Objectives. Venous thromboembolism (VTE) is a major potential complication following orthopaedic surgery. Subcutaneously administered enoxaparin has been used as the benchmark to reduce the incidence of VTE. However, concerns have been raised regarding the long-term administration of enoxaparin and its possible negative effects on bone healing and bone density with an increase of the risk of osteoporotic fractures. New oral anticoagulants such as rivaroxaban have recently been introduced, however, there is a lack of information regarding how these drugs affect bone metabolism and post-operative bone healing. Methods. We measured the migration and proliferation capacity of mesenchymal stem cells (MSCs) under enoxaparin or rivaroxaban treatment for three consecutive weeks, and evaluated effects on MSC mRNA expression of markers for stress and osteogenic differentiation. Results. We demonstrate that enoxaparin, but not rivaroxaban, increases the migration potential of MSCs and increases their cell count in line with elevated mRNA expression of C-X-C chemokine receptor type 4 (CXCR4), tumor necrosis factor alpha (TNFα), and alpha-B-crystallin (CryaB). However, a decrease in early osteogenic markers (insulin-like growth factors 1 and 2 (IGF1, IGF2), bone morphogenetic protein2 (BMP2)) indicated inhibitory effects on MSC differentiation into osteoblasts caused by enoxaparin, but not by rivaroxaban. Conclusions. Our findings may explain the adverse effects of enoxaparin treatment on bone healing. Rivaroxaban has no significant impact on MSC metabolism or capacity for osteogenic differentiation in vitro. Cite this article: Dr H. Pilge. Enoxaparin and rivaroxaban have different effects on human mesenchymal stromal cells in the early stages of bone healing. Bone Joint Res 2016;5:95–100. DOI: 10.1302/2046-3758.53.2000595

Objectives. To review the systemic impact of smoking on bone healing as evidenced within the orthopaedic literature. Methods. A protocol was established and studies were sourced from five electronic databases. Screening, data abstraction and quality assessment was conducted by two review authors. Prospective and retrospective clinical studies were included. The primary outcome measures were based on clinical and/or radiological indicators of bone healing. This review specifically focused on non-spinal orthopaedic studies. Results. Nine tibia studies and eight other orthopaedic studies were considered for systematic review. Of these 17 studies, 13 concluded that smoking negatively influenced bone healing. Conclusions. Smoking has a negative effect on bone healing, in terms of delayed union, nonunion and more complications

Corticosteroids are prescribed for the treatment of many medical conditions and their adverse effects on bone, including steroid-associated osteoporosis and osteonecrosis, are well documented. Core decompression is performed to treat osteonecrosis, but the results are variable. As steroids may affect bone turnover, this study was designed to investigate bone healing within a bone tunnel after core decompression in an experimental model of steroid-associated osteonecrosis. A total of five 28-week-old New Zealand rabbits were used to establish a model of steroid-induced osteonecrosis and another five rabbits served as controls. Two weeks after the induction of osteonecrosis, core decompression was performed by creating a bone tunnel 3 mm in diameter in both distal femora of each rabbit in both the experimental osteonecrosis and control groups. An in vivo micro-CT scanner was used to monitor healing within the bone tunnel at four, eight and 12 weeks postoperatively. At week 12, the animals were killed for histological and biomechanical analysis. In the osteonecrosis group all measurements of bone healing and maturation were lower compared with the control group. Impaired osteogenesis and remodelling within the bone tunnel was demonstrated in the steroid-induced osteonecrosis, accompanied by inferior mechanical properties of the bone. We have confirmed impaired bone healing in a model of bone defects in rabbits with pulsed administration of corticosteroids. This finding may be important in the development of strategies for treatment to improve the prognosis of fracture healing or the repair of bone defects in patients receiving steroid treatment

The re-establishment of vascularity is an early event in fracture healing; upregulation of angiogenesis may therefore promote the formation of bone. We have investigated the capacity of vascular endothelial growth factor (VEGF) to stimulate the formation of bone in an experimental atrophic nonunion model.

Three groups of eight rabbits underwent a standard nonunion operation. This was followed by interfragmentary deposition of 100 μg VEGF, carrier alone or autograft.

After seven weeks, torsional failure tests and callus size confirmed that VEGF-treated osteotomies had united whereas the carrier-treated osteotomies failed to unite. The biomechanical properties of the groups treated with VEGF and autograft were identical. There was no difference in bone blood flow.

