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. 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. 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

We developed a rat model of limb lengthening to study the basic mechanism of distraction osteogenesis, using a small monolateral external fixator. In 11-week-old male rats we performed a subperiosteal osteotomy in the midshaft of the femur with distraction at 0.25 mm every 12 hours from seven days after operation. Radiological and histological examinations showed a growth zone of constant thickness in the middle of the lengthened segment, with formation of new bone at its proximal and distal ends. Osteogenic cells were arranged longitudinally along the tension vector showing the origin and the fate of individual cells in a single section. Typical endochondral bone formation was prominent in the early stage of distraction, but intramembraneous bone formation became the predominant mechanism of ossification at later stages. We also showed a third mechanism of ossification, ‘transchondroid bone formation’. Chondroid bone, a tissue intermediate between bone and cartilage, was formed directly by chondrocyte-like cells, with transition from fibrous tissue to bone occurring gradually and consecutively without capillary invasion. In situ hybridisation using digoxigenin-11-UTP-labelled complementary RNAs showed that the chondroid bone cells temporarily expressed type-II collagen mRNA. They did not show the classical morphological characteristics of chondrocytes, but were assumed to be young chondrocytes undergoing further differentiation into bone-forming cells. We found at least three different modes of ossification during bone lengthening by distraction osteogenesis. We believe that this is the first report of such a rat model, and have shown the validity of in situ hybridisation techniques for the study of the cellular and molecular mechanisms involved in distraction osteogenesis

For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration

The reduced stability of hydroxyapatite (HA)-coated implants in osteopenic conditions is considered to be a major problem. We therefore developed a model of a boosted cementless implantation in osteopenic rats. Twelve-week-old rats were either ovariectomised (OVX) or sham-operated (SO), and after 24 weeks plain or HA-coated implants were inserted. They were treated with either a prostaglandin EP4 receptor agonist (ONO-4819) or saline for one month. The EP4 agonist considerably improved the osteoporosis in the OVX group. Ultrastructural analysis and mechanical testing showed an improvement in the implant-bone attachment in the HA-coated implants, which was further enhanced by the EP4 agonist. Although the stability of the HA-coated implants in the saline-treated OVX rats was less than in the SO normal rats, the administration of the EP4 agonist significantly compensated for this shortage. Our results showed that the osteogenic effect of the EP4 agonist augmented the osteoconductivity of HA and significantly improved the stability of the implant-bone attachment in the osteoporotic rat model

The effects of extracorporeal shock waves (ESWT) on tendon healing were assessed by observing histological and biomechanical parameters in a rat model of injury to the tendo Achillis. The injury was created by inserting an 18-G needle through tendo Achillis in 48 adult Wistar albino rats. The animals were divided into three groups. The first group received radiation only after the operation. The second received no shock waves and the third had 500 15 KV shocks on the second post-operative day. All the rats were killed on the 21st day after surgery. Histopathological analysis showed an increase in the number of capillaries and less formation of adhesions in the study group compared with the control group (p = 0.03). A significantly greater force was required to rupture the tendon in the study group (p = 0.028). Our findings suggest a basis for clinical trials using ESWT

Intermittent treatment with parathyroid hormone (PTH) has an anabolic effect on both intact cancellous and cortical bone. Very little is known about the effect of the administration of PTH on the healing of fractures or the incorporation of orthopaedic implants. We have investigated the spontaneous ingrowth of callus and the formation of bone in a titanium chamber implanted at the medioproximal aspect of the tibial metaphysis of the rat. Four groups of ten male rats weighing approximately 350 g were injected with human PTH (1-34) in a dosage of 0, 15, 60 or 240 μg/kg/day, respectively, for 42 days from the day of implantation of the chamber. During the observation period the chamber became only partly filled with callus and bone and no difference in ingrowth distance into the chamber was found between the groups. The cancellous density was increased by 90%, 132% and 173% in the groups given PTH in a dosage of 15, 60 or 240 μg/kg/day, respectively. There was a linear correlation between bone density and the log PTH doses (r. 2. = 0.6). Our findings suggest that treatment with PTH may have a potential for enhancement of the incorporation of orthopaedic implants as well as a beneficial effect on the healing of fractures when it is given in low dosages

We studied the sensory afferent properties of normal, immobilised and inflamed rat knees by recording the activity of the medial articular nerve (MAN). When the knee was inflamed by kaolin-carrageenan or immobilised for six weeks, MAN activity significantly increased during rest and continuous passive motion (CPM). The maximal discharge rate tended to increase depending on the angular velocity of the CPM. When the knees were then rested for one hour before again starting CPM, activity was further increased at the initial CPM cycle, the ‘post-rest effect’. Analysis of the conduction velocity showed that 94% and 66% of spike units on the recorded discharge of the immobilised and inflamed knees, respectively, belonged to fine nerve fibres. Our findings show that the sensory receptors in the knee are sensitised in a similar manner by immobilisation and by inflammation, suggesting a relationship to pain. The post-rest effect may be related to a characteristic symptom of osteoarthritis called ‘starting pain’

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.

Bone marrow mesenchymal stromal cells were aspirated from immature male green fluorescent protein transgenic rats and cultured in a monolayer. Four weeks after the creation of the osteochondral defect, the rats were divided into three groups of 18: the control group, treated with an intra-articular injection of phosphate-buffered saline only; the drilling group, treated with an intra-articular injection of phosphate-buffered saline with a bone marrow-stimulating procedure; and the bone marrow mesenchymal stromal cells group, treated with an intra-articular injection of bone marrow mesenchymal stromal cells plus a bone marrow-stimulating procedure. The rats were then killed at 4, 8 and 12 weeks after treatment and examined.

The histological scores were significantly better in the bone marrow mesenchymal stromal cells group than in the control and drilling groups at all time points (p < 0.05). The fluorescence of the green fluorescent protein-positive cells could be observed in specimens four weeks after treatment.

Soaking bone grafts in a bisphosphonate solution before implantation can prevent their resorption and increase the local bone density in rats and humans. However, recent studies suggest that pre-treatment of allografts with bisphosphonate can prevent bone ingrowth into impaction grafts. We tested the hypothesis that excessive amounts of bisphosphonate would also cause a negative response in less dense grafts. We used a model where non-impacted metaphyseal bone grafts were randomised into three groups with either no bisphosphonate, alendronate followed by rinsing, and alendronate without subsequent rinsing, and inserted into bone chambers in rats. The specimens were evaluated histologically at one week, and by histomorphometry and radiology at four weeks. At four weeks, both bisphosphonate groups showed an increase in the total bone content, increased newly formed bone, and higher radiodensity than the controls. In spite of being implanted in a chamber with a limited opportunity to diffuse, even an excessive amount of bisphosphonate improved the outcome. We suggest that the negative results seen by others could be due to the combination of densely compacted bone and a bisphosphonate.

We suggest that bisphosphonates are likely to have a negative influence where resorption is a prerequisite to create space for new bone ingrowth.

The nervous system is known to be involved in inflammation and repair. We aimed to determine the effect of physical activity on the healing of a muscle injury and to examine the pattern of innervation. Using a drop-ball technique, a contusion was produced in the gastrocnemius in 20 rats. In ten the limb was immobilised in a plaster cast and the remaining ten had mobilisation on a running wheel. The muscle and the corresponding dorsal-root ganglia were studied by histological and immunohistochemical methods.

