To test and evaluate the effectiveness of local injection of autologous fat-derived mesenchymal stem cells (MSCs) into fracture site to prevent non-union in a clinically relevant model. 5 male Wistar rats underwent the same surgical procedure of inducing non-union. A mid-shaft tibial osteotomy was made with 1mm non-critical gap. Periosteum was stripped around the two fracture ends. Then, the fracture was fixed by ante-grade intramedullary nail. The non-critical gap was maintained by a spacer with minimal effect on the healing surface area. At the same surgical time, subcutaneous fat was collected from the ipsilateral inguinal region and stem cells were isolated and cultured in vitro. Within three weeks postoperatively, the number of expanded stem cells reached 5×10. 6. and were injected into the fracture site. Healing was followed up for 8 weeks and the quality was measured by serial x-rays, microCT, mechanical testing and histologically. Quality of healing was compared with that of previously published allogenic, xenogeneic MSCs and Purified Buffered Saline (PBS) controls. All the five fractures united fully after 8 weeks. There was a progressive increase in the callus radiopacity during the eight-week duration, the average radiopacity in the autologous fat-MSC injected group was significantly higher than that of the allogeneic MSCs, xenogeneic MSCs and the control group, P < 0.0001 for treatment, time after injection, and treatment-time interaction (two-way repeated measure ANOVA). MicroCT, mechanical testing and histology confirmed radiological findings. The autologous fat-MSCs are effective in prevention of atrophic non-union by stimulation of the healing process leading to a solid union. The quality and speed of repair are higher than those of the other types of cell transplantation tested

Fracture healing is a spatially controlled process involving crosstalk of multiple tissues. To precisely capture and understand molecular mechanism underlying impaired healing, there is a need to integrate spatially-resolved molecular analyses into preclinical fracture healing models. I will present our recent data obtained by spatial transcriptomics of musculoskeletal samples from fracture healing studies in mice. Subsequently, I will show how spatial transcriptomics can be integrated into multimodal approaches in preclinical fracture healing models. In combination with established in vivo imaging and emerging omics techniques, spatially-resolved analyses have the potential to elucidate the molecular mechanisms underlying impaired healing with optimization of treatments.

Fractures and related complications are a common challenge in the field of skeletal tissue engineering. Vitamin D and calcium are the only broadly available medications for fracture healing, while zinc has been recognized as a nutritional supplement for healthy bones. Here, we aimed to use polaprezinc, an anti-ulcer drug and a chelate form of zinc and L-carnosine, as a supplement for fracture healing. Polaprezinc induced upregulation of osteogenesis-related genes and enhanced the osteogenic potential of human bone marrow-derived mesenchymal stem cells and osteoclast differentiation potential of mouse bone marrow-derived monocytes. In mouse experimental models with bone fractures, oral administration of polaprezinc accelerated fracture healing and maintained a high number of both osteoblasts and osteoclasts in the fracture areas. Collectively, polaprezinc promotes the fracture healing process efficiently by enhancing the activity of both osteoblasts and osteoclasts. Therefore, we suggest that drug repositioning of polaprezinc would be helpful for patients with fractures.

The primary aim was to assess the reliability of ultrasound in the assessment of humeral shaft fracture healing. The secondary aim was to estimate the accuracy of ultrasound assessment in predicting humeral shaft nonunion.

Twelve patients (mean age 54yrs [20–81], 58% [n=7/12] female) with a non-operatively managed humeral diaphyseal fracture were prospectively recruited and underwent ultrasound scanning at six and 12wks post-injury. Scans were reviewed by seven blinded observers to evaluate the presence of sonographic callus. Intra- and inter-observer reliability were determined using the weighted kappa and intraclass correlation coefficient (ICC). Accuracy of ultrasound assessment in nonunion prediction was estimated by comparing scans for patients that united (n=10/12) with those that developed a nonunion (n=2/12).

At both six and 12wks, sonographic callus was present in 11 patients (10 united, one developed a nonunion) and sonographic bridging callus (SBC) was present in seven patients (all united). Ultrasound assessment demonstrated substantial intra- (6wk kappa 0.75, 95% CI 0.47-1.03; 12wk kappa 0.75, 95% CI 0.46-1.04) and inter-observer reliability (6wk ICC 0.60, 95% CI 0.38-0.83; 12wk ICC 0.76, 95% CI 0.58-0.91). Absence of sonographic callus demonstrated a sensitivity of 50%, specificity 100%, positive predictive value (PPV) 100% and negative predictive value (NPV) 91% in nonunion prediction (accuracy 92%). Absence of SBC demonstrated a sensitivity of 100%, specificity 70%, PPV 40% and NPV 100% (accuracy 75%). Of three patients at risk of nonunion based on reduced radiographic callus formation (Radiographic Union Score for HUmeral fractures <8), one had SBC on 6wk ultrasound (and united) and the other two had non-bridging or absent sonographic callus (both developed a nonunion).