We considered that VEGF stimulated the formation of competent bone in an environment deprived of its normal vascularisation and osteoprogenitor cell supply. It could be used to enhance the healing of fractures predisposed to nonunion.

Adrenomedullin is a peptide hormone that has attracted attention with its proliferative and anti-apoptotic effects on osteoblasts in recent years. We investigated the effect of adrenomedullin on healing of the segmental bone defect in a rat model.

36 Wistar rats were randomly divided in six groups based on follow-up periods and administered dose of adrenomedullin hormone. In each group, a 2 mm bone defect was created at the diaphysis of radius, bilaterally. NaCl solution was administered to sham groups three times a week for 4 and 8 weeks, intraperitoneally. Adrenomedullin was administered to study groups three times a week; 15 µg-4 weeks, 15 µg-8 weeks, 30 µg-4 weeks and 30 µg-8 weeks, respectively. After euthanasia, the segmental defects were evaluated by histomorphometric (new bone area (NBA)) and micro-tomographic (bone volume (BV), bone surface (BS), bone mineral density (BMD)) analysis.

Although 4 and 8 weeks 15 μg administered study groups had higher NBA values than the other study and control groups, histomorphometric analysis did not reveal any statistical difference between the control and study groups in terms of new bone area (p > 0.05). In micro-tomographic analysis, BV was higher in 15 μg – 4 weeks group than 30 μg – 4 weeks group (296.9 vs 208.5, p = 0.003) and BS was lower in 30 μg – 4 weeks than 4 week - control group (695.5 vs 1334.7, p = 0.005) but in overall, no significant difference was found between the control and study groups (p > 0.05). Despite these minor differences in histomorphometric and micro-tomographic criteria indicating new bone formation, BMD values of 15 µg-4 and −8 weeks study groups showed significant increase comparing with the control group (p = 0.04, p = 0.001, respectively).

Adrenomedullin seemed to have a positive effect on BMD at a certain dose (15 µg) but it alone is not considered sufficient for healing of the defect with new bone formation. Further studies are needed to assess its effects on bone tissue trauma.

This study was funded by Hacettepe University Scientific Research Projects Coordination Unit

The use of antifibrinolytic drugs and many other agents have a critical importance in bleeding control. Tranexamic acid [4- (aminomethyl) cyclohexanecarboxylic acid] is a synthetic amino acid lysine derivative with antifibrinolytic activity in humans. There are many studies in the literature that show that it is effective and effective both systemically and locally in spinal surgery. However, all of these studies have investigated the effects of topical tranexamic acid on bleeding and its effect on fusion has not been investigated yet. Aim of this study is to investigate the effect of topical tranexamic acid on fusion using macroscopic, radiologic and microscopic techniques.

After approve of ethics committee with the protocol number 19/2019 for 28 Wistar Albino rats underwent intertransvers fusion. All rats were randomized into four (4) groups, using sealed envelopes. Local tranexamic acid (Transamin® 100 mg/ml, Teva İlaç, İstanbul) doses was determined based on previously conducted studies; 1mg/kg (D1 group), 10mg/kg (D10 group), 100 mg/kg (D100 group) and no tranexamic acid (D0 group). At the end of 8^th weeks all rats were evaluated with manuel palpation, mammography and histopathologic analysis. Radiographic examination was performed two times to evaluate the intra and inter observer differences. 2 rats in-group D0 died after the radiographic examination.

Assessment of fusion with manual palpation revealed that use of local 1mg / kg tranexamic acid had no effect on fusion (p=0.32), however with increasing doses of tranexamic acid had negative effect on fusion (p=0.002). On radiologic examination, spearman's rho correlation coefficient was found to be moderate in the first evaluation (r=0.46) and high in second evaluation (r=0.61). Radiological examination revealed that the control group was the best in fusion (p=0.007), and that tranexamic acid affected fusion adversely, independent of dosage (p=0.27). Among the groups in histopathologic examination, no statistical difference was found (p=0.134).

Local administration of tranexamic acid affects the intertransverse fusion adversely depending on the dosage macroscopically and it also affects fusion adversely independent of the dosage radiologically and histopathologically.