In the mobilisation group, there was a significant reduction in lymphocytes (p = 0.016), macrophages (p = 0.008) and myotubules (p = 0.008) between three and 21 days. The formation of myotubules and the density of nerve fibres was significantly higher (both p = 0.016) compared with those in the immobilisation group at three days, while the density of CGRP-positive fibres was significantly lower (p = 0.016) after 21 days.

Mobilisation after contusional injury to the muscle resulted in early and increased formation of myotubules, early nerve regeneration and progressive reduction in inflammation, suggesting that it promoted a better healing response.

Using in situ hybridisation and the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling (TUNEL) reaction in rats with osteonecrosis of the femoral head we have studied the effect of ischaemia on the gene expression of the stress proteins oxygen-regulated protein 150 (ORP150) and haemoxygenase 1 (HO1) and the death mechanism of the cells involved in osteonecrosis. Both ORP150 and HO1 have been reported to have important roles in the successful adaptation to oxygen deprivation.

ORP150 and HO1 mRNA expression was induced by ischaemia in osteoblasts and osteocytes. In proliferative chondrocytes, these signals were detected constitutively. During the development of ischaemic osteonecrosis, the mechanism of cell death was apoptosis as indicated by DNA fragmentation and the presence of apoptotic bodies in osteocytes, chondrocytes and bone-marrow cells. After the initial ischaemic event, expression of ORP150 and HO1 mRNA, the TUNEL-positive reaction and empty lacunae were found sequentially. These findings were exclusive and may be considered to be markers for each stage in the development of osteonecrosis.

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

The aim of our study was to investigate the effect of platelet-rich plasma on the proliferation and differentiation of rat bone-marrow cells and to determine an optimal platelet concentration in plasma for osseous tissue engineering. Rat bone-marrow cells embedded in different concentrations of platelet-rich plasma gel were cultured for six days. Their potential for proliferation and osteogenic differentiation was analysed. Using a rat limb-lengthening model, the cultured rat bone-marrow cells with platelet-rich plasma of variable concentrations were transplanted into the distraction gap and the quality of the regenerate bone was evaluated radiologically. Cellular proliferation was enhanced in all the platelet-rich plasma groups in a dose-dependent manner. Although no significant differences in the production and mRNA expression of alkaline phosphatase were detected among these groups, mature bone regenerates were more prevalent in the group with the highest concentration of platelets. Our results indicate that a high platelet concentration in the platelet-rich plasma in combination with osteoblastic cells could accelerate the formation of new bone during limb-lengthening procedures

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. 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

Using a rat model the characteristics of the sensory neurones of the dorsal-root ganglia (DRG) innervating the hip were investigated by retrograde neurotransport and immunohistochemistry. Fluoro-Gold solution (FG) was injected into the left hip of ten rats. Seven days later the DRG from both sides between T12 and L6 were harvested. The number of FG-labelled calcitonin gene-related peptide-immunoreactive or isolectin B4-binding neurones were counted. The FG-labelled neurones were distributed throughout the left DRGs between T13 and L5, primarily at L2, L3, and L4. Few FG-labelled isolectin B4-binding neurones were present in the DRGs of either side between T13 and L5, but calcitonin gene-related peptide-immunoreactive neurones made up 30% of all FG-labelled neurones. Our findings may explain the referral of pain from the hip to the thigh or lower leg corresponding to the L2, L3 and L4 levels. Since most neurones are calcitonin gene-related peptide-immunoreactive peptide-containing neurones, they may have a more significant role in the perception of pain in the hip as peptidergic DRG neurones

Thrombin has many biological properties similar to those of growth factors. In a previous study, we showed that thrombin improves healing of the rat tendo Achillis. Low molecular weight heparin (LMWH) inhibits the activity and the generation of thrombin. We therefore considered that LMWH at a thromboprophylactic dose might inhibit tendon repair. Transection of the tendo Achillis was carried out in 86 rats and the healing tested mechanically. Low molecular weight heparin (dalateparin) was either injected a few minutes before the operation and then given continuously with an osmotic mini pump for seven days, or given as one injection before the operation. In another experiment ,we gave LMWH or a placebo by injection twice daily. The anti-factor Xa activity was analysed. Continuous treatment with LMWH impaired tendon healing. After seven days, this treatment caused a 33% reduction in force at failure, a 20% reduction in stiffness and a 67% reduction in energy uptake. However, if injected twice daily, LMWH had no effect on tendon healing. Anti-factor Xa activity was increased by LMWH treatment, but was normal between intermittent injections. Low molecular weight heparin delays tendon repair if given continuously, but not if injected intermittently, probably because the anti-factor Xa activity between injections returns to normal, allowing sufficient thrombin stimulation for repair. These findings indicate the need for caution in the assessment of long-acting thrombin and factor Xa inhibitors

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry. We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008). These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies

A rat model of lumbar root constriction with an additional sympathectomy in some animals was used to assess whether the sympathetic nerves influenced radicular pain. Behavioural tests were undertaken before and after the operation. On the 28th post-operative day, both dorsal root ganglia and the spinal roots of L4 and L5 were removed, frozen and sectioned on a cryostat (8 μm to 10 μm). Immunostaining was then performed with antibodies to tyrosine hydroxylase (TH) according to the Avidin Biotin Complex method. In order to quantify the presence of sympathetic nerve fibres, we counted TH-immunoreactive fibres in the dorsal root ganglia using a light microscope equipped with a micrometer graticule (10 x 10 squares, 500 mm x 500 mm). We counted the squares of the graticule which contained TH-immunoreactive fibres for each of five randomly-selected sections of the dorsal root ganglia. The root constriction group showed mechanical allodynia and thermal hyperalgesia. In this group, TH-immunoreactive fibres were abundant in the ipsilateral dorsal root ganglia at L5 and L4 compared with the opposite side. In the sympathectomy group, mechanical hypersensitivity was attenuated significantly. We consider that the sympathetic nervous system plays an important role in the generation of radicular pain

An injectable material consisting of calcium sulphate mixed with hydroxyapatite was investigated as a possible alternative to autograft in the restoration of bone defects. The material was studied both in vitro in simulated body fluid (SBF) and in vivo when implanted in rat muscles and into the proximal tibiae of rabbits. Variation in the strength and weight of the material during ageing in SBF was measured. Tissue response, material resorption and bone ingrowth were studied in the animal models. A good tissue response was observed in both the rat muscles and rabbit tibiae without inflammatory reactions or the presence of fibrous tissue. Ageing in SBF showed that during the first week carbonated hydroxyapatite precipitated on the surfaces of the material and this may enhance bone ingrowth

We undertook a study of the anti-tumour effects of hyperthermia, delivered via magnetite cationic liposomes (MCLs), on local tumours and lung metastases in a mouse model of osteosarcoma. MCLs were injected into subcutaneous osteosarcomas (LM8) and subjected to an alternating magnetic field which induced a heating effect in MCLs. A control group of mice with tumours received MCLs but were not exposed to an AMF. A further group of mice with tumours were exposed to an AMF but had not been treated with MCLs. The distribution of MCLs and local and lung metastases was evaluated histologically. The weight and volume of local tumours and the number of lung metastases were determined. Expression of heat shock protein 70 was evaluated immunohistologically. Hyperthermia using MCLs effectively heated the targeted tumour to 45°C. The mean weight of the local tumour was significantly suppressed in the hyperthermia group (p = 0.013). The mice subjected to hyperthermia had significantly fewer lung metastases than the control mice (p = 0.005). Heat shock protein 70 was expressed in tumours treated with hyperthermia, but was not found in those tumours not exposed to hyperthermia. The results demonstrate a significant effect of hyperthermia on local tumours and reduces their potential to metastasise to the lung

