Objectives

Recent studies have shown that modulating inflammation-related lipid signalling after a bone fracture can accelerate healing in animal models. Specifically, decreasing 5-lipoxygenase (5-LO) activity during fracture healing increases cyclooxygenase-2 (COX-2) expression in the fracture callus, accelerates chondrogenesis and decreases healing time. In this study, we test the hypothesis that 5-LO inhibition will increase direct osteogenesis.

Objectives. The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy. Methods. Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined. Results. In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased. Conclusion. This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes. Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362–372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2

Objectives. After an injury, the biological reattachment of tendon to bone is a challenge because healing takes place between a soft (tendon) and a hard (bone) tissue. Even after healing, the transition zone in the enthesis is not completely regenerated, making it susceptible to re-injury. In this study, we aimed to regenerate Achilles tendon entheses (ATEs) in wounded rats using a combination of kartogenin (KGN) and platelet-rich plasma (PRP). Methods. Wounds created in rat ATEs were given three different treatments: kartogenin platelet-rich plasma (KGN-PRP); PRP; or saline (control), followed by histological and immunochemical analyses, and mechanical testing of the rat ATEs after three months of healing. Results. Histological analysis showed well organised arrangement of collagen fibres and proteoglycan formation in the wounded ATEs in the KGN-PRP group. Furthermore, immunohistochemical analysis revealed fibrocartilage formation in the KGN-PRP-treated ATEs, evidenced by the presence of both collagen I and II in the healed ATE. Larger positively stained collagen III areas were found in both PRP and saline groups than those in the KGN-PRP group. Chondrocyte-related genes, SOX9 and collagen II, and tenocyte-related genes, collagen I and scleraxis (SCX), were also upregulated by KGN-PRP. Moreover, mechanical testing results showed higher ultimate tensile strength in the KGN-PRP group than in the saline control group. In contrast, PRP treatment appeared to have healed the injured ATE but induced no apparent formation of fibrocartilage. The saline-treated group showed poor healing without fibrocartilage tissue formation in the ATEs. Conclusions. Our results show that injection of KGN-PRP induces fibrocartilage formation in the wounded rat ATEs. Hence, KGN-PRP may be a clinically relevant, biological approach to regenerate injured enthesis effectively. Cite this article: J. Zhang, T. Yuan, N. Zheng, Y. Zhou, M. V. Hogan, J. H-C. Wang. The combined use of kartogenin and platelet-rich plasma promotes fibrocartilage formation in the wounded rat Achilles tendon entheses. Bone Joint Res 2017;6:231–244. DOI: 10.1302/2046-3758.64.BJR-2017-0268.R1

Objectives. In this study, we compared the pain behaviour and osteoarthritis (OA) progression between anterior cruciate ligament transection (ACLT) and osteochondral injury in surgically-induced OA rat models. Methods. OA was induced in the knee joints of male Wistar rats using transection of the ACL or induction of osteochondral injury. Changes in the percentage of high limb weight distribution (%HLWD) on the operated hind limb were used to determine the pain behaviour in these models. The development of OA was assessed and compared using a histological evaluation based on the Osteoarthritis Research Society International (OARSI) cartilage OA histopathology score. Results. Both models showed an increase in joint pain as indicated by a significant (p < 0.05) decrease in the values of %HLWD at one week post-surgery. In the osteochondral injury model, the %HLWD returned to normal within three weeks, while in the ACLT model, a significant decrease in the %HLWD was persistent over an eight-week period. In addition, OA progression was more advanced in the ACLT model than in the osteochondral injury model. Furthermore, the ACLT model exhibited a higher mean OA score than that of the osteochondral injury model at 12 weeks. Conclusion. The development of pain patterns in the ACLT and osteochondral injury models is different in that the OA progression was significant in the ACLT model. Although both can be used as models for a post-traumatic injury of the knee, the selection of appropriate models for OA in preclinical studies should be specified and relevant to the clinical scenario. Cite this article: T. Tawonsawatruk, O. Sriwatananukulkit, W. Himakhun, W. Hemstapat. Comparison of pain behaviour and osteoarthritis progression between anterior cruciate ligament transection and osteochondral injury in rat models. Bone Joint Res 2018;7:244–251. DOI: 10.1302/2046-3758.73.BJR-2017-0121.R2

