Introduction and Objective. Neoangiogenesis drives the replacement of mineralized cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL and the close interaction of progenitors of osteoblasts, chondrocytes, endothelial cells and osteoclasts/chondroclasts. The Heparan sulfate proteoglycan Syndecan-1 (Sdc-1) plays a role in the interaction between osteoclasts and osteoblasts and the development of blood vessels. As the processes of osteogenesis and angiogenesis are closely related to each other in bone, we expected Sdc-1 to have an influence on vessel structure during aging. Therefore, angiogenesis at the growth plate in mice of different ages was compared and the influence of Syndecan-1 deficiency was characterized. Materials and Methods. Animals: C57BL/6 (WT) and Sdc1−/− mice were used for native bone analysis at 4, 12 and 18 month age. Femura were dissected, cryoprotected and embedded. Histology: Embedded bones were sectioned into 80um thick slices so that the 3D network of the vascularization of the bone could be visualized using an anti-Endomucin antibody and DAPI as counter staining. For semi-automatical quantification of the vessel bulbs we used a custom made software. In vitro angiogenesis: For aortic ring assay, aortic tissue was isolated from 4 month old mice, cut into 0.5mm rings and embedded in collagen type I matrix. Microvessel outgrowth was quantified after 6 days of culture. Results. We verified our custom-made software using slices of WT mice and showed that there is no variation of the number of bulbs with regard to the width of the growth plate in periphery versus center zones in all age groups which indicates a homogeneous distribution of angiogenesis throughout the interface of cartilage to newly forming bone. Furthermore, in both, WT and Sdc-1 deficient mice the number of bulbs decreased significantly with age. However, Sdc-1 knockout mice at the age of 4 and 12 month showed a highly significant decrease in angiogenesis close to the growth plate compared to WT mice, whereas in older mice these differences were gone. Quantification of microvessel outgrowth of aortic tissue revealed a significant decrease in number of vessels from rings taken from Syndecan-1 deficient mice compared to WT mice. Conclusions. Syndecan-1 has a significant impact on neoangiogenesis in vitro and in vivo during aging as demonstrated at the chondro-osseous border of the native bone, emphasizing the importance to further characterize the function of Syndecan-1 regulated processes during enchondral ossification

Treatment for delayed wound healing resulting from peripheral vascular diseases and diabetic foot ulcers remain a challenge. A novel surgical technique named Tibial Cortex Transverse Transport has been developed for treating peripheral ischaemia, with encouraging clinical effects. However, its underlying mechanisms remain unclear. In present study, we aimed to explore the wound healing effects after undergoing this novel technique via multiple ways. A novel rat model of Tibial Cortex Transverse Transport was established with a designed external fixator and effects on wound healing were investigated. All rats were randomized into 3 groups, with 12 rats per group: sham group (negative control), fixator group (positive control) and Tibial Cortex Transverse Transport group. Laser speckle perfusion imaging, vessel perfusion, histology and immunohistochemistry were used to evaluate the wound healing processes. Gross and histological examinations showed that Tibial Cortex Transverse Transport technique accelerated wound closure and enhanced the quality of the newly formed skin tissues. In Tibial Cortex Transverse Transport group, HE staining demonstrated a better epidermis and dermis recovery, while immune-histochemical staining showed that Tibial Cortex Transverse Transport technique promoted local collagen deposition. Tibial Cortex Transverse Transport technique also benefited to angiogenesis and immunomodulation. In Tibial Cortex Transverse Transport group, blood flow in the wound area was higher than that ofother groups according to laser speckle imaging with more blood vessels observed. Enhanced neovascularization was seen in the Tibial Cortex Transverse Transport group with double immune-labelling of CD31 and α-SMA. The M2 macrophages at the wound site in the Tibial Cortex Transverse Transport group was also increased. Tibial cortex transverse transport technique accelerated wound healing through enhanced angiogenesis and immunomodulation