In chronically infected fracture non-unions, treatment requires extensive debridement to remove necrotic and infected bone, often resulting in large defects requiring elaborate and prolonged bone reconstruction. One approach includes the induced membrane technique (IMT), although the differences in outcome between infected and non-infectious aetiologies remain unclear. Here we present a new rabbit humerus model for IMT secondary to infection, and, furthermore, we compare bone healing in rabbits with a chronically infected non-union compared to non-infected equivalents. A 5 mm defect was created in the humerus and filled with a polymethylmethacrylate (PMMA) spacer or left empty (n=6 per group). After 3 weeks, the PMMA spacer was replaced with a beta-tricalcium phosphate (chronOs, Synthes) scaffold, which was placed within the induced membrane and observed for a further 10 weeks. The same protocol was followed for the infected group, except that four week prior to treatment, the wound was inoculated with Staphylococcus aureus (4×10. 6. CFU/animal) and the PMMA spacer was loaded with gentamicin, and systemic therapy was applied for 4 weeks prior to chronOs application. All the animals from the infected group were culture positive during the first revision surgery (mean 3×10. 5. CFU/animal, n= 12), while at the second revision, after antibiotic therapy, all the animals were culture negative. The differences in bone healing between the non-infected and infected groups were evaluated by radiography and histology. The initially infected animals showed impaired bone healing at euthanasia, and some remnants of bacteria in histology. The non-infected animals reached bone bridging in both empty and chronOs conditions. We developed a preclinical in vivo model to investigate how bacterial infection influence bone healing in large defects with the future aim to explore new treatment concepts of infected non-union

Growth factors produced by inflammatory cells and mesenchymal progenitors are required for proper bone regeneration. Signaling pathways activated downstream of these proteins work in concert and synergistically to drive osteoblast and/or chondrocyte differentiation. While dysregulation can result in abnormal healing, activating these pathways in the correct spatiotemporal context can enhance healing. Bone morphogenetic protein (BMP) signaling is well-recognized as being required for bone regeneration, and BMP is used clinically to enhance bone healing. However, it is imperative to develop new therapeutics that can be used alone or in conjunction with BMP to drive even more robust healing. Notch signaling is another highly conserved signaling pathway involved in tissue development and regeneration. Our work has explored Notch signaling during osteoblastogenesis and bone healing using both in vitro studies with human primary mesenchymal progenitor cells and in vivo studies with genetically modified mouse models. Notch signaling is required and sufficient for osteoblast differentiation, and is required for proper bone regeneration. Indeed, intact Notch signaling through the Jagged-1 ligand is required for BMP induced bone formation. On-going work continues to explore the intersection between BMP and Notch signaling, and determining cell types that express Notch receptors and Notch ligands during bone healing. Our long-term objective is to develop Notch signaling as a clinical therapy to repair bone

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

Bone healing outcome is highly dependent on the initial mechanical fracture environment [1]. In vivo, direct bone healing requires absolute stability and an interfragmentary strain (IFS) below 2% [2]. In the majority of cases, however, endochondral ossification is engaged where frequency and amplitude of IFS are key factors. Still, at the cellular level, the influence of those parameters remains unknown. Understanding the regulation of naïve hMSC differentiation is essential for developing effective bone healing strategies. Human bone-marrow-derived MSC (KEK-ZH-NR: 2010–0444/0) were embedded in 8% gelatin methacryol. Samples (5mm Ø x 4mm) were subjected to 0, 10 and 30% compressive strain (5sec compression, 2hrs pause sequence for 14 days) using a multi-well uniaxial bioreactor (RISystem) and in presence of chondro-permissive medium (CP, DMEM HG, 1% NEAA, 10 µM ITS, 50 µg/mL ascorbic acid, and 100 mM Dex). Cell differentiation was assessed by qRT-PCR and histo-/immunohistology staining. Experiments were repeated 5 times with cells from 5 donors in duplicate. ANOVA with Tukey post-hoc correction or Kurskal-Wallis test with Dunn's correction was used. Data showed a strong upregulation of hypertrophic related genes COMP, MMP13 and Type 10 collagen upon stimulation when compared to chondrogenic SOX9, ACAN, Type 2 collagen or to osteoblastic related genes Type 1 Collagen, Runx2. When compared to chondrogenic control medium, cells in CP with or without stimulation showed low proteoglycan synthesis as shown by Safranine-O-green staining. In addition, the cells were significantly larger in 10% and 30% strain compared to control medium with 0% strain. Type 1 and 10 collagens immunostaining showed stronger Coll 10 expression in the samples subjected to strain compared to control. Uniaxial deformation seems to mainly promote hypertrophic-like chondrocyte differentiation of MSC. Osteogenic or potentially late hypertrophic related genes are also induced by strain. Acknowledgments: Funded by the AO Foundation, StrainBot sponsored by RISystemAG & PERRENS 101 GmbH