It has been thought that lumbar intervertebral discs were innervated segmentally. We have previously shown that the L5-L6 intervertebral disc in the rat is innervated bilaterally from the L1 and L2 dorsal root ganglia through the paravertebral sympathetic trunks, but the pathways between the disc and the paravertebral sympathetic trunks were unknown. We have now studied the spines of 17 rats to elucidate the exact pathways. We examined serial sections of the lumbar spine using immunohistochemistry for calcitonin gene-related peptide, a sensory nerve marker. We showed that these nerve fibres from the intervertebral disc ran through the sinuvertebral nerve into the rami communicantes, not into the corresponding segmental spinal nerve. In the rat, sensory information from the lumbar intervertebral discs is conducted through rami communicantes. If this innervation pattern applies to man, simple decompression of the corresponding nerve root will not relieve discogenic pain. Anterior interbody fusion, with the denervation of rami communicantes, may be effective for such low back pain

Ciprofloxacin hydrochloride-loaded microspheres were prepared by a spray-drying method using pectin and chitosan. The effects of different polymers and drug ratios were investigated. The most appropriate carriers were selected by in vitro testing. A rat methicillin-resistant Staphylococcus aureus osteomyelitis model was used to evaluate the effects of the loaded microspheres. The drug was released rapidly from the pectin carrier but this was more sustained in the chitosan formulation. Chitosan microspheres loaded with ciprofloxacin hydrochloride were more effective for the treatment of osteomyelitis than equivalent intramuscular antibiotics

This study was undertaken to elucidate the mechanism of biological repair at the tendon-bone junction in a rat model. The stump of the toe flexor tendon was sutured to a drilled hole in the tibia (tendon suture group, n = 23) to investigate healing of the tendon-bone junction both radiologically and histologically. Radiological and histological findings were compared with those observed in a sham control group where the bone alone was drilled (n = 19). The biomechanical strength of the repaired junction was confirmed by pull-out testing six weeks after surgery in four rats in the tendon suture group. Callus formation was observed at the site of repair in the tendon suture group, whereas in the sham group callus formation was minimal. During the pull-out test, the repaired tendon-bone junction did not fail because the musculotendinous junction always disrupted first. In order to understand the factors that influenced callus formation at the site of repair, four further groups were evaluated. The nature of the sutured tendon itself was investigated by analysing healing of a tendon stump after necrosis had been induced with liquid nitrogen in 16 cases. A proximal suture group (n = 16) and a partial tenotomy group (n = 16) were prepared to investigate the effects of biomechanical loading on the site of repair. Finally, a group where the periosteum had been excised at the site of repair (n = 16) was examined to study the role of the periosteum. These four groups showed less callus formation radiologically and histologically than did the tendon suture group. In conclusion, the sutured tendon-bone junction healed and achieved mechanical strength at six weeks after suturing, showing good local callus formation. The viability of the tendon stump, mechanical loading and intact periosteum were all found to be important factors for better callus formation at a repaired tendon-bone junction

We studied the effect of vitamin C on fracture healing in the elderly. A total of 80 elderly Osteogenic Disorder Shionogi rats were divided into four groups with different rates of vitamin C intake. A closed bilateral fracture was made in the middle third of the femur of each rat. Five weeks after fracture the femora were analysed by mechanical and histological testing. The groups with the lower vitamin C intake demonstrated a lower mechanical resistance of the healing callus and a lower histological grade. The vitamin C levels in blood during healing correlated with the torque resistance of the callus formed (r = 0.525). Therefore, the supplementary vitamin C improved the mechanical resistance of the fracture callus in elderly rats. If these results are similar in humans, vitamin C supplementation should be recommended during fracture healing in the elderly

Spinal nerve roots often sustain compression injuries. We used a Wistar rat model of the cauda equina syndrome to investigate such injuries. Rapid transient compression of the cauda equina was produced using a balloon catheter. The results were assessed by daily neurological examination and somatosensory evoked potential (SEP) recording before surgery and ten weeks after decompression. Compression of the spinal nerves induced changes in the SEP which persisted for up to ten weeks after decompression, but it had no effect on the final neurological outcome. Our study shows the importance of early surgical decompression for cauda equina syndrome

Based on a study using a retrograde neurotracer, we have previously found that the dorsal portion of the L5/6 disc in the rat is multisegmentally innervated by dorsal root ganglia (DRG) from the level of T13 to L6, and that sensory nerve fibres from DRG of T13, L1 and L2 pass through the paravertebral sympathetic trunks. In this study in newborn rats, we injected crystals of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylinedocarbocyanine perchlorate (DiI) into the DRG of T13, L1 and L2 and showed DiI-labelled sensory nerve fibres in the dorsal portion of the discs from the level of T13/L1 to L5/6. Our results show that the dorsal portion of the lumbar discs is innervated by the DRG from levels T13 to L2

Ischaemic preconditioning is a process by which exposure of a tissue to a short period of non-damaging ischaemic stress leads to resistance to the deleterious effects of a subsequent prolonged ischaemic stress. It has been extensively described in the heart, but few studies have examined the possibility that it can occur in skeletal muscle. We have used a rat model of ischaemia of one limb to examine this possibility. Exposure of the hind limb to a period of ischaemia of five minutes and reperfusion for five minutes significantly protected the tibialis anterior muscle against the structural damage induced by a subsequent period of limb ischaemia for four hours and reperfusion for one hour. This protection was evident on examination of the muscle by both light and electron microscopy. Longer or shorter times of prior ischaemia had no effect

Synthetic bone substitutes provide an alternative to autograft but do not give equivalent clinical results. Their performance may be enhanced by adding osteogenic growth factors. In this study, TGFβ1 was absorbed on to a carrier of β tricalcium phosphate and Gelfoam® and used to fill a defect around a tibial implant in a rat model of revision arthoplasty. We added 0.0, 0.02 μg, 0.1 μg or 1.0 μg of TGFβ1 to the carrier and then implanted it around an hydroxyapatite-coated stainless-steel pin in the proximal tibia of rats. The tibiae were harvested at three, six or 26 weeks and the amount of bone formation and ceramic resorption were assessed. TGFβ1 had no effect on the amount of bone in the defect, the amount of fluorescent label incorporated or the rate of mineral apposition. The growth factor did not significantly affect the amount of β TCP remaining in the tissue at any of the time points

Using a rat model, we created a bone-to-titanium interface and applied phagocytosable high-density polyethylene pArticles between the bone and implant, either initially or when the interface had matured. No fibrous membrane developed and no bone resorption was found. If sliding movements were initiated at the interface after two weeks, there was formation of a fibrous membrane. The additional application of pArticles did not change the thickness of the membrane, and there were only minor qualitative changes. Creation of a membrane by movement followed by cessation of movement and the application of pArticles caused the membrane to persist, whereas in a pArticle-free control group bone-to-metal contact was re-established. Our findings suggest that mechanical stimuli are of primary importance for prosthetic loosening, and that pArticles may modulate the later stages of the loosening process