Neoangiogenesis drives the replacement of mineralised cartilage by trabecular bone during bone growth regulated by molecules like e.g. VEGF, OPG and RANKL. The Heparan sulfate proteoglycan Syndecan-1 (Sdc1) plays a role in the interaction of osteoclasts and osteoblasts and the development of blood vessels. We expected Sdc1 to have an influence on bone structure and vessel development. Therefore, bone structure and angiogenesis at the growth plate in mice was compared and the influence of Syndecan-1 deficiency was characterised. Animals: Femura of male and female C57BL/6 WT (5♀, 6♂) and Sdc1-/- (9♀, 5♂) mice were used for native bone analysis at 4 month age. Histology: Bone structure was analysed using microCT scans with a resolution of 9µm. Vascularisation was visualised using an anti-Endomucin antibody in 80µm thick cryosections. In vitro angiogenesis: Bone marrow isolates were used to generate endothelial progenitor cells by sequential cultivation on fibronectin. Microvessel development was analysed 4h after plating on matrigel. Bone structure in male Sdc1 deficient mice was significantly reduced compare to male WT, whereas female mice of both genotypes did not differ. Sdc1 deficient mice at the age of 4 month showed a high decrease in the number of vessel bulbs at the chondro-osseous border (growth plate) compared to WT mice. However, no sex related differences were shown. Quantification of microvessel outgrowth of endothelial cells revealed a decreased amount of sprouting, but increased length of microvessels of Sdc1-/- cells compared to WT. Syndecan-1 has a significant impact on neoangiogenesis at the chondro-osseous border of the native bone, but the impact of Syndecan-1 deficiency on the loss of bone structure was significantly higher in male mice. This emphasises the importance to further characterise the function of Syndecan-1 regulated processes during enchondral ossification in a sex dependent manner

Intervertebral disc (IVD) degeneration is the most frequent cause of Low Back Pain (LBP) affecting nearly 80% of the population [1]. Current treatments fail to restore a functional IVD or to provide a long-term solution, so, there is an urgent need for novel therapeutic strategies. We have defined the IVD extracellular matrix (ECM) profile, showing that the pro-regenerative molecules Collagen type XII and XIV, are uniquely expressed during fetal stages [2]. Now we propose the first fetal injectable biomaterial to regenerate the IVD. Fetal decellularized IVD scaffolds were recellularized with adult IVD cells and further implanted in vivo to evaluate their anti-angiogenic potential. Young decellularized IVD scaffolds were used as controls. Finally, a large scale protocol to produce a stable, biocompatible and easily injectable fetal IVD-based hydrogel was developed. Fetal scaffolds were more effective at promoting Aggrecan and Collagen type II expression by IVD cells. In a Chorioallantoid membrane assay, only fetal matrices showed an anti-angiogenic potential. The same was observed in vivo when the angiogenesis was induced by human NP cells. In this context, human NP cells were more effective in GAG synthesis within a fetal microenvironment. Vaccum-assisted perfusion decellularized IVDs were obtained, with high DNA removal and sGAG retention. Hydrogel pre-solution passed through 21-30G needles. IVD cells seeded on the hydrogels initially decreased metabolic activity, but increased up to 70% at day 7, while LDH assay revealed cytotoxicity always below 30%. This study will open new avenues for the establishment of a disruptive treatment for IVD degeneration with a positive impact on the angiogenesis associated with LBP, and on the improvement of patients’ quality of life. Acknowledgements: Financial support was obtained from EUROSPINE, ON Foundation and FCT (Fundação para a Ciência e a Tecnologia)

Bone regeneration is an area of acute medical need, but its clinical success is hampered by the need to ensure rapid vascularization of osteogenic grafts. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and during bone development angiogenesis and osteogenesis are physiologically coupled through so-called angiocrine factors produced by blood vessels. However, how to exploit this process for therapeutic bone regeneration remains a challenge (1). Here we will describe recent work aiming at understanding the cross-talk between vascular growth and osteogenesis under conditions relevant for therapeutic bone regeneration. To this end we take advantage of a unique platform to generate controlled signalling microenvironments, by the covalent decoration of fibrin matrices with tunable doses and combinations of engineered growth factors. The combination of human osteoprogenitors and hydroxyapatite in these engineered fibrin matrices provides a controlled model to investigate how specific molecular signals regulate vascular invasion and bone formation in vivo. In particular, we found that:. 1). Controlling the distribution of VEGF protein in the microenvironment is key to recapitulate its physiologic function to couple angiogenesis and osteogenesis (2);. 2). Such coupling is exquisitely dependent on VEGF dose and on a delicate equilibrium between opposing effects. A narrow range of VEGF doses specifically activates Notch1 signaling in invading blood vessels, inducing a pro-osteogenic functional state called Type H endothelium, that promotes differentiation of surrounding mesenchymal progenitors. However, lower doses are ineffective and higher ones paradoxically inhibit both vascular invasion and bone formation (Figure 1) (3);. 3). Semaphorin3a (Sema3a) acts as a novel pro-osteogenic angiocrine factor downstream of VEGF and it mediates VEGF dose-dependent effects on both vascular invasion and osteogenic progenitor stimulation. In conclusion, vascularization of osteogenic grafts is not simply necessary in order to enable progenitor survival. Rather, blood vessels can actively stimulate bone regeneration in engineered grafts through specific molecular signals that can be harnessed for therapeutic purposes. Acknowledgements: This work was supported in part by the European Union Horizon 2020 Program (Grant agreement 874790 – cmRNAbone). For any figures and tables, please contact the authors directly

