Aims. Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors. Methods. In our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation. Results. TGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply. Conclusion. A combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades. Cite this article: Bone Joint Res 2020;9(7):412–420

Objectives. The injured anterior cruciate ligament (ACL) is thought to exhibit an impaired healing response, and attempts at surgical repair have not been successful. Connective tissue growth factor (CTGF) is reported to be associated with wound healing, probably through transforming growth factor beta 1 (TGF-β1). Methods. A rabbit ACL injury model was used to study the effect of CTGF on ligament recovery. Quantitative real-time PCR (qRT-PCR) was performed for detection of changes in RNA levels of TGF-β1, type 1 collagen (COL1), type 2 collagen (COL2), SRY-related high mobility group-box gene9 (SOX9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase 13 (MMP-13). Expression of related proteins was detected by Western blotting. Results. The current study showed that CTGF could promote the recovery of an injured anterior cruciate ligament. It can upregulate mRNA and expression of TGF-β1, COL1, COL2, SOX9, and tissue inhibitor of TIMP-1, and downregulate mRNA and expression of MMP-13, suggesting that the curative effect of CTGF on injured rabbit ligaments is through regulation of these cellular factors. Conclusions. This finding revealed the healing role of CTGF in injured tissues and provides new possibilities of treating injured tissues and wound healing by using CTGF. Cite this article: X. Sun, W. Liu, G. Cheng, X. Qu, H. Bi, Z. Cao, Q. Yu. The influence of connective tissue growth factor on rabbit ligament injury repair. Bone Joint Res 2017;6:399–404. DOI: 10.1302/2046-3758.67.BJR.2016-0255.R1

Objectives. Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought. Methods. Primary human osteoblasts in in vitro culture were treated with IGF1, BMP2 or FGF2 (10 ng/ml) for 24 hours (IGF1) or 48 hours (BMP2 and FGF2). Experiments were performed during the exponential growth phase with approximately 1e7 cells per 75 cm. 2. flask. mRNA was reverse transcribed directly and analysed using RT-PCR Taqman assays. Expression levels of key genes involved in cell growth and differentiation (CDH11, TNFRSF11B, RUNX2, POSTN, ALP, WNT5A, LEF1, HSPA5, FOS, p21) were monitored using RT-PCR with gene-specific Taqman probes. . Results. Autocrine expression of BMP2 is stimulated by FGF2 and BMP2 itself. BMP2 and FGF2 act as proliferative factors as indicated by reduced expression of ALP and POSTN, whereas IGF1 exhibits a more subtle picture: the Wingless und Int-1 (Wnt) signalling pathway and the Smad pathway, but not p38 mitogen-activated protein (MAP) kinase signalling, were shown to be activated by IGF1, leading to proliferation and differentiation of the cells. . Conclusions. For future use of autologous bone cells in the management of bony defects, new treatment options take advantage of growth factors and differentiation factors. Thus, our results might help to guide the timely application of these factors for the expansion and subsequent differentiation of osteoblastic cells in culture. Cite this article: Bone Joint Res 2014;3:236–40

Objectives. The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known. Methods. In this study, TGF-β1 from osteoclasts and knee joints were analyzed using a co-cultured cell model and an OA rat model, respectively. Five patients with a femoral neck fracture (four female and one male, mean 73.4 years (68 to 79)) were recruited between January 2015 and December 2015. Results showed that TGF-β1 was significantly upregulated in osteoclasts by cyclic loading in a time- and dose-dependent mode. The osteoclasts were subjected to cyclic loading before being co-cultured with chondrocytes for 24 hours. Results. A significant decrease in the survival rate of co-cultured chondrocytes was found. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay demonstrated that mechanical stress-induced apoptosis occurred significantly in co-cultured chondrocytes but administration of the TGF-β1 receptor inhibitor, SB-505124, can significantly reverse these effects. Abdominal administration of SB-505124 can attenuate markedly articular cartilage degradation in OA rats. Conclusion. Mechanical stress-induced overexpression of TGF-β1 from osteoclasts is responsible for chondrocyte apoptosis and cartilage degeneration in OA. Administration of a TGF-β1 inhibitor can inhibit articular cartilage degradation. Cite this article: R-K. Zhang, G-W. Li, C. Zeng, C-X. Lin, L-S. Huang, G-X. Huang, C. Zhao, S-Y. Feng, H. Fang. Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-β1). Bone Joint Res 2018;7:587–594. DOI: 10.1302/2046-3758.711.BJR-2018-0057.R1

Transforming growth factor-beta2 (TGF-β2) is recognized as a versatile cytokine that plays a vital role in regulation of joint development, homeostasis, and diseases, but its role as a biological mechanism is understood far less than that of its counterpart, TGF-β1. Cartilage as a load-resisting structure in vertebrates however displays a fragile performance when any tissue disturbance occurs, due to its lack of blood vessels, nerves, and lymphatics. Recent reports have indicated that TGF-β2 is involved in the physiological processes of chondrocytes such as proliferation, differentiation, migration, and apoptosis, and the pathological progress of cartilage such as osteoarthritis (OA) and rheumatoid arthritis (RA). TGF-β2 also shows its potent capacity in the repair of cartilage defects by recruiting autologous mesenchymal stem cells and promoting secretion of other growth factor clusters. In addition, some pioneering studies have already considered it as a potential target in the treatment of OA and RA. This article aims to summarize the current progress of TGF-β2 in cartilage development and diseases, which might provide new cues for remodelling of cartilage defect and intervention of cartilage diseases

Cite this article: Bone Joint Res 2023;12(1):5–8.

Aims. The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. Methods. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. Results. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. Conclusion. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction. Cite this article: Bone Joint Res 2023;12(1):9–21

Aims. To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration. Methods. The OCD lesion was created on the trochlear groove of left articular cartilage of femur per rat (40 rats in total). The experimental groups were Sham, OCD, and ESWT (0.25 mJ/mm. 2. , 800 impulses, 4 Hz). The animals were euthanized at 2, 4, 8, and 12 weeks post-treatment, and histopathological analysis, micro-CT scanning, and immunohistochemical staining were performed for the specimens. Results. In the histopathological analysis, the macro-morphological grading scale showed a significant increase, while the histological score and cartilage repair scale of ESWT exhibited a significant decrease compared to OCD at the 8- and 12-week timepoints. At the 12-week follow-up, ESWT exhibited a significant improvement in the volume of damaged bone compared to OCD. Furthermore, immunohistochemistry analysis revealed a significant decrease in type I collagen and a significant increase in type II collagen within the newly formed hyaline cartilage following ESWT, compared to OCD. Finally, SRY-box transcription factor 9 (SOX9), aggrecan, and TGF-β, BMP-2, -3, -4, -5, and -7 were significantly higher in ESWT than in OCD at 12 weeks. Conclusion. ESWT promoted the effect of TGF-β/BMPs, thereby modulating the production of extracellular matrix proteins and transcription factor involved in the regeneration of articular cartilage and subchondral bone in an OCD rat model. Cite this article: Bone Joint Res 2024;13(7):342–352

Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT-PCR. ELISA and qRT-PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results. Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS-CM) enhanced MSC chondrogenic differentiation, and mechanically stimulated BMDMs generated more TGF-β1. Further, neutralizing TGF-β1 in MS-CM can attenuate its pro-chondrogenic effect. In vivo, mechanical stimulation promoted TGF-β1 generation, MSC chondrogenesis, and T-B healing, which were abolished following macrophage depletion. Conclusion. Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-β1 to promote MSC chondrogenesis, which subsequently augments T-B healing. Cite this article: Bone Joint Res 2023;12(3):219–230