Ultrasound assessment of humeral shaft fracture healing was reliable and predictive of nonunion, and may be a useful tool in defining the risk of nonunion among patients with reduced radiographic callus formation.

There is still no consensus on which concentration of mesenchymal stem cells (MSCs) to use for promoting fracture healing in a rat model of long bone fracture.

To assess the optimal concentration of MSCs for promoting fracture healing in a rat model.

Wistar rats were divided into four groups according to MSC concentrations: Normal saline (C), 2.5 × 106 (L), 5.0 × 106 (M), and 10.0 × 106 (H) groups. The MSCs were injected directly into the fracture site. The rats were sacrificed at 2 and 6 자 post-fracture. New bone formation [bone volume (BV) and percentage BV (PBV)] was evaluated using micro-computed tomography (CT). Histological analysis was performed to evaluate fracture healing score. The protein expression of factors related to MSC migration [stromal cell-derived factor 1 (SDF-1), transforming growth factor-beta 1 (TGF-β1)] and angiogenesis [vascular endothelial growth factor (VEGF)] was evaluated using western blot analysis. The expression of cytokines associated with osteogenesis [bone morphogenetic protein-2 (BMP-2), TGF-β1 and VEGF] was evaluated using real-time polymerase chain reaction.

Micro-CT showed that BV and PBV was significantly increased in groups M and H compared to that in group C at 6 wk post-fracture (P = 0.040, P = 0.009; P = 0.004, P = 0.001, respectively). Significantly more cartilaginous tissue and immature bone were formed in groups M and H than in group C at 2 and 6 wk post-fracture (P = 0.018, P = 0.010; P = 0.032, P = 0.050, respectively). At 2 wk post fracture, SDF-1, TGF-β1 and VEGF expression were significantly higher in groups M and H than in group L (P = 0.031, P = 0.014; P < 0.001, P < 0.001; P = 0.025, P < 0.001, respectively). BMP-2 and VEGF expression were significantly higher in groups M and H than in group C at 6 wk postfracture (P = 0.037, P = 0.038; P = 0.021, P = 0.010). Compared to group L, TGF-β1 expression was significantly higher in groups H (P = 0.016). There were no significant differences in expression levels of chemokines related to MSC migration, angiogenesis and cytokines associated with osteogenesis between M and H groups at 2 and 6 wk post-fracture.

The administration of at least 5.0 × 106 MSCs was optimal to promote fracture healing in a rat model of long bone fractures.

Introduction

Immunomodulation represents a novel strategy to improve bone healing in combination with low doses of bone morphogenetic growth factors like BMP-2. This study aims to investigate the effect and timing of monoclonal anti-IL-1ß antibody administration with 1μg BMP-2 on bone healing over 14 weeks in a rat femur segmental defect model.

Introduction and Objective

In multiple trauma patients, as well as in the healing of isolated fractures (Fx) with heavy bleeding (trauma haemorrhage, TH), complications occur very often. This is particularly evident in elderly patients over 65 years of age. Since these accompanying circumstances strongly influence the clinical course of treatment, the influence of age on bone regeneration after femoral fracture and severe blood loss was investigated in this study.

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.

Although 80% of fractures typically heal without any problems, there is a small proportion (<20%) that suffer complications such as delayed healing and potential progression to non-union. In patients with healing complications, the coordinated regulation between pro- and anti-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) respectively, is often dysregulated. The aim of this study is to develop a therapeutic strategy based on the local delivery of genes to reparative mesenchymal stromal cells (MSCs) migrating into the local fracture microenvironment, thereby promoting a more favourable healing environment to enhance fracture repair. Our approach involves the local delivery of nanoparticles complexing the non-viral vector polyethyleneimine (PEI) with therapeutic plasmid DNA (pDNA) encoding for IL-1Ra.

pDNA encoding green fluorescent protein and Gaussia luciferase were used as reporter genes to determine the transfection efficiency of both rat and human MSCs using flow cytometry and to assess the transgene expression profile using a luciferase expression assay. The effect of transfection with PEI on the viability of MSCs was assessed using the metabolic assay Cell Titer Blue and dsDNA quantification. Levels of IL-1Ra produced by cells following transfection with nanoparticles encoding IL-1Ra was assessed using enzyme-linked immunosorbent assays (ELISA). HEK-Blue IL-1β reporter cells, which secrete alkaline phosphatase in response to IL-1β stimulation, were used to confirm that the IL-1Ra produced by transfected cells is functionally active, i.e. the successful antagonism of IL-1β bioactivity.