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

Tendinopathy is the most common form of chronic tendon disorders, accounting for up 30% of all musculoskeletal clinic visits [1]. In tendon disease, the largely avascular tendon tissue often becomes hypervascularized and fibrotic [2]. As blood vessel growth and angiogenic signaling molecules are often induced by the lack of adequate nutrients and oxygen, hypoxic signaling is speculated to be a root cause of tendon neovascularization and tendinopathy [3,4,5]. However, how the vascular switch is initiated in tendons, and how vascularization contributes to tendon pathology remains unknown. In this talk, we provide evidence that HIF-1α is implicated in tendon disease and HIF-1α stabilization in human tendon cells induces vascular recruitment of endothelial cells via VEGFa secretion. More interesting, HIF-1α stabilization in tendon cells in vivo, seems to recapitulate all main features of fibrotic human tendon disease, including vascular ingrowth, matrix disorganization, changes in tissue mechanics, cell proliferation and innervation. Surprisingly, in vivo knock-out of VEGFa rescued angiogenesis in the tendon core but it did not affect tendon mechanical properties and tissue pathophysiological changes, suggesting that blood vessels ingrowth might not be a primary cause but a consequence of HIF-1α activation

The pathophysiological basis of alterations in trabecular bone of patients with osteonecrosis of the femoral head (ONFH) remains unclear. ONFH has classically been considered a vascular disease with secondary changes in the subchondral bone. However, there is increasing evidence suggesting that ONFH could be a bone disease, since alterations in the functionality of bone tissue distant from the necrotic lesion have been observed. We comparatively studied the transcriptomic profile of trabecular bone obtained from the intertrochanteric region of patients with ONFH without an obvious aetiological factor, and patients with osteoarthritis (OA) undergoing total hip replacement in our Institution. To explore the biological processes that could be affected by ONFH, we compared the transcriptomic profile of trabecular bone from the intertrochanteric region and the femoral head of patients affected by this condition. Differential gene expression was studied using an Affymetrix microarray platform. Transcriptome analysis showed a differential signature in trabecular bone from the intertrochanteric region between patients with ONFH and those with OA. The gene ontology analyses of the genes overexpressed in bone tissue of patients with ONFH revealed a range of enriched biological processes related to cell adhesion and migration and angiogenesis. In contrast, most downregulated transcripts were involved in cell division. Trabecular bone in the intertrochanteric region and in the femoral head also exhibited a differential expression profile. Among the genes differentially expressed, we highlighted those related with cytokine production and immune response. This study identified a set of differently expressed genes in trabecular bone of patients with idiopathic ONFH, which might underlie the pathophysiology of this condition. Acknowledgements: This work was supported by grants PI18/00643 and PI22/00939 from ISCIII-FEDER, Ministerio de Ciencia, Innovación y Universidades (MICINN)-AES

Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs). Results. The supernatant contained several GFs/CKs, with especially high levels of basic fibroblast growth factor, and CD34+ cells as the stem/progenitor cell fraction. With regard to biological potential, we confirmed that cell proliferation, osteoinduction, and angiogenesis in hMSCs and HUVECs were enhanced by the supernatant. Conclusions. The current study demonstrates the potential of a new point-of-care strategy for regenerative medicine using skeletal muscle supernatant. This attractive approach and readily-available material could be a promising option for tissue repair/regeneration in the clinical setting. Cite this article: M. Yoshikawa, T. Nakasa, M. Ishikawa, N. Adachi, M. Ochi. Evaluation of autologous skeletal muscle-derived factors for regenerative medicine applications. Bone Joint Res 2017;6:277–283. DOI: 10.1302/2046-3758.65.BJR-2016-0187.R1