Objectives. Re-rupture is common after primary flexor tendon repair. Characterization of the biological changes in the ruptured tendon stumps would be helpful, not only to understand the biological responses to the failed tendon repair, but also to investigate if the tendon stumps could be used as a recycling biomaterial for tendon regeneration in the secondary grafting surgery. Methods. A canine flexor tendon repair and failure model was used. Following six weeks of repair failure, the tendon stumps were analyzed and characterized as isolated tendon-derived stem cells (TDSCs). Results. Failed-repair stump tissue showed cellular accumulation of crumpled and disoriented collagen fibres. Compared with normal tendon, stump tissue had significantly higher gene expression of collagens I and III, matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF). The stump TDSCs presented both mesenchymal stem and haematopoietic cell markers with significantly increased expression of CD34, CD44, and CD90 markers. Stump TDSCs exhibited similar migration but a lower proliferation rate, as well as similar osteogenic differentiation but a lower chondrogenic/adipogenic differentiation capability, compared with normal TDSCs. Stump TDSCs also showed increasing levels of SRY-box 2 (Sox2), octamer-binding transcription factor 4 (Oct4), tenomodulin (TNMD), and scleraxis (Scx) protein and gene expression. Conclusion. We found that a failed repair stump had increased cellularity that preserved both mesenchymal and haematopoietic stem cell characteristics, with higher collagen synthesis, MMP, and growth factor gene expression. This study provides evidence that tendon stump tissue has regenerative potential. Cite this article: C-C. Lu, T. Zhang, R. L. Reisdorf, P. C. Amadio, K-N. An, S. L. Moran, A. Gingery, C. Zhao. Biological analysis of flexor tendon repair-failure stump tissue: A potential recycling of tissue for tendon regeneration. Bone Joint Res 2019;8:232–245. DOI: 10.1302/2046-3758.86.BJR-2018-0239.R1

Aims. To investigate the correlations among cytokines and regulatory T cells (T-regs) in ankylosing spondylitis (AS) patients, and their changes after anti-tumour necrosis factor-α (TNF-α) treatment. Methods. We included 72 AS patients with detailed medical records, disease activity score (Bath Ankylosing Spondylitis Disease Activity Index), functional index (Bath Ankylosing Spondylitis Functional Index), and laboratory data (interleukin (IL)-2, IL-4, IL-10, TNF-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, ESR, and CRP). Their peripheral blood mononuclear cells (PBMCs) were marked with anti-CD4, anti-CD25, and anti-FoxP3 antibodies, and triple positive T cells were gated by flow cytometry as T-regs. Their correlations were calculated and the changes after anti-TNF-α therapy were compared. Results. The frequency of T-regs in PBMCs was positively correlated to ESR and CRP in AS (r = 0.35 and 0.43; p = 0.032 and 0.027, respectively), and there was also a significant correlation between serum level of TNF-α and CRP (p = 0.041). The frequency of T-regs in PBMCs positively correlated to serum levels of TNF-α, IL-10, and TGF-β, while IL-2, IL-4, and IFN-γ showed opposite results. After anti-TNF-α treatment, there were significantly lower serum levels of TNF-α, IL-10, TGF-β, and frequency of T-regs in PBMCs among these AS patients (p = 0.026, 0.032, 0.029, and 0.037, respectively). Conclusion. In AS patients, proinflammatory cytokine may give positive feedback to induce more T-reg production and anti-inflammatory cytokine secretion to suppress this inflammatory status, and they can be reversed by anti-TNF-α therapy. However, the detailed interactions among T-regs and complex cytokine networks in autoinflammatory diseases still need more studies and further functional assay. Cite this article: Bone Joint Res 2023;12(2):133–137

Aims. Therapeutic agents that prevent chondrocyte loss, extracellular matrix (ECM) degradation, and osteoarthritis (OA) progression are required. The expression level of epidermal growth factor (EGF)-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) in damaged human cartilage is significantly higher than in undamaged cartilage. However, the effect of EDIL3 on cartilage is still unknown. Methods. We used human cartilage plugs (ex vivo) and mice with spontaneous OA (in vivo) to explore whether EDIL3 has a chondroprotective effect by altering OA-related indicators. Results. EDIL3 protein prevented chondrocyte clustering and maintained chondrocyte number and SOX9 expression in the human cartilage plug. Administration of EDIL3 protein prevented OA progression in STR/ort mice by maintaining the number of chondrocytes in the hyaline cartilage and the number of matrix-producing chondrocytes (MPCs). It reduced the degradation of aggrecan, the expression of matrix metalloproteinase (MMP)-13, the Osteoarthritis Research Society International (OARSI) score, and bone remodelling. It increased the porosity of the subchondral bone plate. Administration of an EDIL3 antibody increased the number of matrix-non-producing chondrocytes (MNCs) in cartilage and exacerbated the serum concentrations of OA-related pro-inflammatory cytokines, including monocyte chemotactic protein-3 (MCP-3), RANTES, interleukin (IL)-17A, IL-22, and GROα. Administration of β1 and β3 integrin agonists (CD98 protein) increased the expression of SOX9 in OA mice. Hence, EDIL3 might activate β1 and β3 integrins for chondroprotection. EDIL3 may also protect cartilage by attenuating the expression of IL-1β-enhanced phosphokinase proteins in chondrocytes, especially glycogen synthase kinase 3 alpha/beta (GSK-3α/β) and phospholipase C gamma 1 (PLC-γ1). Conclusion. EDIL3 has a role in maintaining the cartilage ECM and inhibiting the development of OA, making it a potential therapeutic drug for OA. Cite this article: Bone Joint Res 2023;12(12):734–746

Aims. This study investigated the effects of β-caryophyllene (BCP) on protecting bone from vitamin D deficiency in mice fed on a diet either lacking (D-) or containing (D+) vitamin D. Methods. A total of 40 female mice were assigned to four treatment groups (n = 10/group): D+ diet with propylene glycol control, D+ diet with BCP, D-deficient diet with control, and D-deficient diet with BCP. The D+ diet is a commercial basal diet, while the D-deficient diet contains 0.47% calcium, 0.3% phosphorus, and no vitamin D. All the mice were housed in conditions without ultraviolet light. Bone properties were evaluated by X-ray micro-CT. Serum levels of klotho were measured by enzyme-linked immunosorbent assay. Results. Under these conditions, the D-deficient diet enhanced the length of femur and tibia bones (p < 0.050), and increased bone volume (BV; p < 0.010) and trabecular bone volume fraction (BV/TV; p < 0.010) compared to D+ diet. With a diet containing BCP, the mice exhibited higher BV and bone mineral density (BMD; p < 0.050) than control group. The trabecular and cortical bone were also affected by vitamin D and BCP. In addition, inclusion of dietary BCP improved the serum concentrations of klotho (p < 0.050). In mice, klotho regulates the expression level of cannabinoid type 2 receptor (Cnr2) and fibroblast growth factor 23 (Fgf23) through CD300a. In humans, data suggest that klotho is connected to BMD. The expression of klotho is also associated with bone markers. Conclusion. These data indicate that BCP enhances the serum level of klotho, leading to improved bone properties and mineralization in an experimental mouse model. Cite this article: Bone Joint Res 2022;11(8):528–540

