Re-rupture rates after rotator cuff repair remain high because of inadequate biological healing at the tendon-bone interface. Single-growth factor therapies to augment healing at the enthesis have so far yielded inconsistent results. An emerging approach is to combine multiple growth factors over a spatiotemporal distribution that mimics normal healing. We propose a novel combination treatment of insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1) and parathyroid hormone (PTH) incorporated into a controlled-release tyraminated poly-vinyl-alcohol hydrogel to improve healing after rotator cuff repair. We aimed to evaluate this growth factor treatment in a rat chronic rotator cuff tear model. A total of 30 male Sprague-Dawley rats underwent unilateral supraspinatus tenotomy. Delayed rotator cuff repairs were then performed after 3 weeks, to allow tendon degeneration that resembles the human clinical scenario. Animals were randomly assigned to: [1] a control group with repair alone; or [2] a treatment group in which the hydrogel was applied at the repair site. All animals were euthanized 12 weeks after rotator cuff surgery and the explanted shoulders were analyzed for biomechanical strength and histological quality of healing at the repair site. In the treatment group had significantly higher stress at failure (73% improvement, P=0.003) and Young's modulus (56% improvement, P=0.028) compared to the control group. Histological assessment revealed improved healing with significantly higher overall histological scores (10.1 of 15 vs 6.55 of 15, P=0.032), and lower inflammation and vascularity. This novel combination growth factor treatment improved the quality of healing and strength of the repaired enthesis in a chronic rotator cuff tear model. Further optimization and tailoring of the growth factors hydrogel is required prior to consideration for clinical use in the treatment of rotator cuff tears. This novel treatment approach holds promise for improving biological healing of this clinically challenging problem

Altered mechanical loading is a widely suggested, but poorly understood potential cause of cartilage degeneration in osteoarthritis. In rodents, osteoarthritis is induced following destabilization of the medial meniscus (DMM). This study estimates knee kinematics and contact forces in rats with DMM to gain better insight into the specific mechanisms underlying disease development in this widely-used model. Unilateral knee surgery was performed in adult male Sprague-Dawley rats (n=5 with DMM, n=5 with sham surgery). Radio-opaque beads were implanted on their femur and tibia. 8 weeks following knee surgery, rat gait was recorded using the 3D²YMOX setup (Sanctorum et al. 2019, simultaneous acquisition of biplanar XRay videos and ground reaction forces). 10 trials (1 per rat) were calibrated and processed in XMALab (Knörlein et al. 2016). Hindlimb bony landmarks were labeled on the XRay videos using transfer learning (Deeplabcut, Mathis et al. 2019; Laurence-Chasen et al. 2020). A generic OpenSim musculoskeletal model of the rat hindlimb (Johnson et al. 2008) was adapted to include a 3-degree-of-freedom knee. Inverse kinematics, inverse dynamics, static optimization of muscle forces, and joint reaction analysis were performed. In rats with DMM, knee adduction was lower compared to sham surgery. Ground reaction forces were less variable with DMM, resulting in less variability in joint external moments. The mediolateral ground reaction force was lower, resulting in lower hip adduction moment, thus less force was produced by the rectus femoris. Rats with DMM tended to break rather than propel, resulting in lower hip flexion moment, thus less force was produced by the semimembranosus. These results are consistent with lower knee contact forces in the anteroposterior and axial directions. These preliminary data indicate no overloading of the knee joint in rats with DMM, compared with sham surgery. We are currently expanding our workflow to finite element analysis, to examine mechanical cues in the cartilage of these rats (Fig1G)