Objectives. We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days. Materials and Methods. Unilateral femoral osteotomies were created in 58 male rats and fixed with an intramedullary compression nail. Rats were treated daily either with vehicle, tadalafil or COMB-4. Biomechanical testing of the healed fracture was performed on day 42. The volume, mineral content and bone density of the callus were measured by quantitative CT on days 14 and 42. Expression of iNOS was measured by immunohistochemistry. Results. When compared with the control group, the COMB-4 group exhibited 46% higher maximum strength (t-test, p = 0.029) and 92% higher stiffness (t-test, p = 0.023), but no significant changes were observed in the tadalafil group. At days 14 and 42, there was no significant difference between the three groups with respect to callus volume, mineral content and bone density. Expression of iNOS at day 14 was significantly higher in the COMB-4 group which, as expected, had returned to baseline levels at day 42. Conclusion. This study demonstrates an enhancement in fracture healing by an oral natural product known to augment iNOS expression. Cite this article: R. A. Rajfer, A. Kilic, A. S. Neviaser, L. M. Schulte, S. M. Hlaing, J. Landeros, M. G. Ferrini, E. Ebramzadeh, S-H. Park. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase. Bone Joint Res 2017:6:–97. DOI: 10.1302/2046-3758.62.BJR-2016-0164.R2

Objectives. The aim of this study was to examine whether asymmetric loading influences macrophage elastase (MMP12) expression in different parts of a rat tail intervertebral disc and growth plate and if MMP12 expression is correlated with the severity of the deformity. Methods. A wedge deformity between the ninth and tenth tail vertebrae was produced with an Ilizarov-type mini external fixator in 45 female Wistar rats, matched for their age and weight. Three groups were created according to the degree of deformity (10°, 30° and 50°). A total of 30 discs and vertebrae were evaluated immunohistochemically for immunolocalisation of MMP12 expression, and 15 discs were analysed by western blot and zymography in order to detect pro- and active MMP12. Results. No MMP12 expression was detected in the nucleus pulposus. Expression of MMP12 in the annulus progressively increased from group I to groups II and III, mainly at the concave side. Many growth plate chondrocytes expressed MMP12 in the control group, less in group I and rare in groups II and III. Changes in cell phenotype and reduction of cell number were observed, together with disorganisation of matrix microstructure similar to disc degeneration. ProMMP12 was detected at the area of 54 kDa and active MMP12 at 22 kDa. Conclusions. Expression of MMP12 after application of asymmetric loading in a rat tail increased in the intervertebral disc but decreased in the growth plate and correlated with the degree of the deformity and the side of the wedged disc. Cite this article: Bone Joint Res 2014;3:273–9

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

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine.

Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2.

Aims. This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation. Methods. In this study, 60 rats were included in a titanium rod implantation model and underwent subsequent guanylate cyclase treatment. Imaging, histology, and biomechanics were used to evaluate the osseointegration of rats in each group. First, the impact of VIT on bone integration in aged rats with iron overload was investigated. Subsequently, VIT was employed to modulate the differentiation of MC3T3-E1 cells and RAW264.7 cells under conditions of iron overload. Results. Utilizing an OVX rat model, we observed significant alterations in bone mass and osseointegration due to VIT administration in aged rats with iron overload. The observed effects were concomitant with reductions in bone metabolism, oxidative stress, and inflammation. To elucidate whether these effects are associated with osteoclast and osteoblast activity, we conducted in vitro experiments using MC3T3-E1 cells and RAW264.7 cells. Our findings indicate that iron accumulation suppressed the activity of MC3T3-E1 while enhancing RAW264.7 function. Furthermore, iron overload significantly decreased oxidative stress levels; however, these detrimental effects can be mitigated by VIT treatment. Conclusion. Collectively, our data provide compelling evidence that VIT has the potential to reverse the deleterious consequences of iron overload on osseointegration and bone mass during ageing. Cite this article: Bone Joint Res 2024;13(9):427–440

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

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

Objectives. This study aims to assess the correlation of CT-based structural rigidity analysis with mechanically determined axial rigidity in normal and metabolically diseased rat bone. Methods. A total of 30 rats were divided equally into normal, ovariectomized, and partially nephrectomized groups. Cortical and trabecular bone segments from each animal underwent micro-CT to assess their average and minimum axial rigidities using structural rigidity analysis. Following imaging, all specimens were subjected to uniaxial compression and assessment of mechanically-derived axial rigidity. Results. The average structural rigidity-based axial rigidity was well correlated with the average mechanically-derived axial rigidity results (R. 2. = 0.74). This correlation improved significantly (p < 0.0001) when the CT-based Structural Rigidity Analysis (CTRA) minimum axial rigidity was correlated to the mechanically-derived minimum axial rigidity results (R. 2. = 0.84). Tests of slopes in the mixed model regression analysis indicated a significantly steeper slope for the average axial rigidity compared with the minimum axial rigidity (p = 0.028) and a significant difference in the intercepts (p = 0.022). The CTRA average and minimum axial rigidities were correlated with the mechanically-derived average and minimum axial rigidities using paired t-test analysis (p = 0.37 and p = 0.18, respectively). Conclusions. In summary, the results of this study suggest that structural rigidity analysis of micro-CT data can be used to accurately and quantitatively measure the axial rigidity of bones with metabolic pathologies in an experimental rat model. It appears that minimum axial rigidity is a better model for measuring bone rigidity than average axial rigidity