Aims. Proliferation, migration, and differentiation of anterior cruciate ligament (ACL) remnant and surrounding cells are fundamental processes for ACL reconstruction; however, the interaction between ACL remnant and surrounding cells is unclear. We hypothesized that ACL remnant cells preserve the capability to regulate the surrounding cells’ activity, collagen gene expression, and tenogenic differentiation. Moreover, extracorporeal shock wave (ESW) would not only promote activity of ACL remnant cells, but also enhance their paracrine regulation of surrounding cells. Methods. Cell viability, proliferation, migration, and expression levels of Collagen-I (COL-I) A1, transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF) were compared between ACL remnant cells untreated and treated with ESW (0.15 mJ/mm. 2. , 1,000 impulses, 4 Hz). To evaluate the subsequent effects on the surrounding cells, bone marrow stromal cells (BMSCs)’ viability, proliferation, migration, and levels of Type I Collagen, Type III Collagen, and tenogenic gene (Scx, TNC) expression were investigated using coculture system. Results. ESW-treated ACL remnant cells presented higher cell viability, proliferation, migration, and increased expression of COL-I A1, TGF-β, and VEGF. BMSC proliferation and migration rate significantly increased after coculture with ACL remnant cells with and without ESW stimulation compared to the BMSCs alone group. Furthermore, ESW significantly enhanced ACL remnant cells’ capability to upregulate the collagen gene expression and tenogenic differentiation of BMSCs, without affecting cell viability, TGF-β, and VEGF expression. Conclusion. ACL remnant cells modulated activity and differentiation of surrounding cells. The results indicated that ESW enhanced ACL remnant cells viability, proliferation, migration, and expression of collagen, TGF-β, VEGF, and paracrine regulation of BMSC proliferation, migration, collagen expression, and tenogenesis. Cite this article: Bone Joint Res 2020;9(8):457–467

Aims. There is an increasing concern of osteoporotic fractures in the ageing population. Low-magnitude high-frequency vibration (LMHFV) was shown to significantly enhance osteoporotic fracture healing through alteration of osteocyte lacuno-canalicular network (LCN). Dentin matrix protein 1 (DMP1) in osteocytes is known to be responsible for maintaining the LCN and mineralization. This study aimed to investigate the role of osteocyte-specific DMP1 during osteoporotic fracture healing augmented by LMHFV. Methods. A metaphyseal fracture was created in the distal femur of ovariectomy-induced osteoporotic Sprague Dawley rats. Rats were randomized to five different groups: 1) DMP1 knockdown (KD), 2) DMP1 KD + vibration (VT), 3) Scramble + VT, 4) VT, and 5) control (CT), where KD was performed by injection of short hairpin RNA (shRNA) into marrow cavity; vibration treatment was conducted at 35 Hz, 0.3 g; 20 minutes/day, five days/week). Assessments included radiography, micro-CT, dynamic histomorphometry and immunohistochemistry on DMP1, sclerostin, E11, and fibroblast growth factor 23 (FGF23). In vitro, murine long bone osteocyte-Y4 (MLO-Y4) osteocyte-like cells were randomized as in vivo groupings. DMP1 KD was performed by transfecting cells with shRNA plasmid. Assessments included immunocytochemistry on osteocyte-specific markers as above, and mineralized nodule staining. Results. Healing capacities in DMP1 KD groups were impaired. Results showed that DMP1 KD significantly abolished vibration-enhanced fracture healing at week 6. DMP1 KD significantly altered the expression of osteocyte-specific markers. The lower mineralization rate in DMP1 KD groups indicated that DMP1 knockdown was associated with poor fracture healing process. Conclusion. The blockage of DMP1 would impair healing outcomes and negate LMHFV-induced enhancement on fracture healing. These findings reveal the importance of DMP1 in response to the mechanical signal during osteoporotic fracture healing. Cite this article: Bone Joint Res 2022;11(7):465–476

Aims. Osteoarthritis (OA) is a common degenerative joint disease. The osteocyte transcriptome is highly relevant to osteocyte biology. This study aimed to explore the osteocyte transcriptome in subchondral bone affected by OA. Methods. Gene expression profiles of OA subchondral bone were used to identify disease-relevant genes and signalling pathways. RNA-sequencing data of a bone loading model were used to identify the loading-responsive gene set. Weighted gene co-expression network analysis (WGCNA) was employed to develop the osteocyte mechanics-responsive gene signature. Results. A group of 77 persistent genes that are highly relevant to extracellular matrix (ECM) biology and bone remodelling signalling were identified in OA subchondral lesions. A loading responsive gene set, including 446 principal genes, was highly enriched in OA medial tibial plateaus compared to lateral tibial plateaus. Of this gene set, a total of 223 genes were identified as the main contributors that were strongly associated with osteocyte functions and signalling pathways, such as ECM modelling, axon guidance, Hippo, Wnt, and transforming growth factor beta (TGF-β) signalling pathways. We limited the loading-responsive genes obtained via the osteocyte transcriptome signature to identify a subgroup of genes that are highly relevant to osteocytes, as the mechanics-responsive osteocyte signature in OA. Based on WGCNA, we found that this signature was highly co-expressed and identified three clusters, including early, late, and persistently responsive genes. Conclusion. In this study, we identified the mechanics-responsive osteocyte signature in OA-lesioned subchondral bone. Cite this article: Bone Joint Res 2022;11(6):362–370

Objectives. Osteophytes are products of active endochondral and intramembranous ossification, and therefore could theoretically provide significant efficacy as bone grafts. In this study, we compared the bone mineralisation effectiveness of osteophytes and cancellous bone, including their effects on secretion of growth factors and anabolic effects on osteoblasts. Methods. Osteophytes and cancellous bone obtained from human patients were transplanted onto the calvaria of severe combined immunodeficient mice, with Calcein administered intra-peritoneally for fluorescent labelling of bone mineralisation. Conditioned media were prepared using osteophytes and cancellous bone, and growth factor concentration and effects of each graft on proliferation, differentiation and migration of osteoblastic cells were assessed using enzyme-linked immunosorbent assays, MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assays, quantitative real-time polymerase chain reaction, and migration assays. Results. After six weeks, the area of mineralisation was significantly higher for the transplanted osteophytes than for the cancellous bone (43803 μm. 2. , . sd. 14660 versus 9421 μm. 2. , . sd. 5032, p = 0.0184, one-way analysis of variance). Compared with cancellous bone, the conditioned medium prepared using osteophytes contained a significantly higher amounts of transforming growth factor (TGF)-β1 (471 pg/ml versus 333 pg/ml, p = 0.0001, Wilcoxon rank sum test), bone morphogenetic protein (BMP)-2 (47.75 pg/ml versus 32 pg/ml, p = 0.0214, Wilcoxon rank sum test) and insulin-like growth factor (IGF)-1 (314.5 pg/ml versus 191 pg/ml, p = 0.0418, Wilcoxon rank sum test). The stronger effects of osteophytes towards osteoblasts in terms of a higher proliferation rate, upregulation of gene expression of differentiation markers such as alpha-1 type-1 collagen and alkaline phosphate, and higher migration, compared with cancellous bone, was confirmed. Conclusion. We provide evidence of favourable features of osteophytes for bone mineralisation through a direct effect on osteoblasts. The acceleration in metabolic activity of the osteophyte provides justification for future studies evaluating the clinical use of osteophytes as autologous bone grafts. Cite this article: K. Ishihara, K. Okazaki, T. Akiyama, Y. Akasaki, Y. Nakashima. Characterisation of osteophytes as an autologous bone graft source: An experimental study in vivo and in vitro. Bone Joint Res 2017;6:73–81. DOI: 10.1302/2046-3758.62.BJR-2016-0199.R1