Bone regeneration is a complex but very well organized process in which the immune system has a decisive role. The adaptive immune system and its experience level (percentage of effector and memory T cells) has been proven to influence the healing cascade especially in the early healing phases. This opens the possibility of an early intervention to enhance bone healing during the primary clinical treatment. Patients stratified for possible delayed bone healing could benefit from immunomodulatory treatment approaches. In pre-clinical studies cells and signaling molecules have been identified that could represent promising candidates to help patients in need

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. Method. 2 mm femoral defects were created in 22-27 weeks-old female Fischer F344 rats, internally fixed with a plate (animal license: GR/19/2022) using established protocols for analgesia and anesthesia. Animals (n=4/group) received either a collagen sponge, a collagen sponge+1μg BMP-2 (InductOs, Medtronic) or a collagen sponge+1μg BMP-2 with a monoclonal anti-IL-1ß antibody (BioXCell, 10 mg/ml), administered intravenously under anesthesia every third day until day 15, from day 0 or 3. In vivo micro-CT was performed after surgery and at 2, 3, 4, 6, 8, 10 and 14-weeks post-OP. Mechanical properties of the operated femurs were assessed by 4-point bending (Instron5866) and compared to contralateral femurs (one-way ANOVA, GraphPad Prism8). Histopathological analysis was performed semi-quantitatively on Giemsa-Eosin-stained sections (Olympus BX63) using a six-grade severity grading scale. Result. Operated femurs with BMP-2 reached an average stiffness of 91±37% of contralateral femurs, femurs in IL-1ß groups 105±11% (day 0) and 111±12% (day 3). Administration of anti-IL-1ß+1μg BMP-2 led to faster cortical bridging (3/4 femurs bridged by week 4 for day 0, 4/4 for day 3) than 1μg BMP-2 alone (0/4 by week 4). Micro-CT results confirmed histopathological evaluation, as collagen sponge alone led to non-union, complete bicortical bridging was observed for 3/4 femurs in the BMP-2 group and for 4/4 femurs in the IL-1β groups after 14 weeks. Conclusion. Anti-IL-1ß had a beneficial effect on late fracture healing with faster cortical bridging and new bone formation than 1μg BMP-2 alone. Acknowledgments. AO foundation. We thank Andrea Furter, Alisa Hangartner and Thomas Krüger for technical support