Our aim was to develop a clinically relevant model of atrophic nonunion in the rat to test the hypothesis that the vessel density of atrophic nonunion reaches that of normal healing bone, but at a later time-point. Atrophic nonunion is usually attributed to impaired blood supply and is poorly understood. We determined the number of blood vessels at the site of an osteotomy using immunolocalisation techniques in both normally healing bones and in atrophic nonunion. At one week after operation there were significantly fewer blood vessels in the nonunion group than in the healing group. By eight weeks, the number in the atrophic nonunion group had reached the same level as that in the healing group. Our findings suggest that the number of blood vessels in atrophic nonunion reaches the same level as that in healing bone, but at a later time-point. Diminished vascularity within the first three weeks, but not at a later time-point, may prevent fractures from uniting

Little is known about the tissue reactions to various implant materials which coincide with an inflammatory reaction. We used the avridine arthritis rat model to evaluate the tissue response in the synovial, interstitial and subcutaneous tissues after implant insertion. Quantitative immunohistochemistry showed that normal joint synovial tissue is dominated by ED2-positive resident macrophages. Polyethylene implants induced a much stronger foreign-body reaction than titanium implants, as measured by the number of interfacial ED1-positive macrophages. The tissue response to titanium and polyethylene was also vastly different in arthritic synovial tissue compared with control tissue. It is likely that these biomaterials interact differently with inflammatory cells or intermediary compounds. It may be that arthritic synovial tissue produces reactive oxygen intermediates (free radicals) with which titanium has a unique anti-inflammatory interaction in vitro

Compartment syndrome is a unique form of ischaemia of skeletal muscle which occurs despite patency of the large vessels. Decompression allows the influx of activated leucocytes which cause further injury. Vitamin C is a powerful antioxidant which concentrates preferentially in leucocytes and attenuates reperfusion-induced muscle injury. We have evaluated the use of pretreatment with oral vitamin C in the prevention of injury caused by compartment syndrome in a rat cremasteric muscle model. Acute and delayed effects of pretreatment with vitamin C were assessed at one and 24 hours after decompression of compartment syndrome. Muscle function was assessed electrophysiologically. Vascular, cellular and tissue inflammation was assessed by staining of intercellular adhesion molecule-1 (ICAM-1) and by determination of the activity of myeloperoxidase (MPO) in neutrophils and tissue oedema. Compartment syndrome impaired skeletal muscle function and increased the expression of ICAM-1, activity of MPO and muscle weight increased significantly. Pretreatment with vitamin C preserved muscle function and reduced the expression of ICAM-1, infiltration of the neutrophils and oedema

Immobilisation causes denervation-like changes in the motor endplates, decreases the content of IGF-I, and increases the number of IGF-I receptors in the spinal cord. In the rat we investigated whether similar changes occur after a fracture of the midshaft of the femur which had been treated by intramedullary fixation with adequate or undersized pins. A more pronounced reduction in muscle wet weight was seen after fixation by undersized pins as well as decreased ash density of the ipsilateral tibia which did not completely return to normal within the 12-week experimental period. The nicotinic cholinergic receptors in the motor endplates of tibialis anterior were increased (p < 0.01) and there was a significant increase (p < 0.02) in IGF-I receptors in the lumbar spinal cord ipsilateral to the fracture after treatment by undersized nails. These changes may be associated with the impaired proprioception, co-ordination and motor activity which are sometimes seen after fractures

We used a rat model in vivo to study the effects of the concentration of polyethylene particles on the bone-implant interface around stable implants in the proximal tibia. Intra-articular injections of 10. 4. , 10. 6. or 10. 8. high-density polyethylene (HDPE) particles per joint were given 8, 10 and 12 weeks after surgery. The animals were killed after 14 and 26 weeks and the response at the interface determined. Fibrous tissue was seen at the bone-implant interface when the head of the implant was flush with the top of the tibia but not when it was sunk below the tibial plateau. In the latter case the implant was completely surrounded by a shell of bone. The area of fibrous tissue and that of the gap between the implant and bone was related to the concentration of particles in the 14-week group (p < 0.05). Foreign-body granulomas containing HDPE particles were seen at the bone-implant interface in animals given 10. 8. particles. The pathology resembles that seen around prostheses with aseptic loosening and we suggest that this is a useful model by which to study this process

Abundant implant-derived biomaterial wear particles are generated in aseptic loosening and are deposited in periprosthetic tissues in which they are phagocytosed by mononuclear and multinucleated macrophage-like cells. It has been stated that the multinucleated cells which contain wear particles are not bone-resorbing osteoclasts. To investigate the validity of this claim we isolated human osteoclasts from giant-cell tumours of bone and rat osteoclasts from long bones. These were cultured on glass coverslips and on cortical bone slices in the presence of particles of latex, PMMA and titanium. Osteoclast phagocytosis of these particle types was shown by light microscopy, energy-dispersive X-ray analysis and SEM. Giant cells containing phagocytosed particles were seen to be associated with the formation of resorption lacunae. Osteoclasts containing particles were also calcitonin-receptor-positive and showed an inhibitory response to calcitonin. Our findings demonstrate that osteoclasts are capable of phagocytosing particles of a wide range of size, including particles of polymeric and metallic bio-materials found in periprosthetic tissues, and that after particle phagocytosis, they remain fully functional, hormone-responsive, bone-resorbing cells

We used a rat model in vivo to study the effects of particulate bone cements at the bone-implant interface. A ceramic pin was implanted into the tibiae of 48 rats. Three types of particle of clinically relevant size were produced from one bone-cement base without radio-opacifier, with zirconium dioxide (ZrO. 2. ) and with barium sulphate (BaSO. 4. ). The rats were randomly assigned to four groups to receive one of the three bone cements or normal saline with 2% v/v Sprague-Dawley serum as the control. A total of 10. 9. particles was injected into the knee at 8, 10 and 12 weeks after the original surgery. The animals were killed at 14 weeks and the tibiae processed for histomorphometry. The area of fibrous tissue and the gap between the implant and bone were measured using image analysis. All three types of particle were associated with a larger area of bone resorption than the control. Only in the case of the BaSO. 4. -containing cement did this reach statistical significance (p = 0.01). Particles of bone cement appear to promote osteolysis at the bone-implant interface and this effect is most marked when BaSO. 4. is used as the radiopaque agent

We have previously shown that prior exposure of rat hind limbs to ischaemia for five minutes and reperfusion for five minutes reduced the structural damage to skeletal muscle which followed a subsequent period of ischaemia for four hours and reperfusion for one hour. We have now examined the potential mechanisms by which this ischaemic preconditioning protocol may be effective in reducing damage to skeletal muscle induced by prolonged ischaemia and reperfusion. Prior exposure of the hindlimb to ischaemia for five minutes and reperfusion for five minutes did not prevent the fall in the ATP content of tibialis anterior which occurred after a subsequent period of ischaemia for four hours and reperfusion for one hour. Similarly, no effect of the preconditioning protocol was seen on the elevated muscle myeloperoxidase, indicative of an elevated neutrophil content, or abnormal muscle cation content. Reperfused ischaemic muscle was also found to have an increased content of heat-shock protein (HSP) 72, but the preconditioning protocol did not further increase the content of this or other HSPs indicating that it was not acting by increasing the expression of these cytoprotective proteins. The protective effects of preconditioning appeared to be mimicked by the infusion of adenosine to animals immediately before exposure to the four-hour period, indicating a potential mechanism by which skeletal muscle may be preconditioned to maintain structural viability