Macrophages (Mφ) are immune cells that play a crucial role in both innate and adaptive immunity as they are involved in a wide range of physiological and pathological processes. Depending on the microenvironment and signals present, Mφ can polarize into either M1 or M2 phenotypes, with M1 macrophages exhibiting pro-inflammatory and cytotoxic effects, while M2 macrophages having immunosuppressive and tissue repair properties. Macrophages have been shown to play key roles in the development and progression or inhibition of various diseases, including cancer. For example, macrophages can stimulate tumor progression by promoting immunosuppression, angiogenesis, invasion, and metastasis. This work aimed to investigate the effect of extracellular vesicles (EVs)-derived from polarized macrophages on an osteosarcoma cell line. Monocytes were extracted from buffy coats and cultured in RPMI medium with platelet lysate or M-CSF. After 6 days of seeding, Mφ were differentiated into M1 and M2 with INF-γ/LPS and IL-4/IL-13, respectively. The medium with M1 or M2 derived EVs was collected and EVs were isolated by differential centrifugation and size exclusion chromatography and its morphology and size were characterized with SEM and NTA, respectively. The presence of typical EVs markers (CD9, CD63) was assessed by Western Blot. Finally, EVs from M1 or M2-polarized Mφ were added onto osteosarcoma cell cultures and their effect on cell viability and cell cycle, proliferation, and gene expression was assessed. The EVs showed the typical shape, size and surface markers of EVs. Overall, we observed that osteosarcoma cells responded differentially to EVs isolated from the M1 and M2-polarized Mφ. In summary, the use of Mφ-derived EVs for the treatment of osteosarcoma and other cancers deserves further study as it could benefit from interesting traits of EVs such as low immunogenicity, nontoxicity, and ability to pass through tissue barriers. Acknowledgements: Carlos III Health Institute and the European Social Fund for contract CP21/00136 and project PI22/01686

In this work, we combined tissue engineering and gene therapy technologies to develop a therapeutic platform for bone regeneration. We have developed photothermal fibrin-based hydrogels that incorporate degradable CuS nanoparticles (CuSNP) which transduce incident near-infrared (NIR) light into heat. A heat-activated and rapamycin-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in the photothermal hydrogels. In the presence of rapamycin, photoinduced mild hyperthermia induced the release of BMP-2 from the NIR responsive cell constructs. Transcriptome analysis of BMP-2 expressing cells showed a signature of induced genes related to stem cell proliferation and angiogenesis. We next generated 4 mm diameter calvarial defects in the left parietal bone of immunocompetent mice. The defects were filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the gene circuit. After one and eight days, rapamycin was administered intraperitoneally followed by irradiation with an NIR laser. Ten weeks after implantation, the animals were euthanized and samples from the bone defect zone were processed for histological analysis using Masson's trichrome staining and for immunohistochemistry analyses using specific CD31 and CD105 antibodies. Samples from mice that were only administered rapamycin or vehicle or that were only NIR-irradiated showed the persistence of fibrous tissue bridging the defect. In animals that were treated with rapamycin, NIR irradiation of implants resulted in the formation of new mineralized tissue with a high degree of vascularization, thus indicating the therapeutic potential of the approach. Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033)

Pericytes are contractile, motile cells that surround the capillary. Recent studies have shown that pericytes promoted joint fibrosis and induced subchondral bone angiogenesis, indicating the role of pericytes in osteoarthritis (OA). However, whether pericytes are involved in regulating inflammatory and catabolic response, as well as fibrotic repair of cartilage is still unclear. Here we used 2D and 3D models to investigate the communication of pericytes and chondrocytes under inflammatory osteoarthritis conditions. CD34-CD146+ pericytes were isolated and sorted from human bone marrow. Human OA chondrocytes were isolated from OA joints. In 2D studies, monolayer cultured chondrocytes were treated +/- pericyte conditioned media, +/- 1ng/ml IL1β for 24h. In 3D studies, pericytes and chondrocytes were cultured within fibrin gel in 3D polyurethane scaffolds, separately or combined for 7 days, followed by treatment of +/- IL1β for another 7 days (Fig 2A). The inflammatory response, catabolic activity and expression of fibrosis markers of chondrocytes and pericytes were measured by ELISA and/or q-rtPCR. Pericytes had weak inflammatory, catabolic and fibrotic response to IL1β (data not shown). The 2D study showed that pericyte conditioned media promoted inflammation, catabolism and fibrosis markers of chondrocytes, in the absence of IL1β treatment (Figure 1). However, study in 3D showed that coculture of chondrocytes and pericytes reduced the inflammatory and catabolic response of chondrocytes to IL1β and induced fibrosis markers in chondrocytes (Figure 2). Pericytes are involved in regulating inflammatory response, catabolic response and fibrosis of chondrocytes. The opposite results from 2D and 3D experiments indicate the variety of the regulatory role of pericytes in the interaction with chondrocytes within different culture models. The underlying mechanism is under evaluation with on-going studies. Acknowledgements. This study was funded by SINPAIN project, from European Union's Horizon Europe research and innovation programme under Grant Agreement NO. 101057778. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. For any figures or tables, please contact the authors directly