Abstract. OBJECTIVES. To determine if force measured using a strain gauge in circular external fixation frames is different for 1) different simulated stages of bone healing, and for 2) fractures clinically deemed either united or un-united. METHODS. In a laboratory study, 3 similar Ilizarov frame constructs were assembled using a Perspex bone analogue. Constructs were tested in 10 different clinical situations simulating different stages of bone healing including with the bone analogue intact, with 1,3 and 50mm gaps, and with 6 materials of varying stiffness's within the 50mm gap. A Bluetooth strain gauge was inserted across the simulated fracture focus, replacing one of the 4 threaded rods used to construct the frame. Constructs were loaded to 700N using an Instron testing machine and maximum force during loading was measured by the strain gauge. Testing was repeated with the strain gauge replacing each of the 4 threaded rods in turn, with measurements being repeated 3 times, across all 3 frame constructs for all 10 simulated clinical situations (n=360). Force measurements between the situations were compared using a Kruskal-Wallis test (KW) and a post-hoc Steel test was used for multiple comparison against control (intact bone model). Additionally, a pilot study has been initiated to assess clinical efficacy of the strain gauge measurement in patients with circular frames. The strain gauge replaced the anterior rod across the fracture focus for each patient. Patients were asked to step on a weighing scale with their affected limb, and maximum weight transfer through the limb and maximal force measured in the frame were recorded. This was repeated 3 times and a mean ratio of force to weight through affected limb was calculated for each patient. The clinical situation at each measurement was designated as united or un-united by one of the senior authors for analysis. Force measurements between the situations were compared using a Wilcoxon-Mann-Whitney test. RESULTS. In the laboratory study, including all constructs with the strain gauge in all positions, a statistically significant relationship between model stability and force measured was identified (KW test for overall relationship p<0.0001). The largest force was measured in the model with a 50mm gap (median 170N, IQR 155–192, range 83–213) and the smallest in the intact bone model (median 3N, IQR 1–8, range 0–11). Multiple comparison testing found a significant difference between intact bone and all the unstable situations (p=0.002 or better). Examining initial results from our pilot clinical study, nine measurements were available in seven patients. Three of these were taken in patients with fractures yet to unite, six in patients where union has since been confirmed clinically. The median force measured was significantly greater where the fracture was not united (median 1.66 N/kg, range 1.07–1.99 vs 0.12 N/kg, range 0.05–0.73, p=0.02). CONCLUSIONS. This laboratory study demonstrates that force measurement may be different at different stages of healing, and although only limited data was available, a pilot clinical study showed a significant relationship between the force measured and clinical union of the patient's fracture. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Our previous rat study demonstrated an ex vivo-created “Biomimetic Hematoma” (BH) that mimics the intrinsic structural properties of normal fracture hematoma, consistently and efficiently enhanced the healing of large bone defects at extremely low doses of rhBMP-2 (0.33 μg). The aim of this study was to determine if an extremely low dose of rhBMP-2 delivered within BH can efficiently heal large bone defects in goats. Goat 2.5 cm tibial defects were stabilized with circular fixators, and divided into groups (n=2-3): 2.1 mg rhBMP-2 delivered on an absorbable collagen sponge (ACS); 52.5 μg rhBMP-2 delivered within BH; and an empty group. BH was created using autologous blood with a mixture of calcium and thrombin at specific concentrations. Healing was monitored with X-rays. After 8 weeks, femurs were assessed using microCT. Using 2.1 mg on ACS was sufficient to heal 2.5 cm bone defects. Empty defects resulted in a nonunion after 8 weeks. Radiographic evaluation showed earlier and more robust callus formation with 97.5 % (52.5 μg) less of rhBMP-2 delivered within the BH, and all tibias were fully bridged at 3 weeks. The bone mineral density was significantly higher in defects treated with BH than with ACS. Defects in the BH group had smaller amounts of intramedullary and cortical trabeculation compared to the ACS group, indicating advanced remodeling. The results confirm that the delivery of rhBMP-2 within the BH was much more efficient than on an ACS. Not only did the large bone defects heal consistently with a 40x lower dose of rhBMP-2, but the quality of the defect regeneration was also superior in the BH group. These findings should significantly influence how rhBMP-2 is delivered clinically to maximize the regenerative capacity of bone healing while minimizing the dose required, thereby reducing the risk of adverse effects

Electrical impedance spectroscopy measurements might be used for real-time monitoring of bone healing. Differences in electrical characteristics of different tissues during fracture healing can provide information of the tissue composition within the fracture region. This concept embraces the digital revolution of orthopaedics allowing for a sensor-based and home-based monitoring of bone healing. Furthermore, real-time monitoring will allow individualized and timely treatment adjustments to prevent bone healing complications. However, electrodes must be placed at a distance to the fracture site in order not to interfere with bone healing. Therefore, we investigated whether longitudinal and transverse electrical impedance measurements from electrodes placed at a distance to the bone defect can detect differences between intact bone and bone defects in vivo. Approval was granted from the Inspectorate of the Animal Experimentation under the Danish Ministry of Justice. Six rabbits were anaesthetized and had both tibias subjected to an osteotomy protocol where first the medial, then the lateral, and final the posterior cortex were removed resulting in a complete 2 mm bone defect. Electrical impedance was measured prior to and after each step of the osteotomy protocol. Recordings were obtained at different frequencies (10 Hz to 1 MHz) from an inner electrode placed into the medullary canal and two different electrodes placed extracortical on the lateral and posterior bone with a distance of 5 mm to the defect. For each rabbit, one tibia had measurements with a free inner electrode and the other tibia had measurements both with a nail and an isolated nail. For all tibias, the intact bone resulted in higher impedance compared with the complete defect, and this difference was most pronounced in the frequency range of 1 kHz to 100 kHz. This applied for all types of internal electrodes including electrode, nail, isolated nail. The isolated nail showed the biggest impedance difference between the intact bone and the complete defect. Incomplete bone defects had lower impedance compared with intact bone, but no consistent pattern for differences in impedance was observed between the different applied defects. Consistent impedance differences between intact bone and complete defects were detected in-vivo in rabbits. Further research is needed to explore whether the presented method of electrical impedance measurements can be used to characterize bone healing over time