To evaluate the therapeutic effect of Pulsed Electromagnetic Field (PEMF) in the treatment of meniscal tears in the avascular region. Seventy-two twelve-week-old male Sprague-Dawley rats with full-thickness longitudinal medial meniscal tears in the avascular region were divided into 3 groups: control group (G. con. ), treated with classic signal PEMF (G. classic. ), and high slew rate signal PEMF(G. HSR. ). The HSR signal has the same pulse and burst frequencies as the classic signal, but with a higher slew rate. Macroscopic observation and histological analysis of the meniscus and articular cartilage were performed to evaluate the meniscal healing and progressions of osteoarthritis. The synovium was harvested for histological and immunofluorescent analysis to assess the intra-articular inflammation. The meniscal healing, articular cartilage degeneration, and synovitis were quantitatively evaluated according to their respective scoring system. Dramatic degenerative changes of the meniscus and articular cartilage were noticed during gross observation and histological evaluation in the control group at 8 weeks. However, the menisci in the two treatment groups were restored to normal morphology with a smooth surface and shiny white color. Particularly, the HSR signal remarkably enhanced the fibrochondrogenesis and accelerated the remodeling process of the regenerated tissue. The meniscal healing scores of PEMF treatment groups were significantly higher than those in the control group at 8 weeks. Specifically, the HSR signal showed a significantly higher meniscal repair score than the classic signal at week 8 (P < .01). The degeneration score (G. con. versus G. classic. : P < .0001; Gcon versus G. HSR. : P < .0001) and synovitis score (G. con. versus Gclassic: P < .0001; G. con. versus G. HSR. : P = .0002) of the control groups were significantly higher than those in the two treatment groups. PEMF promoted the healing of meniscal tears in the avascular region and restored the injured meniscus to its structural integrity in a rat model. Compared to the classic signal, the HSR signal showed the increased capability to promote fibrocartilaginous tissue formation and modulate the inflammatory environment and therefore protected the knee joint from post-traumatic osteoarthritis development

Obesity is associated with poor outcomes and increased risk of failure after rotator cuff (RC) repair surgery. The effect of diet-induced obesity (DIO) on enthesis healing has not been well characterised and whether its effects can be reversed with dietary intervention is unknown. We hypothesised that DIO would result in inferior enthesis healing in a rat model of RC repair and that dietary intervention in the peri-operative period would improve enthesis healing. A total of 78 male Sprague-Dawley rats were divided into three weight-matched groups from weaning and fed either: control diet (CD), high-fat diet (HFD), or HFD until surgery, then CD thereafter (HF-CD). After 12 weeks the left supraspinatus tendon was detached, followed by immediate surgical repair. At 2 and 12 weeks post-surgery, animals were cullers and RCs harvested for biomechanical and histological evaluation. Body composition and metabolic markers were assessed via DEXA and plasma analyses, respectively. DIO was established in the HFD and HF-CD groups prior to surgery, and subsequently reversed in the HF-CD group after surgery. At 12 weeks post-surgery, plasma leptin concentrations were higher in the HFD group compared to the CD group (5.28 vs. 2.91ng/ml, P=0.003). Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD compared to the CD group at 12 weeks (overall histological score 6.20 (P=0.008), 4.98 (P=0.001) and 8.68 out of 15, respectively). The repaired entheses in the HF-CD group had significantly lower (26.4 N, P=0.028) load-at-failure 12 weeks post-surgery compared to the CD group (34.4 N); while the HFD group was low, but not significantly different (28.1 N, P=0.096). Body mass at the time of surgery, plasma leptin and body fat percentage were negatively correlated with histological scores and plasma leptin with load-at-failure 12 weeks post-surgery. DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring normal weight with dietary change after surgery did not improve healing outcomes. Exploring interventions that improve the metabolic state of obese patients and counselling patients appropriately about their modest expectations after repair should be considered