Aims. Trained immunity confers non-specific protection against various types of infectious diseases, including bone and joint infection. Platelets are active participants in the immune response to pathogens and foreign substances, but their role in trained immunity remains elusive. Methods. We first trained the innate immune system of C57BL/6 mice via intravenous injection of two toll-like receptor agonists (zymosan and lipopolysaccharide). Two, four, and eight weeks later, we isolated platelets from immunity-trained and control mice, and then assessed whether immunity training altered platelet releasate. To better understand the role of immunity-trained platelets in bone and joint infection development, we transfused platelets from immunity-trained mice into naïve mice, and then challenged the recipient mice with Staphylococcus aureus or Escherichia coli. Results. After immunity training, the levels of pro-inflammatory cytokines (tumour necrosis factor alpha (TNF-α), interleukin (IL)-17A) and chemokines (CCL5, CXCL4, CXCL5, CXCL7, CXCL12) increased significantly in platelet releasate, while the levels of anti-inflammatory cytokines (IL-4, IL-13) decreased. Other platelet-secreted factors (e.g. platelet-derived growth factor (PDGF)-AA, PDGF-AB, PDGF-BB, cathepsin D, serotonin, and histamine) were statistically indistinguishable between the two groups. Transfusion of platelets from trained mice into naïve mice reduced infection risk and bacterial burden after local or systemic challenge with either S. aureus or E. coli. Conclusion. Immunity training altered platelet releasate by increasing the levels of inflammatory cytokines/chemokines and decreasing the levels of anti-inflammatory cytokines. Transfusion of platelets from immunity-trained mice conferred protection against bone and joint infection, suggesting that alteration of platelet releasate might be an important mechanism underlying trained immunity and may have clinical implications. Cite this article: Bone Joint Res 2022;11(2):73–81

Aims. Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH. Methods. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology. Results. More than 1,000 genes were differentially expressed in hip joint capsules between healthy controls and DDH. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that extracellular matrix (ECM) modifications, muscle system processes, and cell proliferation were markedly influenced by the differentially expressed genes. Expression of Collagen Type I Alpha 1 Chain (COL1A1), COL3A1, matrix metalloproteinase-1 (MMP1), MMP3, MMP9, and MMP13 was downregulated in DDH, with the loss of collagen fibres in the joint capsule. Expression of transforming growth factor beta 1 (TGF-β1) was downregulated, while that of TGF-β2, Mothers against decapentaplegic homolog 3 (SMAD3), and WNT11 were upregulated in DDH, and alpha smooth muscle actin (αSMA), a key myofibroblast marker, showed marginal increase. In vitro studies showed that fibroblast proliferation was suppressed in DDH, which was associated with cell cycle arrest in G0/G1 and G2/M phases. Cell cycle regulators including Cyclin B1 (CCNB1), Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), Cyclin-dependent kinase 1 (CDK1), E2F1, cell division cycle 6 (CDC6), and CDC7 were downregulated in DDH. Conclusion. DDH is associated with the loss of collagen fibres and fibroblasts, which may cause loose joint capsule formation. However, the degree of differentiation of fibroblasts to myofibroblasts needs further study. Cite this article: Bone Joint Res 2021;10(9):558–570

Aims. MicroRNA-183 (miR-183) is known to play important roles in osteoarthritis (OA) pain. The aims of this study were to explore the specific functions of miR-183 in OA pain and to investigate the underlying mechanisms. Methods. Clinical samples were collected from patients with OA, and a mouse model of OA pain was constructed by surgically induced destabilization of the medial meniscus (DMM). Reverse transcription quantitative polymerase chain reaction was employed to measure the expression of miR-183, transforming growth factor α (TGFα), C-C motif chemokine ligand 2 (CCL2), proinflammatory cytokines (interleukin (IL)-6, IL-1β, and tumour necrosis factor-α (TNF-α)), and pain-related factors (transient receptor potential vanilloid subtype-1 (TRPV1), voltage-gated sodium 1.3, 1.7, and 1.8 (Nav1.3, Nav1.7, and Nav1.8)). Expression of miR-183 in the dorsal root ganglia (DRG) of mice was evaluated by in situ hybridization. TGFα, CCL2, and C-C chemokine receptor type 2 (CCR2) levels were examined by immunoblot analysis and interaction between miR-183 and TGFα, determined by luciferase reporter assay. The extent of pain in mice was measured using a behavioural assay, and OA severity assessed by Safranin O and Fast Green staining. Immunofluorescent staining was conducted to examine the infiltration of macrophages in mouse DRG. Results. miR-183 was downregulated in tissue samples from patients and mice with OA. In DMM mice, overexpression of miR-183 inhibited the expression of proinflammatory cytokines (IL-6, IL-1β, TNF-α) and pain-related factors (TRPV1, Nav1.3, Nav1.7, Nav1.8) in DRG. OA pain was relieved by miR-183-mediated inhibition of macrophage infiltration, and dual luciferase reporter assay demonstrated that miR-183 directly targeted TGFα. Conclusion. Our data demonstrate that miR-183 can ameliorate OA pain by inhibiting the TGFα-CCL2/CCR2 signalling axis, providing an excellent therapeutic target for OA treatment. Cite this article: Bone Joint Res 2021;10(8):548–557

Objectives. We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells. Materials and Methods. We seeded mouse skeletal muscle cells C2C12 on the hydroxyapatite/calcium sulphate biomaterial and the phenotype of the cells was analysed. To mimic surgical conditions with leakage of extra cellular matrix (ECM) proteins and growth factors, we cultured rat bone cells ROS 17/2.8 in a bioreactor and harvested the secreted proteins. The secretome was added to rat muscle cells L6. The phenotype of the muscle cells after treatment with the media was assessed using immunostaining and light microscopy. Results. C2C12 cells differentiated into osteoblast-like cells expressing prominent bone markers after seeding on the biomaterial. The conditioned media of the ROS 17/2.8 contained bone morphogenetic protein-2 (BMP-2 8.4 ng/mg, standard deviation (. sd. ) 0.8) and BMP-7 (50.6 ng/mg, . sd. 2.2). In vitro, this secretome induced differentiation of skeletal muscle cells L6 towards an osteogenic lineage. Conclusion. Extra cellular matrix proteins and growth factors leaking from a bone cavity, along with a ceramic biomaterial, can synergistically enhance the process of ectopic ossification. The overlaying muscle acts as an osteoinductive niche, and provides the required cells for bone formation. Cite this article: D. B. Raina, A. Gupta, M. M. Petersen, W. Hettwer, M. McNally, M. Tägil, M-H. Zheng, A. Kumar, L. Lidgren. Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial. Bone Joint Res 2016;5:500–511. DOI: 10.1302/2046-3758.510.BJR-2016-0133.R1

Aims. For cementless implants, stability is initially attained by an interference fit into the bone and osteo-integration may be encouraged by coating the implant with bioactive substances. Blood based autologous glue provides an easy, cost-effective way of obtaining high concentrations of growth factors for tissue healing and regeneration with the intention of spraying it onto the implant surface during surgery. The aim of this study was to incorporate nucleated cells from autologous bone marrow (BM) aspirate into gels made from the patient’s own blood, and to investigate the effects of incorporating three different concentrations of platelet rich plasma (PRP) on the proliferation and viability of the cells in the gel. Methods. The autologous blood glue (ABG) that constituted 1.25, 2.5, and 5 times concentration PRP were made with and without equal volumes of BM nucleated cells. Proliferation, morphology, and viability of the cells in the glue was measured at days 7 and 14 and compared to cells seeded in fibrin glue. Results. Overall, 2.5 times concentration of PRP in ABG was capable of supporting the maximum growth of cells isolated from the BM aspirate and maintain their characteristics. Irrespective of PRP concentration, cells in ABG had statistically significantly higher viability compared to cells in fibrin glue. Conclusion. In vitro this novel autologous gel is more capable of supporting the growth of cells in its structure for up to 14 days, compared to commercially available fibrin-based sealants, and this difference was statistically significant. Cite this article: Bone Joint Res 2020;9(7):402–411