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. Osteoarthritis (OA) is the most common form of arthritis, affecting approximately 15% of the human population. Recently, increased concentration of nitric oxide in serum and synovial fluid in patients with OA has been observed. However, the exact role of nitric oxide in the initiation of OA has not been elucidated. The aim of the present study was to investigate the role of nitric oxide in innate immune regulation during OA initiation in rats. Methods. Rat OA was induced by performing meniscectomy surgery while cartilage samples were collected 0, 7, and 14 days after surgery. Cartilage cytokine levels were determined by using enzyme-linked immunosorbent assay, while other proteins were assessed by using Western blot. Results. In the time course of the study, nitric oxide was increased seven and 14 days after OA induction. Pro-inflammatory cytokines including tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were decreased. L-NG-Nitroarginine methyl ester (L-NAME, a non-specific nitric oxide synthase inhibitor) significantly decreased cartilage nitric oxide and blocked immune suppression. Further, L-NAME decreased Matrix metalloproteinase (MMPs) and increased tissue inhibitor of metalloproteinase (TIMP) expression in meniscectomised rats. Conclusion. Nitric oxide-dependent innate immune suppression protects cartilage from damage in the early stages of OA initiation in rats. Cite this article: C-C. Hsu, C-L. Lin, I-M. Jou, P-H. Wang, J-S. Lee. The protective role of nitric oxide-dependent innate immunosuppression in the early stage of cartilage damage in rats: Role of nitric oxide in ca rtilage da mage. Bone Joint Res 2017;6:253–258. DOI: 10.1302/2046-3758.64.BJJ-2016-0161.R1

Objectives. This study aims to evaluate if micro-CT can work as a method for the 3D assessment and analysis of cancellous bone by comparing micro-CT with undecalcified histological sections in OVX rats. Methods. The mandible and tibia of sham, ovariectomised (OVX) and zoledronate-injected ovariectomised (OVX-ZOL) rats were assessed morphometrically. Specimens were scanned by micro-CT. Undecalcified histological sections were manufactured from the specimen scanned by micro-CT and stained with haematoxylin and eosin. Bivariate linear regressions and one-way analysis of variance were undertaken for statistics using SPSS 16.0.1 software. Results. There were highly significant correlations between undecalcified histological sections and micro-CT for all parameters (bone volume density (BV/TV), bone surface density (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp))in the mandible and tibia. Bone histomorphometric parameters analysed by both methods exhibited significant differences among sham, OVX, and OVX-ZOL groups. There were significant correlations between mandible and tibia in BV/TV, BS/BV, and Tb.Sp. Conclusions. Micro-CT is a complementary tool to histological sections in basic research that could improve our understanding of bone histomorphometry. The mandible can be used as an effective site to assess bone morphometry of OVX or metabolic bone disease rat models. Cite this article: H. Liu, W. Li, Y. S. Liu, Y. S. Zhou. Bone micro-architectural analysis of mandible and tibia in ovariectomised rats: A quantitative structural comparison between undecalcified histological sections and micro-CT. Bone Joint Res 2016;5:253–262

Objectives. To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Methods. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. Results. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. Conclusions. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis. Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1

Objectives. In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells. Methods. BM cells obtained from a total of 13 Sprague-Dawley (SD) green fluorescent protein transgenic (GFP-Tg) rats were culture-expanded in an osteogenic differentiation medium for three weeks. Osteoblast-like cells were then locally transplanted with collagen scaffolds to the rat model of segmental bone defect. Donor cells were also intravenously infused to the normal Sprague-Dawley (SD) rats for systemic biodistribution. The flow cytometric and histological analyses were performed for cellular tracking after transplantation. Results. Locally transplanted donor cells remained within the vicinity of the transplantation site without migrating to other organs. Systemically administered large amounts of osteoblast-like cells were cleared from various organ tissues within three days of transplantation and did not show any adverse effects in the transplanted rats. Conclusions. We demonstrated a precise assessment of donor cell biodistribution that further augments prospective utility of regenerative cell therapy. Cite this article: Bone Joint Res 2014;3:76–81

