechanical stabilisation has been a hallmark of orthopaedic surgical management, orthobiologics are now playing an increasing role. Platelet-rich plasma (PRP) is a volume of plasma fraction of autologous blood having platelet concentrations above baseline. The platelet α granules are rich in growth factors that play an essential role in tissue healing, such as transforming growth factor-β, vascular endothelial growth factor, and platelet-derived growth factor. PRP is used in various surgical fields to enhance bone and soft-tissue healing by placing supraphysiological concentrations of autologous platelets at the site of tissue damage. The easily obtainable PRP and its possible beneficial outcome hold promise for new regenerative treatment approaches.

The aim of this literature review was to describe the bioactivities of PRP, to elucidate the different techniques for PRP preparation, to review animal and human studies, to evaluate the evidence regarding the use of PRP in trauma and orthopaedic surgery, to clarify risks, and to provide guidance for future research.

We investigated the eventual diagnosis in patients referred to a tertiary centre with a possible diagnosis of a primary bone malignancy.

We reviewed our database from between 1986 and 2010, during which time 5922 patients referred with a suspicious bone lesion had a confirmed diagnosis. This included bone sarcoma in 2205 patients (37%), benign bone tumour in 1309 (22%), orthopaedic conditions in 992 (17%), metastatic disease in 533 (9%), infection in 289 (5%) and haematological disease in 303 (5%). There was a similar frequency of all diagnoses at different ages except for metastatic disease. Only 0.6% of patients (17 of 2913) under the age of 35 years had metastatic disease compared with 17.1% (516 of 3009) of those over 35 years (p < 0.0001). Of the 17 patients under 35 years with metastatic disease, only four presented with an isolated lesion, had no past history of cancer and were systematically well.

Patients under the age of 35 years should have suitable focal imaging (plain radiography, CT or MRI) and simple systemic studies (blood tests and chest radiography). Reduction of the time to biopsy can be achieved by avoiding an unnecessary investigation for a primary tumour to rule out metastatic disease.

Smoking is known to have an adverse effect on wound healing and musculoskeletal conditions. This case-controlled study looked at whether smoking has a deleterious effect in the outcome of autologous chondrocyte implantation for the treatment of full thickness chondral defects of the knee.

The mean Modified Cincinatti Knee score was statistically significantly lower in smokers (n = 48) than in non-smokers (n = 66) both before and after surgery (p < 0.05). Smokers experienced significantly less improvement in the knee score two years after surgery (p < 0.05). Graft failures were only seen in smokers (p = 0.016). There was a strong negative correlation between the number of cigarettes smoked and the outcome following surgery (Pearson’s correlation coefficient −0.65, p = 0.004).

These results suggest that patients who smoke have worse pre-operative function and obtain less benefit from this procedure than non-smokers. The counselling of patients undergoing autologous chondrocyte implantation should include smoking, not only as a general cardiopulmonary risk but also because poorer results can be expected in smokers following this procedure.

We have previously shown that joint distraction and movement with a hinged external fixation device for 12 weeks was useful for repairing a large articular cartilage defect in a rabbit model. We have now investigated the results after six months and one year. The device was applied to 16 rabbits who underwent resection of the articular cartilage and subchondral bone from the entire tibial plateau. In group A (nine rabbits) the device was applied for six months. In group B (seven rabbits) it was in place for six months, after which it was removed and the animals were allowed to move freely for an additional six months. The cartilage remained sound in all rabbits. The areas of type II collagen-positive staining and repaired soft tissue were larger in group B than in group A. These findings provide evidence of long-term persistence of repaired cartilage with this technique and that weight-bearing has a positive effect on the quality of the cartilage.

Heterotopic ossification occurring after the use of commercially available bone morphogenetic proteins has not been widely reported. We describe four cases of heterotopic ossification in patients treated with either recombinant bone morphogenetic protein 2 or recombinant bone morphogenetic protein 7. We found that while some patients were asymptomatic, heterotopic ossification which had occurred around a joint often required operative excision with good results.

Conventional non-steroidal anti-inflammatory drugs (NSAIDs) and newer specific cyclo-oxygenase-2 (cox-2) inhibitors are commonly used in musculoskeletal trauma and orthopaedic surgery to reduce the inflammatory response and pain. These drugs have been reported to impair bone metabolism. In reconstruction of the anterior cruciate ligament the hamstring tendons are mainly used as the graft of choice, and a prerequisite for good results is healing of the tendons in the bone tunnel. Many of these patients are routinely given NSAIDs or cox-2 inhibitors, although no studies have elucidated the effects of these drugs on tendon healing in the bone tunnel.