Aims. Abnormal lipid metabolism is involved in the development of osteoarthritis (OA). Growth differentiation factor 11 (GDF11) is crucial in inhibiting the differentiation of bone marrow mesenchymal stem cells into adipocytes. However, whether GDF11 participates in the abnormal adipogenesis of chondrocytes in OA cartilage is still unclear. Methods. Six-week-old female mice were subjected to unilateral anterior crossbite (UAC) to induce OA in the temporomandibular joint (TMJ). Histochemical staining, immunohistochemical staining (IHC), and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. Primary condylar chondrocytes of rats were stimulated with fluid flow shear stress (FFSS) and collected for oil red staining, immunofluorescence staining, qRT-PCR, and immunoprecipitation analysis. Results. Abnormal adipogenesis, characterized by increased expression of CCAAT/enhancer-binding protein α (CEBPα), fatty acid binding protein 4 (FABP4), Perilipin1, Adiponectin (AdipoQ), and peroxisome proliferator-activated receptor γ (PPARγ), was enhanced in the degenerative cartilage of TMJ OA in UAC mice, accompanied by decreased expression of GDF11. After FFSS stimulation, there were fat droplets in the cytoplasm of cultured cells with increased expression of PPARγ, CEBPα, FABP4, Perilipin1, and AdipoQ and decreased expression of GDF11. Exogenous GDF11 inhibited increased lipid droplets and expression of AdipoQ, CEBPα, and FABP4 induced by FFSS stimulation. GDF11 did not affect the change in PPARγ expression under FFSS, but promoted its post-translational modification by small ubiquitin-related modifier (SUMOylation). Local injection of GDF11 alleviated TMJ OA-related cartilage degeneration and abnormal adipogenesis in UAC mice. Conclusion. Abnormal adipogenesis of chondrocytes and decreased GDF11 expression were observed in degenerative cartilage of TMJ OA. GDF11 supplementation effectively inhibits the adipogenesis of chondrocytes and thus alleviates TMJ condylar cartilage degeneration. GDF11 may inhibit the abnormal adipogenesis of chondrocytes by affecting the SUMOylation of PPARγ. Cite this article: Bone Joint Res 2022;11(7):453–464

Aims. The anterior cruciate ligament (ACL) is known to have a poor wound healing capacity, whereas other ligaments outside of the knee joint capsule such as the medial collateral ligament (MCL) apparently heal more easily. Plasmin has been identified as a major component in the synovial fluid that varies among patients. The aim of this study was to test whether plasmin, a component of synovial fluid, could be a main factor responsible for the poor wound healing capacity of the ACL. Methods. The effects of increasing concentrations of plasmin (0, 0.1, 1, 10, and 50 µg/ml) onto the wound closing speed (WCS) of primary ACL-derived ligamentocytes (ACL-LCs) were tested using wound scratch assay and time-lapse phase-contrast microscopy. Additionally, relative expression changes (quantitative PCR (qPCR)) of major LC-relevant genes and catabolic genes were investigated. The positive controls were 10% fetal calf serum (FCS) and platelet-derived growth factor (PDGF). Results. WCS did not differ significantly among no plasmin versus each of the tested concentrations (six donors). The positive controls with PDGF and with FCS differed significantly from the negative controls. However, we found a trend demonstrating that higher plasmin concentrations up-regulate the expression of matrix metalloproteinase 13 (MMP13), 3 (MMP3), and tenomodulin (TNMD). Conclusion. The clinical relevance of this study is the possibility that it is not solely the plasmin, but also additional factors in the synovial fluid of the knee, that may be responsible for the poor healing capacity of the ACL. Cite this article: Bone Joint Res 2020;9(9):543–553

Aims. The lack of disease-modifying treatments for osteoarthritis (OA) is linked to a shortage of suitable biomarkers. This study combines multi-molecule synovial fluid analysis with machine learning to produce an accurate diagnostic biomarker model for end-stage knee OA (esOA). Methods. Synovial fluid (SF) from patients with esOA, non-OA knee injury, and inflammatory knee arthritis were analyzed for 35 potential markers using immunoassays. Partial least square discriminant analysis (PLS-DA) was used to derive a biomarker model for cohort classification. The ability of the biomarker model to diagnose esOA was validated by identical wide-spectrum SF analysis of a test cohort of ten patients with esOA. Results. PLS-DA produced a streamlined biomarker model with excellent sensitivity (95%), specificity (98.4%), and reliability (97.4%). The eight-biomarker model produced a fingerprint for esOA comprising type IIA procollagen N-terminal propeptide (PIIANP), tissue inhibitor of metalloproteinase (TIMP)-1, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), monocyte chemoattractant protein (MCP)-1, interferon-γ-inducible protein-10 (IP-10), and transforming growth factor (TGF)-β3. Receiver operating characteristic (ROC) analysis demonstrated excellent discriminatory accuracy: area under the curve (AUC) being 0.970 for esOA, 0.957 for knee injury, and 1 for inflammatory arthritis. All ten validation test patients were classified correctly as esOA (accuracy 100%; reliability 100%) by the biomarker model. Conclusion. SF analysis coupled with machine learning produced a partially validated biomarker model with cohort-specific fingerprints that accurately and reliably discriminated esOA from knee injury and inflammatory arthritis with almost 100% efficacy. The presented findings and approach represent a new biomarker concept and potential diagnostic tool to stage disease in therapy trials and monitor the efficacy of such interventions. Cite this article: Bone Joint Res 2020;9(9):623–632

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

Aims. Periprosthetic joint infection (PJI) is a debilitating condition with a substantial socioeconomic burden. A novel autologous blood glue (ABG) has been developed, which can be prepared during surgery and sprayed onto prostheses at the time of implantation. The ABG can potentially provide an antimicrobial coating which will be effective in preventing PJI, not only by providing a physical barrier but also by eluting a well-known antibiotic. Hence, this study aimed to assess the antimicrobial effectiveness of ABG when impregnated with gentamicin and stem cells. Methods. Gentamicin elution from the ABG matrix was analyzed and quantified in a time-dependent manner. The combined efficiency of gentamicin and ABG as an anti-biofilm coating was investigated on titanium disks. Results. ABG-gentamicin was bactericidal from 10 μg/ml and could release bactericidal concentrations over seven days, preventing biofilm formation. A concentration of 75 μg/ml of gentamicin in ABG showed the highest bactericidal effect up to day 7. On titanium disks, a significant bacterial reduction on ABG-gentamicin coated disks was observed when compared to both uncoated (mean 2-log reduction) and ABG-coated (mean 3-log reduction) disks, at days 3 and 7. ABG alone exhibited no antimicrobial or anti-biofilm properties. However, a concentration of 75 μg/ml gentamicin in ABG sustains release over seven days and significantly reduced biofilm formation. Its use as an implant coating in patients with a high risk of infection may prevent bacterial adhesion perioperatively and in the early postoperative period. Conclusion. ABG’s use as a carrier for stem cells was effective, as it supported cell growth. It has the potential to co-deliver compatible cells, drugs, and growth factors. However, ABG-gentamicin’s potential needs to be further justified using in vivo studies. Cite this article: Bone Joint Res 2020;9(12):848–856