The intermittent administration of parathyroid hormone (PTH) increases the formation of bone by stimulating osteoblastic activity. Our study evaluates the possibility that intermittent treatment with PTH (1-34) may also enhance the implant-bone fixation of stainless-steel screws. Twenty-eight rats received one screw in either one (n = 8) or in both (n = 20) proximal tibiae. We administered either PTH (1-34) in a dosage of 60 μg/kg/day (n = 14) or vehicle (n = 14) over a period of four weeks. At the end of this time, the degree of fixation was assessed by measuring the removal torque on one screw in each rat (n = 28) and the pull-out strength on the contralateral screw (n = 20). PTH increased the mean removal torque from 1.1 to 3.5 Ncm (p = 0.001) and the mean pull-out strength from 66 to 145 N (p = 0.002). No significant differences in body-weight or ash weight of the femora were seen. Histological examination showed that both groups had areas of soft tissue at the implant-bone interface, but these appeared less in the PTH group. These results indicate that intermittent treatment with PTH may enhance the early fixation of orthopaedic implants

Colchicine is often used in the treatment of diseases such as familial Mediterranean fever (FMF) and gout. We have previously reported that patients with FMF who had colchicine on a daily basis and who had a total hip arthroplasty showed no heterotopic ossification after surgery. The mechanism by which colchicine causes this clinical phenomenon has never been elucidated. We therefore evaluated the effect of various concentrations of colchicine on cell proliferation and mineralisation in tissue culture, using rat and human cells with and without osteogenic potential. Cell proliferation was assessed by direct cell counts and uptake of (. 3. H)thymidine, and mineralisation by measuring the amount of staining by Alizarin Red. Our findings indicate that concentrations of colchicine of up to 3 ng/ml did not affect cell proliferation but inhibition was observed at 10 to 30 ng/ml. Mineralisation decreased to almost 50%, which was the maximum inhibition observed, at concentrations of colchicine of 2.5 ng/ml. These results indicate that colchicine at low concentrations, of up to 3 ng/ml, has the capacity to inhibit selectively bone-like cell mineralisation in culture, without affecting cell proliferation. Further clinical and laboratory studies are necessary to evaluate the effects of colchicine on biological processes involving the proliferation of osteoblasts and tissue mineralisation in vivo, such as the healing of fractures, the formation of heterotopic bone and neoplastic bone growth

Mononuclear osteoclast precursors are present in the wear-particle-associated macrophage infiltrate found in the membrane surrounding loose implants. These cells are capable of differentiating into osteoclastic bone-resorbing cells when co-cultured with the rat osteoblast-like cell line, UMR 106, in the presence of 1,25(OH). 2. vitamin D. 3. In order to develop an in vitro model of osteoclast differentiation which more closely parallels the cellular microenvironment at the bone-implant interface in situ, we determined whether osteoblast-like human bone-derived cells were capable of supporting the differentiation of osteoclasts from arthroplasty-derived cells and analysed the humoral conditions required for this to occur. Long-term co-culture of arthroplasty-derived cells and human trabecular-bone-derived cells (HBDCs) resulted in the formation of numerous tartrate-resistant-acid-phosphatase (TRAP) and vitronectin-receptor (VNR)-positive multinucleated cells capable of extensive resorption of lacunar bone. The addition of 1,25(OH). 2. vitamin D. 3. was not required for the formation of osteoclasts and bone resorption. During the formation there was release of substantial levels of M-CSF and PGE. 2. Exogenous PGE. 2. (10. −8. to 10. −6. M) was found to stimulate strongly the resorption of osteoclastic bone. Our study has shown that HBDCs are capable of supporting the formation of osteoclasts from mononuclear phagocyte precursors present in the periprosthetic tissues surrounding a loose implant. The release of M-CSF and PGE. 2. by activated cells at the bone-implant interface may be important for the formation of osteoclasts at sites of pathological bone resorption associated with aseptic loosening

The multifunctional adhesion molecule CD44 is a major cell-surface receptor for hyaluronic acid (HUA). Recent data suggest that it may also bind the ubiquitous bone-matrix protein, osteopontin (OPN). Because OPN has been shown to be a potentially important protein in bone remodelling, we investigated the hypothesis that OPN interactions with the CD44 receptor on bone cells participate in the regulation of the healing of fractures. We examined the spatial and temporal patterns of expression of OPN and CD44 in healing fractures of rat femora by in situ hybridisation and immunohistochemistry. We also localised HUA in the fracture callus using biotinylated HUA-binding protein. OPN was expressed in remodelling areas of the hard callus and was found in osteocytes, osteoclasts and osteoprogenitor cells, but not in cuboidal osteoblasts which were otherwise shown to express osteocalcin. The OPN signal in osteocytes was not uniformly distributed, but was restricted to specific regions near sites where OPN mRNA-positive osteoclasts were attached to bone surfaces. In the remodelling callus, intense immunostaining for CD44 was detected in osteocyte lacunae, along canaliculi, and on the basolateral plasma membrane of osteoclasts, but not in the cuboidal osteoblasts. HUA staining was detected in fibrous tissues but little was observed in areas of hard callus where bone remodelling was progressing. Our findings suggest that OPN, rather than HUA, is the major ligand for CD44 on bone cells in the remodelling phase of healing of fractures. They also raise the possibility that such interactions may be involved in the communication of osteocytes with each other and with osteoclasts on bone surfaces. The interactions between CD44 and OPN may have important clinical implications in the repair of skeletal tissues

Extracorporeal shock-wave (ESW) treatment hasbeen shown to be effective in promoting the healing of fractures. We aimed to determine whether ESW could enhance the growth of bone-marrow osteoprogenitor cells. We applied ESW to the left femur of rats 10 mm above the knee at 0.16 mJ/mm. 2. in a range of between 250 and 2000 impulses. Bone-marrow cells were harvested after ESW for one day and subjected to assessment of colony-forming unit (CFU) granulocytes, monocytes, erythocytes, megakaryocytes (CFU-Mix), CFU-stromal cells (CFU-S) and CFU-osteoprogenitors (CFU-O). We found that the mean value for the CFU-O colonies after treatment with 500 impulses of ESW was 168.2 CFU-O/well (. sem. 11.3) compared with 88.2 CFU-O/well (. sem. 7.2) in the control group. By contrast, ESW treatment did not affect haematopoiesis as shown by the CFU-Mix (p = 0.557). Treatment with 250 and 500 impulses promoted CFU-O, but not CFU-Mix formations whereas treatment with more than 750 impulses had an inhibiting effect. Treatment with 500 impulses also enhanced the activity of bone alkaline phosphatase in the subculture of CFU-O (p< 0.01), indicating a selective promotion of growth of osteoprogenitor cells. Similarly, formation of bone nodules in the long-term culture of bone-marrow osteoprogenitor cells was also significantly enhanced by ESW treatment with 500 impulses. The mean production of TGF-β1 was 610 pg/ml (. sem. 84.6) in culture supernatants from ESW-treated rats compared with 283 pg/ml (. sem. 36.8) in the control group. Our findings suggest that optimal treatment with ESW could enhance rat bone-marrow stromal growth and differentiation towards osteoprogenitors presumably by induction of TGF-β1

Objectives

The treatment of osteoporotic fractures is a major challenge, and the enhancement of healing is critical as a major goal in modern fracture management. Most osteoporotic fractures occur at the metaphyseal bone region but few models exist and the healing is still poorly understood. A systematic review was conducted to identify and analyse the appropriateness of current osteoporotic metaphyseal fracture animal models.