In our study 60 female Wistar rats were randomly allocated into three groups of 20. One received parecoxib, one indometacin and one acted as a control. In all the rats the tendo-Achillis was released proximally from the calf muscles. It was then pulled through a drill hole in the distal tibia and sutured anteriorly. The rats were given parecoxib, indometacin or saline intraperitoneally twice daily for seven days. After 14 days the tendon/bone-tunnel interface was subjected to mechanical testing.

Significantly lower maximum pull-out strength (p < 0.001), energy absorption (p < 0.001) and stiffness (p = 0.035) were found in rats given parecoxib and indometacin compared with the control group, most pronounced with parecoxib.

The haematoma occurring at the site of a fracture is known to play an important role in bone healing. We have recently shown the presence of progenitor cells in human fracture haematoma and demonstrated that they have the capacity for multilineage mesenchymal differentiation. There have been many studies which have shown that low-intensity pulsed ultrasound (LIPUS) stimulates the differentiation of a variety of cells, but none has investigated the effects of LIPUS on cells derived from human fracture tissue including human fracture haematoma-derived progenitor cells (HCs). In this in vitro study, we investigated the effects of LIPUS on the osteogenic activity of HCs. Alkaline phosphatase activity, osteocalcin secretion, the expression of osteoblast-related genes and the mineralisation of HCs were shown to be significantly higher when LIPUS had been applied but without a change in the proliferation of the HCs. These findings provide evidence in favour of the use of LIPUS in the treatment of fractures.

Perilesional changes of chronic focal osteochondral defects were assessed in the knees of 23 sheep. An osteochondral defect was created in the main load-bearing region of the medial condyle of the knees in a controlled, standardised manner. The perilesional cartilage was evaluated macroscopically and biopsies were taken at the time of production of the defect (T0), during a second operation one month later (T1), and after killing animals at three (T3; n = 8), four (T4; n = 8), and seven (T7; n = 8) months. All the samples were histologically assessed by the International Cartilage Repair Society grading system and Mankin histological scores. Biopsies were taken from human patients (n = 10) with chronic articular cartilage lesions and compared with the ovine specimens. The ovine perilesional cartilage presented with macroscopic and histological signs of degeneration. At T1 the International Cartilage Repair Society ‘Subchondral Bone’ score decreased from a mean of 3.0 (sd 0) to a mean of 1.9 (sd 0.3) and the ‘Matrix’ score from a mean of 3.0 (sd 0) to a mean of 2.5 (sd 0.5). This progressed further at T3, with the International Cartilage Repair Society ‘Surface’ grading, the ‘Matrix’ grading, ‘Cell Distribution’ and ‘Cell Viability’ grading further decreasing and the Mankin score rising from a mean of 1.3 (sd 1.4) to a mean of 5.1 (sd 1.6). Human biopsies achieved Mankin grading of a mean of 4.2 (sd 1.6) and were comparable with the ovine histology at T1 and T3.

The perilesional cartilage in the animal model became chronic at one month and its histological appearance may be considered comparable with that seen in human osteochondral defects after trauma.

Highly active anti-retroviral therapy has transformed HIV into a chronic disease with a long-term asymptomatic phase. As a result, emphasis is shifting to other effects of the virus, aside from immunosuppression and mortality. We have reviewed the current evidence for an association between HIV infection and poor fracture healing.

The increased prevalence of osteoporosis and fragility fractures in HIV patients is well recognised. The suggestion that this may be purely as a result of highly active anti-retroviral therapy has been largely rejected. Apart from directly impeding cellular function in bone remodelling, HIV infection is known to cause derangement in the levels of those cytokines involved in fracture healing (particularly tumour necrosis factor-α) and appears to impair the blood supply of bone.

Many other factors complicate this issue, including a reduced body mass index, suboptimal nutrition, the effects of anti-retroviral drugs and the avoidance of operative intervention because of high rates of wound infection. However, there are sound molecular and biochemical hypotheses for a direct relationship between HIV infection and impaired fracture healing, and the rewards for further knowledge in this area are extensive in terms of optimised fracture management, reduced patient morbidity and educated resource allocation. Further investigation in this area is overdue.