Aims. This study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss. Methods. TNF-α immunoexpression was examined in periprosthetic tissues of patients with failed metal-on-metal hip arthroplasties and also in myeloid MM6 cells after treatment with cobalt ions. Viability and function of human osteoblast-like SaOs-2 cells treated with recombinant TNF-α were studied by immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Results. Macrophages, lymphocytes, and endothelial cells displayed strong TNF-α immunoexpression in periprosthetic tissues containing metal wear debris. Colocalization of TNF-α with the macrophage marker CD68 and the pan-T cell marker CD3 confirmed TNF-α expression in these cells. Cobalt-treated MM6 cells secreted more TNF-α than control cells, reflecting the role of metal wear products in activating the TNF-α pathway in the myeloid cells. While TNF-α did not alter the immunoexpression of the TNF-receptor 1 (TNF-R1) in SaOs-2 cells, it increased the release of the soluble TNF-receptor 1 (sTNF-R1). There was also evidence for TNF-α-induced apoptosis. TNF-α further elicited the expression of the endoplasmic reticulum stress markers inositol-requiring enzyme (IRE)-1α, binding-immunoglobulin protein (BiP), and endoplasmic oxidoreductin1 (Ero1)-Lα. In addition, TNF-α decreased pro-collagen I α 1 secretion without diminishing its synthesis. TNF-α also induced an inflammatory response in SaOs-2 cells, as evidenced by the release of reactive oxygen and nitrogen species and the proinflammatory cytokine vascular endothelial growth factor. Conclusion. The results suggest a novel osteoblastic mechanism, which could be mediated by TNF-α and may be involved in metal wear debris-induced periprosthetic bone loss. Cite this article: Bone Joint Res 2020;9(11):827–839

Aims. Cigarette smoking has a negative impact on the skeletal system, causes a decrease in bone mass in both young and old patients, and is considered a risk factor for the development of osteoporosis. In addition, it disturbs the bone healing process and prolongs the healing time after fractures. The mechanisms by which cigarette smoking impairs fracture healing are not fully understood. There are few studies reporting the effects of cigarette smoking on new blood vessel formation during the early stage of fracture healing. We tested the hypothesis that cigarette smoke inhalation may suppress angiogenesis and delay fracture healing. Methods. We established a custom-made chamber with airflow for rats to inhale cigarette smoke continuously, and tested our hypothesis using a femoral osteotomy model, radiograph and microCT imaging, and various biomechanical and biological tests. Results. In the smoking group, Western blot analysis and immunohistochemical staining revealed less expression of vascular endothelial growth factor (VEGF) and von Willebrand factor (vWF). The smoking group also had a lower microvessel density than the control group. Image and biochemical analysis also demonstrated delayed bone healing. Conclusion. Cigarette smoke inhalation was associated with decreased expression of angiogenic markers in the early bone healing phase and with impaired bone healing. Cite this article:Bone Joint Res. 2020;9(3):99–107

Objectives. The present study describes a novel technique for revitalising allogenic intrasynovial tendons by combining cell-based therapy and mechanical stimulation in an ex vivo canine model. Methods. Specifically, canine flexor digitorum profundus tendons were used for this study and were divided into the following groups: (1) untreated, unprocessed normal tendon; (2) decellularised tendon; (3) bone marrow stromal cell (BMSC)-seeded tendon; and (4) BMSC-seeded and cyclically stretched tendon. Lateral slits were introduced on the tendon to facilitate cell seeding. Tendons from all four study groups were distracted by a servohydraulic testing machine. Tensile force and displacement data were continuously recorded at a sample rate of 20 Hz until 200 Newton of force was reached. Before testing, the cross-sectional dimensions of each tendon were measured with a digital caliper. Young’s modulus was calculated from the slope of the linear region of the stress-strain curve. The BMSCs were labeled for histological and cell viability evaluation on the decellularized tendon scaffold under a confocal microscope. Gene expression levels of selected extracellular matrix tendon growth factor genes were measured. Results were reported as mean ± SD and data was analyzed with one-way ANOVAs followed by Tukey’s post hoc multiple-comparison test. Results. We observed no significant difference in cross-sectional area or in Young’s modulus among the four study groups. In addition, histological sections showed that the BMSCs were aligned well and viable on the tendon slices after two-week culture in groups three and four. Expression levels of several extracellular matrix tendon growth factors, including collagen type I, collagen type III, and matrix metalloproteinase were significantly higher in group four than in group three (p < 0.05). Conclusion. Lateral slits introduced into de-cellularised tendon is a promising method of delivery of BMSCs without compromising cell viability and tendon mechanical properties. In addition, mechanical stimulation of a cell-seeded tendon can promote cell proliferation and enhance expression of collagen types I and III in vitro. Cite this article: J. H. Wu, A. R. Thoreson, A. Gingery, K. N. An, S. L. Moran, P. C. Amadio, C. Zhao. The revitalisation of flexor tendon allografts with bone marrow stromal cells and mechanical stimulation: An ex vivo model revitalising flexor tendon allografts. Bone Joint Res 2017;6:179–185. DOI: 10.1302/2046-3758.63.BJR-2016-0207.R1

Objectives. Ligaments which heal spontaneously have a healing process that is similar to skin wound healing. Menopause impairs skin wound healing and may likewise impair ligament healing. Our purpose in this study was to investigate the effect of surgical menopause on ligament healing in a rabbit medial collateral ligament model. Methods. Surgical menopause was induced with ovariohysterectomy surgery in adult female rabbits. Ligament injury was created by making a surgical gap in the midsubstance of the medial collateral ligament. Ligaments were allowed to heal for six or 14 weeks in the presence or absence of oestrogen before being compared with uninjured ligaments. Molecular assessment examined the messenger ribonucleic acid levels for collagens, proteoglycans, proteinases, hormone receptors, growth factors and inflammatory mediators. Mechanical assessments examined ligament laxity, total creep strain and failure stress. Results. Surgical menopause in normal medial collateral ligaments initiated molecular changes in all the categories evaluated. In early healing medial collateral ligaments, surgical menopause resulted in downregulation of specific collagens, proteinases and inflammatory mediators at 6 weeks of healing, and proteoglycans, growth factors and hormone receptors at 14 weeks of healing. Surgical menopause did not produce mechanical changes in normal or early healing medial collateral ligaments. With or without surgical menopause, healing ligaments exhibited increased total creep strain and decreased failure stress compared with uninjured ligaments. Conclusions. Surgical menopause did not affect the mechanical properties of normal or early healing medial collateral ligaments in a rabbit model. The results in this preclinical model suggest that menopause may result in no further impairment to the ligament healing process. . Cite this article: Bone Joint Res 2015;4:38–44

Objectives. Long bone defects often require surgical intervention for functional restoration. The ‘gold standard’ treatment is autologous bone graft (ABG), usually from the patient’s iliac crest. However, autograft is plagued by complications including limited supply, donor site morbidity, and the need for an additional surgery. Thus, alternative therapies are being actively investigated. Autologous bone marrow (BM) is considered as a candidate due to the presence of both endogenous reparative cells and growth factors. We aimed to compare the therapeutic potentials of autologous bone marrow aspirate (BMA) and ABG, which has not previously been done. Methods. We compared the efficacy of coagulated autologous BMA and ABG for the repair of ulnar defects in New Zealand White rabbits. Segmental defects (14 mm) were filled with autologous clotted BM or morcellized autograft, and healing was assessed four and 12 weeks postoperatively. Harvested ulnas were subjected to radiological, micro-CT, histological, and mechanical analyses. Results. Comparable results were obtained with autologous BMA clot and ABG, except for the quantification of new bone by micro-CT. Significantly more bone was found in the ABG-treated ulnar defects than in those treated with autologous BMA clot. This is possibly due to the remnants of necrotic autograft fragments that persisted within the healing defects at week 12 post-surgery. Conclusion. As similar treatment outcomes were achieved by the two strategies, the preferred treatment would be one that is associated with a lower risk of complications. Hence, these results demonstrate that coagulated BMA can be considered as an alternative autogenous therapy for long bone healing. Cite this article: Z. X. H. Lim, B. Rai, T. C. Tan, A. K. Ramruttun, J. H. Hui, V. Nurcombe, S. H. Teoh, S. M. Cool. Autologous bone marrow clot as an alternative to autograft for bone defect healing. Bone Joint Res 2019;8:107–117. DOI: 10.1302/2046-3758.83.BJR-2018-0096.R1