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass and deterioration of bone microarchitecture, which results in increased bone fragility and fracture risk. Casein kinase 2-interacting protein-1 (CKIP-1) is a protein that plays an important role in regulation of bone formation. The effect of CKIP-1 on bone formation is mainly mediated through negative regulation of the bone morphogenetic protein pathway. In addition, CKIP-1 has an important role in the progression of osteoporosis. This review provides a summary of the recent studies on the role of CKIP-1 in osteoporosis development and treatment.

Cite this article: X. Peng, X. Wu, J. Zhang, G. Zhang, G. Li, X. Pan. The role of CKIP-1 in osteoporosis development and treatment. Bone Joint Res 2018;7:173–178. DOI: 10.1302/2046-3758.72.BJR-2017-0172.R1.

Objectives

Cellular movement and relocalisation are important for many physiologic properties. Local mesenchymal stem cells (MSCs) from injured tissues and circulating MSCs aid in fracture healing. Cytokines and chemokines such as Stromal cell-derived factor 1(SDF-1) and its receptor chemokine receptor type 4 (CXCR4) play important roles in maintaining mobilisation, trafficking and homing of stem cells from bone marrow to the site of injury. We investigated the differences in migration of MSCs from the femurs of young, adult and ovariectomised (OVX) rats and the effect of CXCR4 over-expression on their migration.

Objectives

This study aimed to examine the effects of SRT1720, a potent SIRT1 activator, on osteoarthritis (OA) progression using an experimental OA model.

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.

Objectives

Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics.

Objectives

Distraction osteogenesis (DO) mobilises bone regenerative potential and avoids the complications of other treatments such as bone graft. The major disadvantage of DO is the length of time required for bone consolidation. Mesenchymal stem cells (MSCs) have been used to promote bone formation with some good results.

Aims

We aimed to evaluate the temperature around the nerve root during drilling of the lamina and to determine whether irrigation during drilling can reduce the chance of nerve root injury.

Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect.

Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1.

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.

Objectives

Microindentation has the potential to measure the stiffness of an individual patient’s bone. Bone stiffness plays a crucial role in the press-fit stability of orthopaedic implants. Arming surgeons with accurate bone stiffness information may reduce surgical complications including periprosthetic fractures. The question addressed with this systematic review is whether microindentation can accurately measure cortical bone stiffness.

Objectives

This study aimed to investigate the functional effects of microRNA (miR)-214-5p on osteoblastic cells, which might provide a potential role of miR-214-5p in bone fracture healing.

Objectives

Ultraviolet (UV) light-mediated photofunctionalisation is known to improve osseointegration of pure titanium (Ti). However, histological examination of titanium alloy (Ti6Al4V), which is frequently applied in orthopaedic and dental surgery, has not yet been performed. This study examined the osseointegration of photofunctionalised Ti6Al4V implants.

Objectives

Infection of implants is a major problem in elective and trauma surgery. Heating is an effective way to reduce the bacterial load in food preparation, and studies on hyperthermia treatment for cancer have shown that it is possible to heat metal objects with pulsed electromagnetic fields selectively (PEMF), also known as induction heating. We therefore set out to answer the following research question: is non-contact induction heating of metallic implants effective in reducing bacterial load in vitro?

Objectives

This systematic review aimed to assess the in vivo and clinical effect of strontium (Sr)-enriched biomaterials in bone formation and/or remodelling.

This review is aimed at clinicians appraising preclinical trauma studies and researchers investigating compromised bone healing or novel treatments for fractures. It categorises the clinical scenarios of poor healing of fractures and attempts to match them with the appropriate animal models in the literature.

We performed an extensive literature search of animal models of long bone fracture repair/nonunion and grouped the resulting studies according to the clinical scenario they were attempting to reflect; we then scrutinised them for their reliability and accuracy in reproducing that clinical scenario.

Models for normal fracture repair (primary and secondary), delayed union, nonunion (atrophic and hypertrophic), segmental defects and fractures at risk of impaired healing were identified. Their accuracy in reflecting the clinical scenario ranged greatly and the reliability of reproducing the scenario ranged from 100% to 40%.

It is vital to know the limitations and success of each model when considering its application.

Objectives

The major problem with repair of an articular cartilage injury is the extensive difference in the structure and function of regenerated, compared with normal cartilage. Our work investigates the feasibility of repairing articular osteochondral defects in the canine knee joint using a composite lamellar scaffold of nano-ß-tricalcium phosphate (ß-TCP)/collagen (col) I and II with bone marrow stromal stem cells (BMSCs) and assesses its biological compatibility.

Objectives

Triamcinolone acetonide (TA) is widely used for the treatment of rotator cuff injury because of its anti-inflammatory properties. However, TA can also produce deleterious effects such as tendon degeneration or rupture. These harmful effects could be prevented by the addition of platelet-rich plasma (PRP), however, the anti-inflammatory and anti-degenerative effects of the combined use of TA and PRP have not yet been made clear. The objective of this study was to determine how the combination of TA and PRP might influence the inflammation and degeneration of the rotator cuff by examining rotator cuff-derived cells induced by interleukin (IL)-1ß.

Objectives

The cytotoxicity induced by cobalt ions (Co²⁺) and cobalt nanoparticles (Co-NPs) which released following the insertion of a total hip prosthesis, has been reported. However, little is known about the underlying mechanisms. In this study, we investigate the toxic effect of Co²⁺ and Co-NPs on liver cells, and explain further the potential mechanisms.

Objectives

Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues.

Objectives

Recent studies have shown that systemic injection of rapamycin can prevent the development of osteoarthritis (OA)-like changes in human chondrocytes and reduce the severity of experimental OA. However, the systemic injection of rapamycin leads to many side effects. The purpose of this study was to determine the effects of intra-articular injection of Torin 1, which as a specific inhibitor of mTOR which can cause induction of autophagy, is similar to rapamycin, on articular cartilage degeneration in a rabbit osteoarthritis model and to investigate the mechanism of Torin 1’s effects on experimental OA.

Objectives

To investigate the appropriate dose and interval for the administration of triamcinolone acetonide (TA) in treating tendinopathy to avoid adverse effects such as tendon degeneration and rupture.

Objectives

Electromagnetic fields (EMF) are widely used in musculoskeletal disorders. There are indications that EMF might also be effective in the treatment of osteoporosis. To justify clinical follow-up experiments, we examined the effects of EMF on bone micro-architectural changes in osteoporotic and healthy rats. Moreover, we tested the effects of EMF on fracture healing.

The treatment of osteochondral lesions and osteoarthritis remains an ongoing clinical challenge in orthopaedics. This review examines the current research in the fields of cartilage regeneration, osteochondral defect treatment, and biological joint resurfacing, and reports on the results of clinical and pre-clinical studies. We also report on novel treatment strategies and discuss their potential promise or pitfalls. Current focus involves the use of a scaffold providing mechanical support with the addition of chondrocytes or mesenchymal stem cells (MSCs), or the use of cell homing to differentiate the organism’s own endogenous cell sources into cartilage. This method is usually performed with scaffolds that have been coated with a chemotactic agent or with structures that support the sustained release of growth factors or other chondroinductive agents. We also discuss unique methods and designs for cell homing and scaffold production, and improvements in biological joint resurfacing. There have been a number of exciting new studies and techniques developed that aim to repair or restore osteochondral lesions and to treat larger defects or the entire articular surface. The concept of a biological total joint replacement appears to have much potential.