Abstract. OBJECTIVE. Knee varus malalignment increases medial knee compartment loading and is associated with knee osteoarthritis (OA) progression and severity. 1. Altered biomechanical loading and dysregulation of joint tissue biology drive OA progression, but mechanistic links between these factors are lacking. Subchondral bone structural changes are biomechanically driven, involve bone resorption, immune cell influx, angiogenesis, and sensory nerve invasion, and contribute to joint destruction and pain. 2. We have investigated mechanisms underlying this involving RANKL and alkaline phosphatase (ALP), which reflect bone resorption and mineralisation respectively. 3. and the axonal guidance factor Sema3A. Sema3A is osteotropic, expressed by mechanically sensitive osteocytes, and an inhibitor of sensory nerve, blood vessel and immune cell invasion. 4. Sema3A is also differentially expressed in human OA bone. 5. HYPOTHESIS: Medial knee compartment overloading in varus knee malalignment patients causes dysregulation of bone derived Sema3A signalling directly linking joint biomechanics to pathology and pain. METHODS. Synovial fluid obtained from 30 subjects with medial knee OA (KL grade II-IV) undergoing high tibial osteotomy surgery (HTO) was analysed by mesoscale discovery and ELISA analysis for inflammatory, neural and bone turnover markers. 11 of these patients had been previously analysed in a published patient-specific musculoskeletal model. 6. of gait estimating joint contact location, pressure, forces, and medial-lateral condyle load distribution in a published data set included in analyses. Data analysis was performed using Pearson's correlation matrices and principal component analyses. Principal Components (PCs) with eigenvalues greater than 1 were analysed. RESULTS. PC1 (32.94% of variation) and PC2 (25.79% of variation) from PCA analysis and correlation matrices separated patients according to correlated clusters of established inflammatory markers of OA pain and progression (IL6/IL8, r=0.754, p<0.001) and anti-inflammatory mediators (IL4/IL10, r=0.469, p=0.005). Bone turnover marker ALP was positively associated with KL grade (r=0.815, p=0.002) and negatively associated with IL10 (r=−0.402, p=0.018) and first peak knee loading pressures (r=−0.688, p=0.019). RANKL was positively associated with IL4 (r=0.489, p=0.003). Synovial fluid Sema3A concentrations showed separate clustering from all OA progression markers and was inversely correlated with TNF-α (r=−0.423, p=0.022) in HTO patients. Sema3A was significantly inversely correlated with total predicted force in the medial joint compartment (r=−0.621, p=0.041), mean (r=−0.63, p=0.038) and maximum (r=−0.613, p=0.045) calculated medial compartment joint pressures during the first phase and mean (r=−0.618, p=0.043) and maximum (r=−0.641, p=0.034) medial compartment joint pressures during midstance outputs of patient-specific musculoskeletal model. CONCLUSIONS. This study shows joint inflammatory status and mechanical overloading influence subchondral bone-remodelling. Synovial Sema3A concentrations are inversely correlated to patient-specific musculoskeletal model estimations of pathological medial overloading. This study reveals Sema3A as a biological mediator with capacity to induce OA pain and disease progression that is directly regulated by gait mechanical loading. 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