Objectives. Adult mice lacking the transcription factor NFAT1 exhibit osteoarthritis (OA). The precise molecular mechanism for NFAT1 deficiency-induced osteoarthritic cartilage degradation remains to be clarified. This study aimed to investigate if NFAT1 protects articular cartilage (AC) against OA by directly regulating the transcription of specific catabolic and anabolic genes in articular chondrocytes. Methods. Through a combined approach of gene expression analysis and web-based searching of NFAT1 binding sequences, 25 candidate target genes that displayed aberrant expression in Nfat1. -/-. AC at the initiation stage of OA, and possessed at least four NFAT1 binding sites in the promoter of each gene, were selected and tested for NFAT1 transcriptional activities by chromatin immunoprecipitation (ChIP) and promoter luciferase reporter assays using chondrocytes isolated from the AC of three- to four-month-old wild-type mice or Nfat1. -/-. mice with early OA phenotype. Results. Chromatin immunoprecipitation assays revealed that NFAT1 bound directly to the promoter of 21 of the 25 tested genes encoding cartilage-matrix proteins, growth factors, inflammatory cytokines, matrix-degrading proteinases, and specific transcription factors. Promoter luciferase reporter assays of representative anabolic and catabolic genes demonstrated that NFAT1-DNA binding functionally regulated the luciferase activity of specific target genes in wild-type chondrocytes, but not in Nfat1. -/-. chondrocytes or in wild-type chondrocytes transfected with plasmids containing mutated NFAT1 binding sequences. Conclusion. NFAT1 protects AC against degradation by directly regulating the transcription of target genes in articular chondrocytes. NFAT1 deficiency causes defective transcription of specific anabolic and catabolic genes in articular chondrocytes, leading to increased matrix catabolism and osteoarthritic cartilage degradation. Cite this article: M. Zhang, Q. Lu, T. Budden, J. Wang. NFAT1 protects articular cartilage against osteoarthritic degradation by directly regulating transcription of specific anabolic and catabolic genes. Bone Joint Res 2019;8:90–100. DOI: 10.1302/2046-3758.82.BJR-2018-0114.R1

Aims. The study aimed to determine whether the microRNA miR21-5p (MiR21) mediates temporomandibular joint osteoarthritis (TMJ-OA) by targeting growth differentiation factor 5 (Gdf5). Methods. TMJ-OA was induced in MiR21 knockout (KO) mice and wild-type (WT) mice by a unilateral anterior crossbite (UAC) procedure. Mouse tissues exhibited histopathological changes, as assessed by: Safranin O, toluidine blue, and immunohistochemistry staining; western blotting (WB); and quantitative real-time polymerase chain reaction (RT-qPCR). Mouse condylar chondrocytes were transfected with a series of MiR21 mimic, MiR21 inhibitor, Gdf5 siRNA (si-GDF5), and flag-GDF5 constructs. The effects of MiR-21 and Gdf5 on the expression of OA related molecules were evaluated by immunofluorescence, alcian blue staining, WB, and RT-qPCR. Results. UAC altered the histological structure and extracellular matrix content of cartilage in the temporomandibular joint (TMJ), and KO of MiR21 alleviated this effect (p < 0.05). Upregulation of MiR21 influenced the expression of TMJ-OA related molecules in mandibular condylar chondrocytes via targeting Gdf5 (p < 0.05). Gdf5 overexpression significantly decreased matrix metalloproteinase 13 (MMP13) expression (p < 0.05) and reversed the effects of MiR21 (p < 0.05). Conclusion. MiR21, which acts as a critical regulator of Gdf5 in chondrocytes, regulates TMJ-OA related molecules and is involved in cartilage matrix degradation, contributing to the progression of TMJ-OA. Cite this article: Bone Joint Res 2020;9(10):689–700

Objectives. Metabolic syndrome and low-grade systemic inflammation are associated with knee osteoarthritis (OA), but the relationships between these factors and OA in other synovial joints are unclear. The aim of this study was to determine if a high-fat/high-sucrose (HFS) diet results in OA-like joint damage in the shoulders, knees, and hips of rats after induction of obesity, and to identify potential joint-specific risks for OA-like changes. Methods. A total of 16 male Sprague-Dawley rats were allocated to either the diet-induced obesity group (DIO, 40% fat, 45% sucrose, n = 9) or a chow control diet (n = 7) for 12 weeks. At sacrifice, histological assessments of the shoulder, hip, and knee joints were performed. Serum inflammatory mediators and body composition were also evaluated. The total Mankin score for each animal was assessed by adding together the individual Modified Mankin scores across all three joints. Linear regression modelling was conducted to evaluate predictive relationships between serum mediators and total joint damage. Results. The HFS diet, in the absence of trauma, resulted in increased joint damage in the shoulder and knee joints of rats. Hip joint damage, however, was not significantly affected by DIO, consistent with findings in human studies. The total Mankin score was increased in DIO animals compared with the chow group, and was associated with percentage of body fat. Positive significant predictive relationships for total Mankin score were found between body fat and two serum mediators (interleukin 1 alpha (IL-1α) and vascular endothelial growth factor (VEGF)). Conclusion. Systemic inflammatory alterations from DIO in this model system may result in a higher risk for development of knee, shoulder, and multi-joint damage with a HFS diet. Cite this article: K. H. Collins, D. A. Hart, R. A. Seerattan, R. A. Reimer, W. Herzog. High-fat/high-sucrose diet-induced obesity results in joint-specific development of osteoarthritis-like degeneration in a rat model. Bone Joint Res 2018;7:274–281. DOI: 10.1302/2046-3758.74.BJR-2017-0201.R2

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

Objectives. Mesenchymal stem cells have the ability to differentiate into various cell types, and thus have emerged as promising alternatives to chondrocytes in cell-based cartilage repair methods. The aim of this experimental study was to investigate the effect of bone marrow derived mesenchymal stem cells combined with platelet rich fibrin on osteochondral defect repair and articular cartilage regeneration in a canine model. Methods. Osteochondral defects were created on the medial femoral condyles of 12 adult male mixed breed dogs. They were either treated with stem cells seeded on platelet rich fibrin or left empty. Macroscopic and histological evaluation of the repair tissue was conducted after four, 16 and 24 weeks using the International Cartilage Repair Society macroscopic and the O’Driscoll histological grading systems. Results were reported as mean and standard deviation (. sd. ) and compared at different time points between the two groups using the Mann-Whitney U test, with a value < 0.05 considered statistically significant. Results. Higher cumulative macroscopic and histological scores were observed in stem cell treated defects throughout the study period with significant differences noted at four and 24 weeks (9.25, . sd. 0.5 vs 7.25, . sd. 0.95, and 10, . sd. 0.81 vs 7.5, . sd. 0.57; p < 0.05) and 16 weeks (16.5, . sd. 4.04 vs 11, . sd. 1.15; p < 0.05), respectively. Superior gross and histological characteristics were also observed in stem cell treated defects. Conclusion. The use of autologous culture expanded bone marrow derived mesenchymal stem cells on platelet rich fibrin is a novel method for articular cartilage regeneration. It is postulated that platelet rich fibrin creates a suitable environment for proliferation and differentiation of stem cells by releasing endogenous growth factors resulting in creation of a hyaline-like reparative tissue. Cite this article: D. Kazemi, K. Shams Asenjan, N. Dehdilani, H. Parsa. Canine articular cartilage regeneration using mesenchymal stem cells seeded on platelet rich fibrin: Macroscopic and histological assessments. Bone Joint Res 2017;6:98–107. DOI: 10.1302/2046-3758.62.BJR-2016-0188.R1

Aims

Dupuytren’s contracture is characterized by increased fibrosis of the palmar aponeurosis, with eventual replacement of the surrounding fatty tissue with palmar fascial fibromatosis. We hypothesized that adipocytokines produced by adipose tissue in contact with the palmar aponeurosis might promote fibrosis of the palmar aponeurosis.