Cite this article: Bone Joint Res 2013;2:193–9.

In order to clarify the role of cytokines in the remodelling of the grafted tendon for ligament reconstruction we compared the responses to interleukin (IL)-1β, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-β1 of extrinsic fibroblasts infiltrating the frozen-thawed patellar tendon in rats with that of the normal tendon fibroblasts, in regard to the gene expression of matrix metalloproteinase (MMP)-13, using Northern blot analysis. We also examined, immunohistologically, the local expression of IL-1β, PDGF-BB, and TGF-β1 in fibroblasts infiltrating the frozen-thawed patellar tendon.

Northern blot analysis showed that fibroblasts derived from the patellar tendon six weeks after the freeze-thaw procedure in situ showed less response to IL-1β than normal tendon fibroblasts with respect to MMP-13 mRNA gene expression. The immunohistological findings revealed that IL-1β was over-expressed in extrinsic fibroblasts which infiltrated the patellar tendon two and six weeks after the freeze-thaw procedure in situ, but neither PDGF-BB nor TGF-β1 was over-expressed in these extrinsic fibroblasts. Our findings indicated that IL-1β had a close relationship to matrix remodelling of the grafted tendon for ligament reconstruction, in addition to the commencement of inflammation during the tissue-healing process.

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic.

Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation.

While the evolution of the bony skeleton of the shoulder girdle is well described, there is little information regarding the soft tissues, in particular of the rotator cuff. We dissected the shoulders of 23 different species and compared the anatomical features of the tendons of the rotator cuff. The alignment and orientation of the collagen fibres of some of the tendons were also examined histologically. The behaviour of the relevant species was studied, with particular reference to the extent and frequency of forward-reaching and overhead activity of the forelimb.

In quadrupedal species, the tendons of supraspinatus, infraspinatus and teres minor were seen to insert into the greater tuberosity of the humerus separately. They therefore did not form a true rotator cuff with blending of the tendons. This was only found in advanced primates and in one unusual species, the tree kangaroo. These findings support the suggestion that the appearance of the rotator cuff in the evolutionary process parallels anatomical adaptation to regular overhead activity and the increased use of the arm away from the sagittal plane.

This study was designed to test the hypothesis that the sensory innervation of bone might play an important role in sensing and responding to low-intensity pulsed ultrasound and explain its effect in promoting fracture healing. In 112 rats a standardised mid-shaft tibial fracture was created, supported with an intramedullary needle and divided into four groups of 28. These either had a sciatic neurectomy or a patellar tendon resection as control, and received the ultrasound or not as a sham treatment. Fracture union, callus mineralisation and remodelling were assessed using plain radiography, peripheral quantitative computed tomography and histomorphology.

Daily ultrasound treatment significantly increased the rate of union and the volumetric bone mineral density in the fracture callus in the neurally intact rats (p = 0.025), but this stimulating effect was absent in the rats with sciatic neurectomy. Histomorphology demonstrated faster maturation of the callus in the group treated with ultrasound when compared with the control group. The results supported the hypothesis that intact innervation plays an important role in allowing low-intensity pulsed ultrasound to promote fracture healing.

Ketamine has been used in combination with a variety of other agents for intra-articular analgesia, with promising results. However, although it has been shown to be toxic to various types of cell, there is no available information on the effects of ketamine on chondrocytes.

We conducted a prospective randomised controlled study to evaluate the effects of ketamine on cultured chondrocytes isolated from rat articular cartilage. The cultured cells were treated with 0.125 mM, 0.250 mM, 0.5 mM, 1 mM and 2 mM of ketamine respectively for 6 h, 24 hours and 48 hours, and compared with controls. Changes of apoptosis were evaluated using fluorescence microscopy with a 490 nm excitation wavelength. Apoptosis and eventual necrosis were seen at each concentration. The percentage viability of the cells was inversely proportional to both the duration and dose of treatment (p = 0.002 and p = 0.009). Doses of 0.5 mM, 1 mM and 2mM were absolutely toxic.

We concluded that in the absence of solid data to support the efficacy of intra-articular ketamine for the control of pain, and the toxic effects of ketamine on cultured chondrocytes shown by this study, intra-articular ketamine, either alone or in combination with other agents, should not be used to control pain.

Cite this article: Bone Joint J 2014; 96-B:989–94.

The haematoma occurring at the site of a fracture is known to play an important role in bone healing. We have recently shown the presence of progenitor cells in human fracture haematoma and demonstrated that they have the capacity for multilineage mesenchymal differentiation. There have been many studies which have shown that low-intensity pulsed ultrasound (LIPUS) stimulates the differentiation of a variety of cells, but none has investigated the effects of LIPUS on cells derived from human fracture tissue including human fracture haematoma-derived progenitor cells (HCs). In this in vitro study, we investigated the effects of LIPUS on the osteogenic activity of HCs. Alkaline phosphatase activity, osteocalcin secretion, the expression of osteoblast-related genes and the mineralisation of HCs were shown to be significantly higher when LIPUS had been applied but without a change in the proliferation of the HCs. These findings provide evidence in favour of the use of LIPUS in the treatment of fractures.

We investigated the effect of progesterone on the nerve during lengthening of the limb in rats. The sciatic nerves of rats were elongated by leg lengthening for ten days at 3 mm per day. On alternate days between the day after the operation and nerve dissection, the progesterone-treated group received subcutaneous injections of 1 mg progesterone in sesame oil and the control group received oil only. On the fifth, tenth and 17th day, the sciatic nerves were excised at the midpoint of the femur and the mRNA expression level of myelin protein P0 was analysed by quantitative real time polymerase chain reaction. On day 52 nodal length was examined by electron microscopy, followed by an examination of the compound muscle action potential (C-MAP) amplitude and the motor conduction velocity (MCV) of the tibial nerve on days 17 and 52. The P0 (a major myelin glycoprotein) mRNA expression level in the progesterone-treated group increased by 46.6% and 38.7% on days five and ten, respectively. On day 52, the nodal length in the progesterone-treated group was smaller than that in the control group, and the MCV of the progesterone-treated group had been restored to normal.

Progesterone might accelerate the restoration of demyelination caused by nerve elongation by activating myelin synthesis.

We investigated the effect of mitomycin-C on the reduction of the formation of peritendinous fibrous adhesions after tendon repair. In 20 Wistar albino rats the tendo Achillis was cut and repaired using a modified Kessler technique. The rats were divided into two equal groups. In group 1, an injection of mitomycin-C was placed between the tendon and skin of the right leg. In group 2, an identical volume of sterile normal saline was injected on the left side in a similar fashion. All the rats received mitomycin-C or saline for four weeks starting from the day of operation. The animals were killed after 30 days. The formation of peritendinous fibrous tissue, the inflammatory reaction and tendon healing were evaluated. The tensile strength of the repaired tendons was measured biomechanically. Microscopic evidence of the formation of adhesions and inflammation was less in group 1. There was no significant difference in the tensile load required to rupture the repaired tendons in the two groups.