We have determined that using PEI-based nanoparticles we can achieve a transfection efficiency of 14.8 + 1.8% in rat MSCs. Transgene expression was found to be transient, with a peak in expression at 7 days post-transfection and a gradual decrease over time, which was maintained for up to 4 weeks. Using an optimized concentration of PEI, the impact of the nanoparticles on MSC viability was limited, with no significant difference in cellular metabolic activity compared to non-transfected cells at 10 days post-transfection. We have additionally demonstrated the capacity to successfully transfect both rat and human MSCs with pDNA encoding for IL-1Ra, resulting in enhanced levels of IL-1Ra, which is functionally active.

The use of non-viral gene therapy to locally deliver immunomodulatory genes, such as IL-1Ra, to MSCs presents a promising strategy to enhance bone healing. Specifically, the transgene expression levels achieved with such an approach can remain therapeutically effective and are transient in nature, presenting an advantage over other methods such as recombinant protein delivery and viral-based gene delivery methodologies.

Background

Sclerostin is a secreted glycoprotein that inhibits the intracellular Wnt signaling pathway, which when inactivated bone formation is stimulated. This stimulation has been proven in fracture studies, showing larger and stronger calluses with accelerated fracture healing, both in sclerostin knockout and sclerostin antibody injection models. The effects of these two mechanisms have not been compared to assess the accurate effect of the Scl-Ab injections. Therefore we designed a study to compare the effect of sclerostin depletion (sclerostin knockout) and inhibition (Scl-Ab injection).

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.

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.

Introduction and Objective

It is widely accepted that interfragmentary strain stimulus promotes callus formation during secondary bone healing. However, the impact of the temporal variation of mechanical stimulation on fracture healing is still not well understood. Moreover, the minimum strain value that initiates callus formation is unknown. The goal of this study was to develop an active fixation system that allows for in vivo testing of varying temporal distribution of mechanical stimulation and that enables detection of the strain limit that initiates callus formation.

Fracture fixation has advanced significantly with the introduction of locked plating and minimally invasive surgical techniques. However, healing complications occur in up to 10% of cases, of which a significant portion may be attributed to unfavorable mechanical conditions at the fracture. Moreover, state-of-the-art plates are prone to failure from excessive loading or fatigue. A novel biphasic plating concept has been developed to create reliable mechanical conditions for timely bone healing and simultaneously improve implant strength. The goal of this study was to test the feasibility and investigate the robustness of fracture healing with a biphasic plate in a large animal experiment. Twenty-four sheep underwent a 2mm mid-diaphyseal tibia osteotomy stabilized with either the novel biphasic plate or a control locking plate.

Different fracture patterns in terms of defect location and orientation were investigated. Animals were free to fully bear weight during the post-operative period. After 12 weeks, the healing fractures were evaluated for callus formation using micro-computer tomography and strength and stiffness using biomechanical testing. No plate deformation or failures were observed under full weight bearing with the biphasic plate. Osteotomies stabilized with the biphasic plate demonstrated robust callus formation. Torsion tests after plate removal revealed no statistical difference in peak torsion to failure and stiffness for the different fracture patterns stabilized with the biphasic plate. However, the biphasic plate group specimens were 45% stronger (p=0.002) and 48% stiffer (p=0.007) than the controls.

The results of this large animal study demonstrate the clinical potential of this novel stabilization concept.

Abstract

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.

The course of secondary fracture healing typically consists of four major phases including inflammation, soft and hard callus formation, and bone remodeling. Callus formation is promoted by mechanical stimulation, yet little is known about the healing tissue response to strain stimuli over shorter timeframes on hourly and daily basis. The aim of this study was to explore the hourly, daily and weekly variations in bone healing progression and to analyze the short-term response of the repair tissue to well-controlled mechanical stimulation.

A system for continuous monitoring of fracture healing was designed for implantation in sheep tibia. The experimental model was adapted from Tufekci et al. 2018 and consisted of 3 mm transverse osteotomy and 30 mm bone defect resulting in an intermediate mobile bone fragment in the tibial shaft. Whereas the distal and proximal parts of the tibia were fixed with external fixator, the mobile fragment was connected to the proximal part via a second, active fixator. A linear actuator embedded in the active fixator moved the mobile fragment axially, thus stimulating mechanically the tissue in the osteotomy gap via well-controlled displacement being independent from the sheep's functional weightbearing. A load sensor was integrated in the active fixation to measure the force acting in the osteotomy gap. During each stimulation cycle the displacement and force magnitudes were recorded to determine in vivo fracture stiffness. Following approval of the local ethics committee, experiments were conducted on four skeletally mature sheep. Starting from the first day after surgery, the daily stimulation protocols consisted of 1000 loading events equally distributed over 12 hours from 9:00 to 21:00 resulting in a single loading event every 44 seconds. No stimulation was performed overnight.