Objectives. Oxidative stress plays a major role in the onset and progression of involutional osteoporosis. However, classical antioxidants fail to restore osteoblast function. Interestingly, the bone anabolism of parathyroid hormone (PTH) has been shown to be associated with its ability to counteract oxidative stress in osteoblasts. The PTH counterpart in bone, which is the PTH-related protein (PTHrP), displays osteogenic actions through both its N-terminal PTH-like region and the C-terminal domain. Methods. We examined and compared the antioxidant capacity of PTHrP (1-37) with the C-terminal PTHrP domain comprising the 107-111 epitope (osteostatin) in both murine osteoblastic MC3T3-E1 cells and primary human osteoblastic cells. Results. We showed that both N- and C-terminal PTHrP peptides at 100 nM decreased reactive oxygen species production and forkhead box protein O activation following hydrogen peroxide (H. 2. O. 2. )-induced oxidation, which was related to decreased lipid oxidative damage and caspase-3 activation in these cells. This was associated with their ability to restore the deleterious effects of H. 2. O. 2. on cell growth and alkaline phosphatase activity, as well as on the expression of various osteoblast differentiation genes. The addition of Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine triethylammonium salt (a cyclic 3',5'-adenosine monophosphate antagonist) and calphostin C (a protein kinase C inhibitor), or a PTH type 1 receptor antagonist, abrogated the effects of N-terminal PTHrP, whereas protein phosphatase 1 (an Src kinase activity inhibitor), SU1498 (a vascular endothelial growth factor receptor 2 inhibitor), or an anti osteostatin antiserum, inhibited the effects of C-terminal PTHrP. Conclusion. These findings indicate that the antioxidant properties of PTHrP act through its N- and C-terminal domains and provide novel insights into the osteogenic action of PTHrP. Cite this article: S. Portal-Núñez, J. A. Ardura, D. Lozano, I. Martínez de Toda, M. De la Fuente, G. Herrero-Beaumont, R. Largo, P. Esbrit. Parathyroid hormone-related protein exhibits antioxidant features in osteoblastic cells through its N-terminal and osteostatin domains. Bone Joint Res 2018;7:58–68. DOI: 10.1302/2046-3758.71.BJR-2016-0242.R2

Objectives. Several genome-wide association studies (GWAS) of bone mineral density (BMD) have successfully identified multiple susceptibility genes, yet isolated susceptibility genes are often difficult to interpret biologically. The aim of this study was to unravel the genetic background of BMD at pathway level, by integrating BMD GWAS data with genome-wide expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (meQTLs) data. Method. We employed the GWAS datasets of BMD from the Genetic Factors for Osteoporosis Consortium (GEFOS), analysing patients’ BMD. The areas studied included 32 735 femoral necks, 28 498 lumbar spines, and 8143 forearms. Genome-wide eQTLs (containing 923 021 eQTLs) and meQTLs (containing 683 152 unique methylation sites with local meQTLs) data sets were collected from recently published studies. Gene scores were first calculated by summary data-based Mendelian randomisation (SMR) software and meQTL-aligned GWAS results. Gene set enrichment analysis (GSEA) was then applied to identify BMD-associated gene sets with a predefined significance level of 0.05. Results. We identified multiple gene sets associated with BMD in one or more regions, including relevant known biological gene sets such as the Reactome Circadian Clock (GSEA p-value = 1.0 × 10. -4. for LS and 2.7 × 10. -2. for femoral necks BMD in eQTLs-based GSEA) and insulin-like growth factor receptor binding (GSEA p-value = 5.0 × 10. -4. for femoral necks and 2.6 × 10. -2. for lumbar spines BMD in meQTLs-based GSEA). Conclusion. Our results provided novel clues for subsequent functional analysis of bone metabolism, and illustrated the benefit of integrating eQTLs and meQTLs data into pathway association analysis for genetic studies of complex human diseases. Cite this article: W. Wang, S. Huang, W. Hou, Y. Liu, Q. Fan, A. He, Y. Wen, J. Hao, X. Guo, F. Zhang. Integrative analysis of GWAS, eQTLs and meQTLs data suggests that multiple gene sets are associated with bone mineral density. Bone Joint Res 2017;6:572–576

Aims

Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants.

Osteoarthritis (OA) is mainly caused by ageing, strain, trauma, and congenital joint abnormalities, resulting in articular cartilage degeneration. During the pathogenesis of OA, the changes in subchondral bone (SB) are not only secondary manifestations of OA, but also an active part of the disease, and are closely associated with the severity of OA. In different stages of OA, there were microstructural changes in SB. Osteocytes, osteoblasts, and osteoclasts in SB are important in the pathogenesis of OA. The signal transduction mechanism in SB is necessary to maintain the balance of a stable phenotype, extracellular matrix (ECM) synthesis, and bone remodelling between articular cartilage and SB. An imbalance in signal transduction can lead to reduced cartilage quality and SB thickening, which leads to the progression of OA. By understanding changes in SB in OA, researchers are exploring drugs that can regulate these changes, which will help to provide new ideas for the treatment of OA.

Cite this article: Bone Joint Res 2023;12(9):536–545.

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This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD).

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Several artificial bone grafts have been developed but fail to achieve anticipated osteogenesis due to their insufficient neovascularization capacity and periosteum support. This study aimed to develop a vascularized bone-periosteum construct (VBPC) to provide better angiogenesis and osteogenesis for bone regeneration.

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage degradation, synovial membrane inflammation, osteophyte formation, and subchondral bone sclerosis. Pathological changes in cartilage and subchondral bone are the main processes in OA. In recent decades, many studies have demonstrated that activin-like kinase 3 (ALK3), a bone morphogenetic protein receptor, is essential for cartilage formation, osteogenesis, and postnatal skeletal development. Although the role of bone morphogenetic protein (BMP) signalling in articular cartilage and bone has been extensively studied, many new discoveries have been made in recent years around ALK3 targets in articular cartilage, subchondral bone, and the interaction between the two, broadening the original knowledge of the relationship between ALK3 and OA. In this review, we focus on the roles of ALK3 in OA, including cartilage and subchondral bone and related cells. It may be helpful to seek more efficient drugs or treatments for OA based on ALK3 signalling in future.

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The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice.

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Adenosine, lidocaine, and Mg²⁺ (ALM) therapy exerts differential immuno-inflammatory responses in males and females early after anterior cruciate ligament (ACL) reconstruction (ACLR). Our aim was to investigate sex-specific effects of ALM therapy on joint tissue repair and recovery 28 days after surgery.

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Degenerative cervical spondylosis (DCS) is a common musculoskeletal disease that encompasses a wide range of progressive degenerative changes and affects all components of the cervical spine. DCS imposes very large social and economic burdens. However, its genetic basis remains elusive.

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