Mitomycin-C may therefore provide a simple and inexpensive means of preventing of post-operative adhesions.

We have examined the deterioration of implant fixation after withdrawal of parathyroid hormone (PTH) in rats. First, the pull-out force for stainless-steel screws in the proximal tibia was measured at different times after withdrawal. The stimulatory effect of PTH on fixation was lost after 16 days. We then studied whether bisphosphonates could block this withdrawal effect. Mechanical and histomorphometric measurements were conducted for five weeks after implantation. Subcutaneous injections were given daily. Specimens treated with either PTH or saline during the first two weeks showed no difference in the mechanical or histological results (pull-out force 76 N vs 81 N; bone volume density 19% vs 20%). Treatment with PTH for two weeks followed by pamidronate almost doubled the pull-out force (152 N; p < 0.001) and the bone volume density (37%; ANOVA, p < 0.001). Pamidronate alone did not have this effect (89 N and 25%, respectively). Thus, the deterioration can be blocked by bisphosphonates. The clinical implications are discussed.

We investigated the effect of locally administered bisphosphonate on distraction osteogenesis in a rabbit model and evaluated its systemic effect. An osteotomy on the right tibia followed by distraction for four weeks was performed on 47 immature rabbits. They were divided into seven equal groups, with each group receiving a different treatment regime. Saline and three types of dosage of alendronate (low, 0.75 μg/kg; mid, 7.5 μg/kg and high 75 μg/kg) were given by systemic injection in four groups, and saline and two dosages (low and mild) were delivered by local injection to the distraction gap in the remaining three groups. The injections were performed five times weekly during the period of distraction.

After nine weeks the animals were killed and image analysis and mechanical testing were performed on the distracted right tibiae and the left tibiae which served as a control group. The local low-dose alendronate group showed a mean increase in bone mineral density of 124.3 mg/cm³ over the local saline group (analysis of variance, p < 0.05) without any adverse effect on the left control tibiae.

The findings indicate that the administration of local low-dose alendronate could be an effective pharmacological means of improving bone formation in distraction osteogenesis.

Using the transverse processes of fresh porcine lumbar spines as an experimental model we evaluated the heat generated by a rotating burr of a high-speed drill in cutting the bone. The temperature at the drilled site reached 174°C with a diamond burr and 77°C with a steel burr. With water irrigation at a flow rate of 540 ml/hr an effective reduction in the temperature was achieved whereas irrigation with water at 180 ml/hr was much less effective. There was a significant negative correlation between the thickness of the residual bone and the temperature measured at its undersurface adjacent to the drilling site (p < 0.001).

Our data suggest that tissues neighbouring the drilled bone, especially nerve roots, can be damaged by the heat generated from the tip of a high-speed drill. Nerve-root palsy, one of the most common complications of cervical spinal surgery, may be caused by thermal damage to nerve roots arising in this manner.

We studied the effects of coating titanium implants with teicoplanin and clindamycin in 30 New Zealand White rabbits which were randomly assigned to three groups. The intramedullary canal of the left tibia of each rabbit was inoculated with 500 colony forming units of Staphylococcus aureus. Teicoplanin-coated implants were implanted into rabbits in group 1, clindamycin-coated implants into rabbits in group 2, and uncoated implants into those in group 3. All the rabbits were killed one week later. The implants were removed and cultured together with pieces of tibial bone and wound swabs. The rate of colonisation of the organisms in the three groups was compared.

Organisms were cultured from no rabbits in group 1, one in group 2 but from all in group 3. There was no significant difference between groups 1 and 2 (p = 1.000). There were significant differences between groups 1 and 3 and groups 2 and 3 (p < 0.001). Significant protection against bacterial colonisation and infection was found with teicoplanin- and clindamycin-coated implants in this experimental model.

Objectives

The period of post-operative treatment before surgical wounds are completely closed remains a key window, during which one can apply new technologies that can minimise complications. One such technology is the use of negative pressure wound therapy to manage and accelerate healing of the closed incisional wound (incisional NPWT).

We have developed an animal model to examine the formation of heterotopic ossification using standardised muscular damage and implantation of a beta-tricalcium phosphate block into a hip capsulotomy wound in Wistar rats. The aim was to investigate how cells originating from drilled femoral canals and damaged muscles influence the formation of heterotopic bone. The femoral canal was either drilled or left untouched and a tricalcium phosphate block, immersed either in saline or a rhBMP-2 solution, was implanted. These implants were removed at three and 21 days after the operation and examined histologically, histomorphometrically and immunohistochemically.

Bone formation was seen in all implants in rhBMP-2-immersed, whereas in those immersed in saline the process was minimal, irrespective of drilling of the femoral canals. Bone mineralisation was somewhat greater in the absence of drilling with a mean mineralised volume to mean total volume of 18.2% (sd 4.5) versus 12.7% (sd 2.9, p < 0.019), respectively.

Our findings suggest that osteoinductive signalling is an early event in the formation of ectopic bone. If applicable to man the results indicate that careful tissue handling is more important than the prevention of the dissemination of bone cells in order to avoid heterotopic ossification.

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff.

We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis).

The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears.

These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.

Conventional non-steroidal anti-inflammatory drugs (NSAIDs) and newer specific cyclo-oxygenase-2 (cox-2) inhibitors are commonly used in musculoskeletal trauma and orthopaedic surgery to reduce the inflammatory response and pain. These drugs have been reported to impair bone metabolism. In reconstruction of the anterior cruciate ligament the hamstring tendons are mainly used as the graft of choice, and a prerequisite for good results is healing of the tendons in the bone tunnel. Many of these patients are routinely given NSAIDs or cox-2 inhibitors, although no studies have elucidated the effects of these drugs on tendon healing in the bone tunnel.

In our study 60 female Wistar rats were randomly allocated into three groups of 20. One received parecoxib, one indometacin and one acted as a control. In all the rats the tendo-Achillis was released proximally from the calf muscles. It was then pulled through a drill hole in the distal tibia and sutured anteriorly. The rats were given parecoxib, indometacin or saline intraperitoneally twice daily for seven days. After 14 days the tendon/bone-tunnel interface was subjected to mechanical testing.

Significantly lower maximum pull-out strength (p < 0.001), energy absorption (p < 0.001) and stiffness (p = 0.035) were found in rats given parecoxib and indometacin compared with the control group, most pronounced with parecoxib.

Objectives

An experimental rabbit model was used to test the null hypothesis, that there is no difference in new bone formation around uncoated titanium discs compared with coated titanium discs when implanted into the muscles of rabbits.

Injuries to the spinal cord may be associated with increased healing of fractures. This can be of benefit, but excessive bone growth can also cause considerable adverse effects.

We evaluated two groups of patients with fractures of the spinal column, those with neurological compromise (n = 10) and those without (n = 15), and also a control group with an isolated fracture of a long bone (n = 12). The level of transforming growth factor-beta (TGF-β), was measured at five time points after injury (days 1, 5, 10, 42 and 84).

The peak level of 142.79 ng/ml was found at day 84 in the neurology group (p < 0.001 vs other time points). The other groups peaked at day 42 and had a decrease at day 84 after injury (p ≤ 0.001).

Our findings suggest that TGF-β may have a role in the increased bone turnover and attendant complications seen in patients with acute injuries to the spinal cord.