Summary Statement. Osteonecrosis of the femoral head (ONFH) is a multifactorial skeletal disorder. S100A9 represseses angiogenesis and vessel integrity in ONFH. It also may function as a marker of diagnosis in ONFH. Introduction. Osteonecrosis of the femoral head (ONFH) is a multifactorial skeletal disorder characterised by ischemic deterioration, bone marrow edema and eventually femoral head collapse and joint destruction. Several surgical, pharmaceutical and non-invasive biophysical modalities have been employed to alleviate this joint disorder. Our proteomic analysis showed that ONFH patients displayed increased expression of S100A9 protein when compared with healthy volunteers. This study is designed to evaluate the pathogenesis of S100A9 on the patients of ONFH. Patients & Methods. We collected 56 patients with ONFH including stage I, II, III and IV and 14 health volunteers. 20 ml of peripheral venous blood is drawn from each subject or prior to general anesthesia for hip arthroplasty. We compared the ELISA of S100A9, Osteocalcin, TRAP-5b, sVCAM-1. Immunohistochemistry of S100A9, vWF and VEGF are compared using femoral head harvested from late stages of ONFH and femoral neck fracture when received hip arthroplasty. In vitro angiogenic assay was performed by tube formation assay. Results. There were significant elevation of S100A9 in the serum of ONFH patients then in healthy volunteers. sVCAM-1 and TRAP-5b were increased and Osteocalcin was decreased in ONFH patient when comapred with healthy volunteers. The expression of S100A9 protein in ONFH tissue was significantly higher than femoral neck fracture tissue. In tube formation assay, we found S100A9 and the serum of ONFH patient supressed angiogenesis in vascular endothelial cell culture. Discussion/Conclusion. The expression of S100A9 significantly increased in the serum and femoral head tissue of patients with ONFH. S100A9 also supressed angiogenesis expression. The results indicated that S100A9 represseses angiogenesis and vessel integrity in ONFH. It also may function as a marker of diagnosis in ONFH

The bone infection osteomyelitis (typically Staphylococcus aureus) requires a multistep treatment process including: surgical debridement, long-term systemic high-dose antibiotics, and often bone grafting. With antibiotic resistance becoming increasingly concerning, alternative approaches are urgently needed. Herein, we develop a one-step treatment for osteomyelitis that combines local, controlled release of non-antibiotic antibacterials (copper) within a proven regenerative scaffold. To maximise efficacy we utilised bioactive glass – an established material with immense osteogenic capacity – as a copper ion delivery reservoir. Copper ions have also been shown to stimulate angiogenesis and induce MSC differentiation down an osteogenic lineage. To eliminate grafting requirements, the copper-doped BG was incorporated into our previously developed collagen scaffolds to produce multifunctional antibacterial, osteogenic, and angiogenic scaffolds. Scaffolds were fabricated by freeze-drying a co-suspension of collagen and bioactive glass particles (+/− copper doping, referred to as CuBG and BG, respectively) at a range of different concentrations (0–300% w/w bioactive glass/collagen). Scaffolds demonstrated a 2.7-fold increase in compressive modulus (300% CuBG vs. 0%; p≤0.01), whilst maintaining >98% porosity. The 300% CuBG scaffolds showed significant antibacterial activity against Staphylococcus aureus (p≤0.001). In terms of osteogenesis, both 100% and 300% CuBG scaffolds increased cell-mediated calcium deposition on the scaffolds at day 14 and 28 (p≤0.05 and p≤0.001), as confirmed by alizarin red staining. 100% CuBG scaffolds significantly enhanced angiogenesis by increased tubule formation (p≤0.01) and VEGF protein production (p≤0.001) (all ≥n=3). In summary, this single-stage, off-the-shelf treatment for osteomyelitis shows potential to minimise bone grafting and antibiotic dependence, while reducing hospital stays and costs