The use of allograft struts and cerclage wire, possibly augmented by plate fixation, for the treatment of Vancouver type-B1 peri-prosthetic fractures around a total hip replacement has been strongly advocated. We examined our results using plate fixation without allograft struts and compared them with the results of the use of struts alone or when combined with plate fixation. Of 20 consecutive patients with type-B1 fractures treated by open reduction and plate fixation, 19 were available for follow-up. The fractures healed in 18 patients with a mean time to weight-bearing of ten weeks (4 to 19). There were no cases of infection or malunion. Nonunion occurred in one patient and required a second plate fixation to achieve union.

Safe, cost-effective treatment of Vancouver type-B1 fractures can be performed by plate fixation without the addition of cortical struts. This procedure may allow earlier weight-bearing than allograft strut fixation alone.

We have evaluated the effect of the short-term administration of low therapeutic doses of modern COX-2 inhibitors on the healing of fractures.

A total of 40 adult male New Zealand rabbits were divided into five groups. A mid-diaphyseal osteotomy of the right ulna was performed and either normal saline, prednisolone, indometacin, meloxicam or rofecoxib was administered for five days. Radiological, biomechanical and histomorphometric evaluation was performed at six weeks.

In the group in which the highly selective anti-COX-2 agent, rofecoxib, was used the incidence of radiologically-incomplete union was similar to that in the control group. All the biomechanical parameters were statistically significantly lower in both the prednisolone and indometacin (p = 0.01) and in the meloxicam (p = 0.04) groups compared with the control group. Only the fracture load values were found to be statistically significantly lower (p = 0.05) in the rofecoxib group. Histomorphometric parameters were adversely affected in all groups with the specimens of the rofecoxib group showing the least negative effect.

Our findings indicated that the short-term administration of low therapeutic doses of a highly selective COX-2 inhibitor had a minor negative effect on bone healing.

The feasibility of bone transport with bone substitute and the factors which are essential for a successful bone transport are unknown. We studied six groups of 12 Japanese white rabbits. Groups A to D received cylindrical autologous bone segments and groups E and F hydroxyapatite prostheses. The periosteum was preserved in group A so that its segments had a blood supply, cells, proteins and scaffold. Group B had no blood supply. Group C had proteins and scaffold and group D had only scaffold. Group E received hydroxyapatite loaded with recombinant human bone morphogenetic protein-2 and group F had hydroxyapatite alone.

Distraction osteogenesis occurred in groups A to C and E which had osteo-conductive transport segments loaded with osteo-inductive proteins. We conclude that scaffold and proteins are essential for successful bone transport, and that bone substitute can be used to regenerate bone.

We retrospectively reviewed 101 consecutive patients with 114 femoral tumours treated by massive bone allograft at our institution between 1986 and 2005. There were 49 females and 52 males with a mean age of 20 years (4 to 74). At a median follow-up of 9.3 years (2 to 19.8), 36 reconstructions (31.5%) had failed. The allograft itself failed in 27 reconstructions (24%).

Mechanical complications such as delayed union, fracture and failure of fixation were studied. The most adverse factor on the outcome was the use of intramedullary nails, followed by post-operative chemotherapy, resection length > 17 cm and age > 18 years at the time of intervention. The simultaneous use of a vascularised fibular graft to protect the allograft from mechanical complications improved the outcome, but the use of intramedullary cementing was not as successful.

In order to improve the strength of the reconstruction and to advance the biology of host–graft integration, we suggest avoiding the use of intramedullary nails and titanium plates, but instead using stainless steel plates, as these gave better results. The use of a supplementary vascularised fibular graft should be strongly considered in adult patients with resection > 17 cm and in those who require post-operative chemotherapy.

A percutaneous supramalleolar osteotomy with multiple drill holes and closed osteoclasis was used to correct rotational deformities of the tibia in patients with cerebral palsy. The technique is described and the results in 247 limbs (160 patients) are reported. The mean age at the time of surgery was 10.7 years (4 to 20). The radiographs were analysed for time to union, loss of correction, and angulation at the site of the osteotomy.

Bone healing was obtained in all patients except one in a mean period of seven weeks (5 to 12). Malunion after loss of reduction at the site of the osteotomy developed in one tibia.

Percutaneous supramalleolar osteotomy of the tibia is a safe and simple surgical procedure.

This paper reviews the current literature concerning the main clinical factors which can impair the healing of fractures and makes recommendations on avoiding or minimising these in order to optimise the outcome for patients. The clinical implications are described.