In a consecutive retrospective analysis of 190 patients treated with the Masquelet technique at the BG Klinikum Hamburg from January 2012 to January 2022, defect-specific features such as the extent and morphology of the defect were recorded, and their influence on the time to reach full weight-bearing of the affected limb was investigated. A total of 217 defects were treated in 190 patients using the Masquelet technique. 70% of all defects were located in the tibia, followed by 22% in the femur and only about 7% in the upper extremity. The average length of all defects was 58 mm (+/−31 mm), with the largest defect measuring 180 mm and the smallest measuring 20 mm. 89% of the patients achieved full weight-bearing at the end of therapy. The average time from initiation of therapy to reaching safe full weight-bearing was 589 days. There was a significant correlation between defect length and time to reach full weight-bearing (p = 0.0134). These results could serve as a basis for creating a score for prognostics and evaluation of bone healing after treatment with the Masquelet technique. Additionally, the results could help guide indications for secondary stabilization using internal fixation

Abstract. Objectives. Review the evidence of low intensity pulsed ultrasound (LIPUS) for fracture non-union treatment and the potential to treat fractures in patients with co-morbidities at risk of fracture non-union. Methods. Data was gathered from both animal and human studies of fracture repair to provide an overview of the LIPUS in bone healing applications to provide in-depth evidence to substantiate the use in treatment of non-union fractures and to propose a scientific rational to develop a clinical development programme. Results. LIPUS is an effective method for treating fracture non-union, with most studies showing heal rates in the mid 80%. In the UK NICE has published MTG-12 guidance for non-union treatment, which demonstrates that LIPUS is an effective and cost effective method as an alternative to surgery to treat non-union fractures. Basic science studies and evaluation of clinical trial data has led to the understanding that LIPUS can mitigate co-morbidities related to failure of bone healing such as diabetes, advancing age and tobacco use. Future clinical trials will evaluate the use of LIPUS in acute fractures in patients with high risk of low bone healing capacity to prevent the development of a non-union. As with all medical treatments, LIPUS for fracture repair needs to be used appropriately, with poorly fixed fractures or large fracture gaps, being unsuitable for LIPUS treatment. In addition, considerations such as targeting the fracture site in deep-seated bones and clinician / patient engagement to ensure good compliant usage are vital factors to ensure good clinical outcomes. Conclusion. Using basic science research, a thorough knowledge of the mechanism of action has been established, which has elucidated that co-morbidities related to the development of fracture non-union can be mitigated by the LIPUS technology. A pragmatic clinical trial in the United States is currently ongoing to test these hypothesises clinically. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Introduction and Objective. The early pro-inflammatory hematoma phase of bone healing is characterized by platelet activation followed by growth factor release. Bone marrow mesenchymal stromal cells (MSC) play a critical role in bone regeneration. However, the impact of the pro-inflammatory hematoma environment on the function of MSC is not fully understood. We here applied platelet-rich plasma (PRP) hydrogels to study how platelet-derived factors modulate functional properties of MSC in comparison to a non-inflammatory control environment simulated by fibrin (FBR) hydrogels. Materials and Methods. MSC were isolated from acetabular bone marrow of patients undergoing hip arthroplasty. PRP was collected from pooled apheresis thrombocyte concentrates. The phenotype of MSC was analyzed after encapsulation in hydrogels or exposure with platelet-derived factors with regards to gene expression changes, cell viability, extracellular vesicle (EV) release and immunomodulatory effects utilizing cellular and molecular, flow cytometry, RT-PCR, western blot and immunofluorescence stainings. Results. Our results showed that encapsulation of MSC in PRP induced changes in cell metabolism increasing lactate production and reducing mitochondria membrane potential. This was followed by significantly decreased mTOR phosphorylation and differential gene regulation. While PRP-released factors could support EV-biogenesis and immunoregulation-related gene expression, FBR hydrogel reduced CD63+ and CD81+ EV release by MSC. In co-cultures with mitogen stimulated PBMC, pre-exposure of MSC with PRP reduced the proliferation rate and frequency of peripheral blood CD4. +. and favored the persistence of FOXP3. +. regulatory T lymphocytes (32±4.7% compared to 9±2.3% in control co-cultures where MSC were exposed to FBR). Conclusions. Our data indicate that exposure of MSC with a hematoma environment causes metabolic adaptation of MSC followed by increased immune regulatory functions, which in turn might contribute to resolution of inflammation required for successful bone healing