It is supposed that disturbed vascularization is a major cause for the development of an atrophic non-union. However, an actual study revealed normal vessel formation in human non-union tissues [1]. An animal study using an atrophic non-union model should clarify the influence of the inhibition of angiogenesis by the inhibitor Fumagillin on bone healing and the underlying processes including inflammation, chondrogenesis, angiogenesis and osteogenesis. For each group and time point (3, 7, 14, 21 and 42 days) 5–6 adult female Sprague Dawley rats were analyzed. The tibia was osteotomized and stabilized intramedullary with a k-wire coated with the drug carrier PDLLA (control group) or PDLLA +10% Fumagillin (atrophy group). Microarrays: Total-RNA were pooled per group, labeled with the Agilent single-color Quick-Amp Labeling Kit Cy3 and hybridized on Agilent SurePrint G3 Rat Gene Expression microarrays. After feature extraction and quantile normalization, relevant biological processes were identified using GeneOntology. Genes with an expression value below the 25. percentile were excluded. Heatmaps were used for visualization. The analysis of inflammatory genes revealed an upregulation of monocyte/macrophage- relevant factors such as the chemokines Ccl2 and Ccl12 and the surface marker CD14. Other factors involved in the early inflammation process such as Il1a, Tnf and Il6 were not affected. Chondrogenic markers including Collagen Type II, -IX, -X, Mmp9, Mmp13, Hapln1, Ucma, Runx2, Sox5 and -9 were downregulated in this group. Furthermore, osteogenic factors were less regulated within the middle stage of healing (day 14–21). This gene panel included Bmps, Bmp antagonists, Bmp- and Tgfb receptors, integrines and matrix proteins. qPCR analysis of angiogenic genes showed an upregulation of Angpt2, Fgf1 and -2, but not for Vegfa over the later healing time points. We demonstrated in a previous study that inhibiting angiogenesis in an osteotomy model led to a reduction in vessel formation and to the development of an atrophic non-union phenotype [2]. The microarray analysis indicated no prolonged inflammatory reaction in the atrophy group. But the upregulation of chemokines together with a delay in hematoma degradation signs to a mismatch between recruitment and demand of macrophages from the vessel system. Furthermore, chondrogenesis was completely blocked, which was shown by a downregulation of chondrogenic but also osteogenic markers being involved in chondrogenic processes. A reduced recruitment of MSCs might be a possible explanation. Although, microarray data revealed only minor expression changes regarding angiogenic genes, validation by q-PCR showed an upregulation of Angpt2, Fgf1 and -2 over the later healing time points. Due to the heterogeneity of the callus tissue it might be that variations of gene expression of a single tissue type will be masked by the expression levels of other tissue types. This issue is even more pronounced when analyzing different time points and by pooling the samples

The large bone defects with high risk of delayed bone union and pseudoarthrosis remain significant clinical challenge. Aim of the present study was the investigation of the critical size fracture healing process in transgenic mice using a novel beta-TCP scaffold. The luciferase transgenic mice strains (BALB/C-Tg(NF-kappaB-RE-luc)-Xen) and FVB/N-Tg(Vegfr2-luc)-Xen were used. Critical size fracture on femur was performed and stabilized using external fixation (RISystem). The fracture was bridged with a synthetic scaffold with and without Strontium. In consequence, the expression levels of NF-kappaB and VEGFR2 could be monitored in a longitudinal fashion using the Xenogen imaging system for two months. Animals were euthanized, serial section of femur were prepared, and the fracture sites were histologically examined. Sr reduced inflammation in the early phase of healing (15th days), but it was increased in the late healing stage. The level of VEGFR2 activity increases in the Sr doped beta-TCP group at the 15th day, the luciferase activity starts to decrease in this group and show significantly less activity compared to other groups in the second half. In the group without scaffold a connective tissue formation were observed. In both, beta-TCP and beta-TCP+Sr, the connection of newly formed tissue within integrated canals in scaffold was visible. Tissue formation in beta-TCP+Sr group was significantly higher than in the beta-TCP group, whereas the percentage of osseous tissue in relation to the newly formed tissue was in beta-TCP scaffold much more than in beta-TCP+ Sr groups. This study presents the first data regarding VEGFR2 and NF-kappB and angiogenesis activity profiles during fracture healing. The collected longitudinal data reduces the number of experimental animals in the study. Addition of strontium in scaffolds influenced the inflammation in different stage of the healing. This effect might influence the healing process and may prove to be advantageous for osteoporosis fracture healing

Objectives. The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing. Methods. Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically. Results. The total number of PBMNCs was decreased after QQ-culture, however, the number of CD34+ and CD206+ cells were found to have increased as assessed by flow cytometry analysis. In addition, gene expression of angiogenic factors was upregulated in QQMNCs. In the animal model, the rate of bone union was higher in the QQMNC group than in the other groups. Radiographic scores and bone volume were significantly associated with the enhancement of angiogenesis in the QQMNC group. Conclusion. We have demonstrated that QQMNCs have superior potential to accelerate fracture healing compared with PBMNCs. The QQMNCs could be a promising option for fracture nonunion. Cite this article: K. Mifuji, M. Ishikawa, N. Kamei, R. Tanaka, K. Arita, H. Mizuno, T. Asahara, N. Adachi, M. Ochi. Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing. Bone Joint Res 2017;6: 489–498. DOI: 10.1302/2046-3758.68.BJR-2016-0338.R1