Objectives. To elucidate the effects of age on the expression levels of the receptor activator of the nuclear factor-κB ligand (RANKL) and osteoclasts in the periodontal ligament during orthodontic mechanical loading and post-orthodontic retention. Materials and Methods. The study included 20 male Sprague-Dawley rats, ten in the young group (aged four to five weeks) and ten in the adult group (aged 18 to 20 weeks). In each rat, the upper-left first molar was subjected to a seven-day orthodontic force loading followed by a seven-day retention period. The upper-right first molar served as a control. The amount of orthodontic tooth movement was measured after seven-day force application and seven-day post-orthodontic retention. The expression levels of RANKL and the tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were evaluated on day 7 (end of mechanical force loading) and day 14 (after seven days of post-orthodontic retention). Statistical analysis was performed using the t-test, and significance was set at p < 0.05. Results. There was no significant difference between the amount of tooth movement in the young group (0.96, standard deviation (. sd. ) 0.30mm) and that in the adult group (0.80mm, . sd. 0.28) (p > 0.05) after the seven-day force application. On the compression side, the expression of RANKL and TRAP-positive osteoclasts in both the young and the adult groups increased after the application of force for seven days, and then decreased at the end of the seven-day retention period. However, by the end of the period, the expression of RANKL on the compression side dropped to the control level in the young group (p > 0.05), while it was still higher than that on the control side in the adult group (p < 0.05). The expression of RANKL on the compression side did not show significant difference between the young and the adult groups after seven-day force application (p > 0.05), but it was significantly higher in the adult group than that in the young group after seven-day post-orthodontic retention (p < 0.05). Similarly, the decreasing trend of TRAP-positive osteoclasts during the retention period in the adult group was less obvious than that in the young group. Conclusions. The bone-resorptive activity in the young rats was more dynamic than that in the adult rats. The expression of RANKL and the number of osteoclasts in adult rats did not drop to the control level during the post-orthodontic retention period while RANKL expression and the number of osteoclasts in young rats had returned to the baseline. Cite this article: X. Li, M. Li, J. Lu, Y. Hu, L. Cui, D. Zhang, Y. Yang. Age-related effects on osteoclastic activities after orthodontic tooth movement. Bone Joint Res 2016;5:492–499. DOI: 10.1302/2046-3758.510.BJR-2016-0004.R2

Objectives. Temperature is known to influence muscle physiology, with the velocity of shortening, relaxation and propagation all increasing with temperature. Scant data are available, however, regarding thermal influences on energy required to induce muscle damage. Methods. Gastrocnemius and soleus muscles were harvested from 36 male rat limbs and exposed to increasing impact energy in a mechanical test rig. Muscle temperature was varied in 5°C increments, from 17°C to 42°C (to encompass the in vivo range). The energy causing non-recoverable deformation was recorded for each temperature. A measure of tissue elasticity was determined via accelerometer data, smoothed by low-pass fifth order Butterworth filter (10 kHz). Data were analysed using one-way analysis of variance (ANOVA) and significance was accepted at p = 0.05. Results. The energy required to induce muscle failure was significantly lower at muscle temperatures of 17°C to 32°C compared with muscle at core temperature, i.e., 37°C (p < 0.01). During low-energy impacts there were no differences in muscle elasticity between cold and warm muscles (p = 0.18). Differences in elasticity were, however, seen at higher impact energies (p < 0.02). Conclusion. Our findings are of particular clinical relevance, as when muscle temperature drops below 32°C, less energy is required to cause muscle tears. Muscle temperatures of 32°C are reported in ambient conditions, suggesting that it would be beneficial, particularly in colder environments, to ensure that peripheral muscle temperature is raised close to core levels prior to high-velocity exercise. Thus, this work stresses the importance of not only ensuring that the muscle groups are well stretched, but also that all muscle groups are warmed to core temperature in pre-exercise routines. Cite this article: Professor A. H. R. W. Simpson. Increased risk of muscle tears below physiological temperature ranges. Bone Joint Res 2016;5:61–65. doi: 10.1302/2046-3758.52.2000484

Objectives . Rotator cuff tears are among the most common and debilitating upper extremity injuries. Chronic cuff tears result in atrophy and an infiltration of fat into the muscle, a condition commonly referred to as ‘fatty degeneration’. While stem cell therapies hold promise for the treatment of cuff tears, a suitable immunodeficient animal model that could be used to study human or other xenograft-based therapies for the treatment of rotator cuff injuries had not previously been identified. Methods . A full-thickness, massive supraspinatus and infraspinatus tear was induced in adult T-cell deficient rats. We hypothesised that, compared with controls, 28 days after inducing a tear we would observe a decrease in muscle force production, an accumulation of type IIB fibres, and an upregulation in the expression of genes involved with muscle atrophy, fibrosis and inflammation. Results . Chronic cuff tears in nude rats resulted in a 30% to 40% decrease in muscle mass, a 23% reduction in production of muscle force, and an induction of genes that regulate atrophy, fibrosis, lipid accumulation, inflammation and macrophage recruitment. Marked large lipid droplet accumulation was also present. Conclusions . The extent of degenerative changes in nude rats was similar to what was observed in T-cell competent rats. T cells may not play an important role in regulating muscle degeneration following chronic muscle unloading. The general similarities between nude and T-cell competent rats suggest the nude rat is likely an appropriate preclinical model for the study of xenografts that have the potential to enhance the treatment of chronically torn rotator cuff muscles. Cite this article: Bone Joint Res 2014;3:262–72