Periosteal mesenchymal stem cells (PMSC) are an emerging niche of stem cells to enhance bone healing by tissue engineering process. They have to be differentiated into osteoprogenitors in order to synthesize new bone matrix. In vitro differentiation with specific differentiation medium (DM) is not exactly representative of what occurs in vivo. The interaction between PMSC and growth factors (GF) present in biological matrix is somewhat less understood. The goal of this study is to explore the possibility of spontaneous PMSC differentiation in contact with different biological matrices without DM. 500.000 porcine PMSC were seeded on 6-well plates and cultured with proliferation medium (PM). When reaching 80% confluence, biological samples (n=3) of demineralized bone matrix (DBM), decellularized porcine bone allograft (AOp), human bone allograft (AOh), human periosteum (HP) and human fascia lata (HFL) were added. Negative and positive control wells included cells with only PM or DM, respectively. The differentiation progress was assessed by Alizarin Red staining at days 7, 14 and 21. Bone morphogenetic protein content (BMP 2, 4, 5, 6, 7, 8, 9 and 11) of each sample was also investigated by western blot. Alizarin red highlighted bone nodules neoformation on wells containing AOp, AOh and DBM, like positive controls. HP and HFL wells did not show any nodules. These results are correlated to a global higher BMP expression profile in AOp than in HP and HFL but not statistically significant (p=0.38 and p>.99, respectively). The highest expression in each tissue was that of BMP2 and BMP7, which play an important role in osteoinduction. PMSC are well known to participate to bone formation but, despite BMP presence in HP and HFL, they did not permit to achieve osteogenesis alone. The bone contact seems to be essential to induce in vitro differentiation into osteoprogenitors

Nitric oxide is a free radical which in vivo is solely produced during the conversion of the amino acid arginine into citrulline by nitric oxide synthase enzymes. Recently, the importance of nitric oxide on inflammation and bone metabolism has been investigated. However, the knowledge regarding possible in vitro effects of arginine supplementation on chondrogenic differentiation is limited. ATDC5, a cell line which is derived from mouse teratocarcinoma cells and which is characterized as chondrogenic cell line, were proliferated in Dulbecco's Modified Eagle Medium (DMEM)/F12 and subsequently differentiated in proliferation medium supplemented with insulin, transferrin and sodium-selenite and where arginine was added in four different concentrations (0, 7.5, 15 and 30 mM). Samples were harvested after 7 or 10 days and were stored at −80 °C for subsequent RNA isolation for qPCR analysis. To determine chondrogenic differentiation, Alcian Blue staining was performed to stain the proteoglycan aggrecan, which is secreted by differentiated ATDC5 cells. All measurements were performed in triplo. Alcian Blue staining showed a qualitative increase of proteoglycan aggrecan secretion in differentiated ATDC5 cells after treatment with 7 and 15 mM arginine, with additional increased expression of ColII, ColX, Bmp4 and Bmp6. Treatment with 30 mM arginine inhibited chondrogenic differentiation and expression of aforementioned genes, however, Cox-2 and Vegfa gene expression were increased in these samples. Bmp7 was not significantly expressed in any experimental condition. The obtained results are suggestive for a dose-dependent effect of arginine supplementation on chondrogenic differentiation and associated gene expression, with 7.5 and 15 mM as most optimal concentrations and implications for apoptosis after incubation with 30 mM arginine. A future recommendation would be to investigate the effects of citrulline in a similar experiment, as this shows even more promising results to enhance the nitric oxide metabolism in sepsis and bone healing