Summary Statement. Bone stress fracture triggers a rapid increase in blood flow in association with mast cell production of inducible nitric oxide synthase (iNOS). NOS inhibition blocks the increase in blood flow and reduces woven bone formation needed for stress fracture healing. Introduction. Vascular-bone interactions are critical in skeletal development and fracture healing. We recently showed that angiogenesis is required for stress fracture healing. However, the changes in vascularity that occur in the first 72 hours after stress fracture can not be explained by angiogenesis. Here, we evaulated early changes in blood flow and vasodilation after either damaging (stress fracture) or non-damaging mechanical loading in rats. Methods. The right forelimbs of adult rats were subjected to cyclic axial compression in vivo. We used two established protocols: damaging loading that creates a stress fracture and leads to woven bone formation (WBF loading), or non-damaging loading that stimulates lamellar bone formation (LBF loading). PET imaging was used to evaluate blood flow and fluoride kinetics based on uptake of . 15. O water and . 18. F fluoride radioisotopes, respectively, at the site of bone formation. To quantify vasodilation, the area of the anterior interosseous artery was measured. Inducible nitric oxide synthase (iNOS) expression was evaluated by immunostaining. Finally, NO production was impaired by administration of L-NAME (N. ω. -nitro-L-arginine methyl ester), a NOS inhibitor. Results. PET Imaging: Damaging WBF loading induced early and persistent increases in blood flow. Blood flow rate was increased ∼30% at 4 hours through 14 days in WBF loaded limbs. Fluoride uptake peaked 7 days after WBF loading, then declined from 7 to 14 days, consistent with the dynamics of woven bone formation described previously. Non-damaging LBF loading did not affect blood flow or fluoride kinetics. Histology: WBF loaded limbs had significantly increased arterial area (+50%) compared to non-loaded limbs at days 1 and 3, with return to normal by day 7. LBF loading did not affect arterial area. Since mast cells are a possible effector of vasodilation, mast cell infiltration and iNOS expression were quantified following loading. iNOS+ mast cells in WBF-loaded limbs were significantly increased on days 1 and 3, with return to normal by day 7. LBF loading was not associated with increases in iNOS+ mast cells. NOS Inhibition: L-NAME blocked the expression of iNOS in mast cells following WBF loading. Additionally, L-NAME treatment abolished the increase in blood flow rate at days 1 and 3, and diminished fluoride uptake at day 3. Finally, L-NAME treatment decreased woven bone formation, with significant decreases in woven bone volume (−27%) and BMD (−26%), compared to vehicle controls. Discussion/Conclusion. Damaging loading produces a stress fracture and leads to woven bone formation (WBF). Prior to bone formation, there is a rapid increase in blood flow rate in association with vasodilation and infiltration of iNOS+ mast cells in the expanded periosteum. Inhibition of NOS blocks the increase in blood flow rate, and ultimately impairs woven bone formation. In contrast, non-damaging (LBF) loading does not affect blood flow rate, vasodilation, or iNOS expression in mast cells. Thus, the vascular response after stress fracture involves an early increase in blood flow by vasodilation, followed by angiogenesis to maintain increased blood flow. Disruption of either response affects subsequent bone formation during stress fracture healing

Distraction histogenesis (DH) techniques have been widely accepted and practiced in orthopaedics, traumatology, and craniofacial surgery over the last two decades. Using DH methods, many previously untreatable conditions have been successfully managed with outstanding clinical outcomes. The biological mechanisms underlying DH have been studied and the tension-stress principles of tissue regeneration are attributed to upregulated gene expression, enhanced cell proliferation, angiogenesis and tissue remodelling and endogenous stem cell mobilization. The new methods of enhancing bone consolidation in DH are proposed and need further clinical studies. The novel applications of DH have now been extended for the treatment of vascular diseases, cranial defect (with neuronal disorders), hip and spinal deformity corrections and soft-tissue defects in addition to various bone defects and deformities. There are more surprises and novel mechanisms yet to be discovered for these novel applications of DH