One animal had to be excluded due to inconsistencies in the load sensor data. The onset of tissue stiffening was detected around the eleventh day post-op. However, on a daily basis, the stiffness was not steadily increasing, but instead, an abrupt drop was observed in the beginning of the daily stimulations. Following this initial drop, the stiffness increased until the last stimulation cycle of the day.

The continuous measurements enabled resolving the tissue response to strain stimuli over hours and days. The presented data contributes to the understanding of the influence of patient activity on daily variations in tissue stiffness and can serve to optimize rehabilitation protocols post fractures.

Abstract

In the course of uneventful secondary bone healing, a fracture gap is progressively overgrown by callus which subsequently calcifies and remodels into new bone. It is widely accepted that callus formation is promoted by mechanical stimulation of the tissue in the fracture gap. However, the optimal levels of the interfragmentary motion's amplitude, frequency and timing remain unknown. The aim of this study was to develop an active fixation system capable of installing a well-controlled mechanical environment in the fracture gap with continuous monitoring of the bone healing progression.

The experimental model was adapted from Tufekci et al. 2018 and required creation of a critical size defect and an osteotomy in a sheep tibia. They were separated by a mobile bone fragment. The distal and proximal parts of the tibia were fixed with an external fixator, whereas the mobile fragment was connected to the proximal part with an active fixator equipped with a linear actuator to move it axially for mechanical stimulation of the tissue in the fracture gap. This configuration installed well-controlled mechanical conditions in the osteotomy, dependent only on the motion of the active fixator and shielded from the influence of the sheep's functional weightbearing. A load sensor was integrated to measure the force acting in the fracture gap during mechanical stimulation. The motion of the bone fragment was controlled by means of a custom-made controller allowing to program stimulation protocols of various profiles, amplitudes and frequencies of loading events. Following in vitro testing, the system was tested in two Swiss White Alpine Sheep. It was configured to simulate immediate weightbearing for one of the animals and delayed weightbearing for the other. The applied loading protocol consisted of 1000 loading events evenly distributed over 12 hours resulting in in a single loading event every 44 seconds.

Bench testing confirmed the ability of the system to operate effectively with frequencies up to 1Hz over a range of stimulation amplitudes from 0.1 to 1.5 mm. Continuous measurements of in vivo callus stiffness revealed progressive fracture consolidation in the course of each experiment. A delayed onset of fracture healing was observed in the sheep with simulated delayed weightbearing.

The conducted preclinical experiments demonstrated its robustness and reliability. The system can be applied for further preclinical research and comprehensive in-depth investigation of fracture healing.

Confirming clinical evidence, we recently demonstrated in a rodent model that a severe trauma which induces an acute systemic inflammation considerably impairs fracture healing. Interleukin-6 (IL-6) is a key cytokine in posttraumatic inflammation as its serum level correlates with injury severity and mortality. IL-6 signals are transmitted by the transmembrane glycoprotein 130 (gp130) via two distinct mechanisms: firstly, through classic signalling via the membrane-anchored IL-6 receptor and secondly, through trans-signalling using a soluble IL-6 receptor. Whereas IL-6 trans-signalling is considered a danger signal driving inflammation, classic signalling may mediate anti-inflammatory, pro-regenerative processes. The role of the two distinct pathways in bone healing has not yet been elucidated. Here, we studied the function of IL-6 in the pathophysiology of compromised bone healing induced by severe trauma.

Male C57BL/6J mice received an osteotomy of the right femur stabilized with an external fixator. Systemic inflammation was induced by additional blunt chest trauma (TxT) applied immediately after the osteotomy. Mice were injected with either fusion protein sgp130Fc, which selectively inhibits IL-6 trans-signalling, or a neutralizing anti-IL-6 antibody (IL-6 Ab), blocking both signalling pathways. Control mice received vehicle solution. Animals were euthanised 21 days after surgery. Fracture healing was analysed by biomechanical testing, μCT, and histomorphometry (n= 6–9; p=0.05; ANOVA/Fisher LSD post hoc).

Thoracic trauma significantly impaired fracture healing [bending stiffness (EI) −57%, p<0.00]. Treatment with sgp130Fc significantly attenuated bone regeneration as demonstrated by an increased EI (+110%, p<0.00) and a trend of augmented apparent Young”s modulus (+69%, p=0.13) compared to TxT control. Histomorphometric analysis could not detect differences in the amount of bone, confirming µCT results, but revealed a significantly decreased cartilage area after treatment with sgp130Fc (−76%, p=0.01). Inhibition of both signalling pathways with IL-6 Ab, however, did not have any effects.

In conclusion, severe trauma significantly impaired fracture healing, confirming previous studies. Treatment with sgp130Fc ameliorated the negative effects providing evidence that IL-6 trans-signalling triggers the excessive immune response after trauma impairing bone regeneration. Injection of IL-6 Ab did not improve fracture healing thereby implying that classic signalling may rather have beneficial effects.