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

Objectives. 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

In a rabbit model we investigated the efficacy of a silk fibroin/hydroxyapatite (SF/HA) composite on the repair of a segmental bone defect. Four types of porous SF/HA composites (SF/HA-1, SF/HA-2, SF/HA-3, SF/HA-4) with different material ratios, pore sizes, porosity and additives were implanted subcutaneously into Sprague-Dawley rats to observe biodegradation. SF/HA-3, which had characteristics more suitable for a bone substitite based on strength and resorption was selected as a scaffold and co-cultured with rabbit bone-marrow stromal cells (BMSCs). A segmental bone defect was created in the rabbit radius. The animals were randomised into group 1 (SF/HA-3 combined with BMSCs implanted into the bone defect), group 2 (SF/HA implanted alone) and group 3 (nothing implanted). They were killed at four, eight and 12 weeks for visual, radiological and histological study. The bone defects had complete union for group 1 and partial union in group 2, 12 weeks after operation. There was no formation of new bone in group 3. We conclude that SF/HA-3 combined with BMSCs supports bone healing and offers potential as a bone-graft substitute

Background. Multiple Myeloma is a hematological malignancy of terminally differentiated plasma cells associated with increased osteoclast activity and decreased osteoblast functions. Systemic antiproliferative treatment includes proteasome inhibitors such as bortezomib, a clinical potent antimyeloma agent. Local delivery of biological active molecules via biomaterial composite implants to the site of the lesion has been shown to be beneficial for bone and implant-associated infections. In anticancer treatment local delivery of anticancer agents to the neoplasia via biomaterial carriers has never been reported before. The purpose of the current is to present the concepts and the first in vivo results for proteasome inhibitor composite biomaterials for local delivery of bortezomib to proliferative multiple myeloma bone lesions including concentration measurements at different anatomical regions in a rat model. Methods. 80 female Sprague-Dawley rats were randomised into five different treatment groups (n=16/group): 1) Empty (2) Xerogel-granulat: XG (3) Xerogel-granulat+100mgbortezomib [b]: XG100b (4) Xerogel-granulat+500mgb:XG500b (5) Xerogel-granulat+2500mgb:XG2500b. A 2.5 mm drill hole was then created in the metaphysis of the left femur. The defect was then either filled with the previously mentioned substitutes or left empty to serve as a control. After 4 weeks femora were harvested followed by histological, histomorphometrical and immunohistochemical (BMP2; bone-morphogenic protein 2, OPG; osteoprotegerin, RANKL; Receptor activator of nuclear factor kappa-B ligand, ASMA; alpha smooth muscle actin, ED1;CD68 antibody). TOF-SIMS was used to assess the distribution of released strontium ions. Statistical analysis was done using SPSS software. Data was not found normally distributed and hence Mann-Whitney U with bonferroni correction was used. To avoid type I errors due to unequal variances and group sizes Games-Howell test was also performed

The biomechanical evaluation of tendon repair with collagen-based scaffolds in rat model is a common method to determine the functional outcome of the tested material. We introduced a magnetic resonance imaging (MRI) approach to verify the biomechanical test data. In present study different collagen scaffolds for tendon repair were examined. Two collagen test materials: based on bovine stabilized collagen, chemically cross-linked with oriented collagenous fibres (material 1) and based on porcine dermal extracellular matrix, with no cross-linking (material 2) were compared. The animal study was approved by the local review board. Surgery was performed on male Sprague-Dawley rats with a body weight of 400 ± 19 g. Each rat underwent a 5 mm transection of the right Achilles tendon. The M. plantaris tendon was removed. The remaining tendon ends were re-joined with a 5 mm scaffold of either the material 1 or 2. Each scaffold material was sutured into place with two single stiches (Vicryl 4–0, Ethicon) each end. A total of 16 rats (n= 8 each group) were observed for 28 days follow up. The animals were sacrificed and hind limbs were transected proximal to the knee joint. MRI was performed using a 7 Tesla scanner (BioSpec 70/30, Bruker). T2-weighted TurboRARE sequences with an in-plane resolution of 0.12 mm and a slice thickness of 0.7 mm were analysed. All soft and hard tissues were removed from the Achilles tendon-calcaneus-foot complex before biomechanical testing. Subsequently, the specimens were fixed in a materials testing machine (Z1.0, Zwick, Ulm, Germany) for tensile testing. All tendons were preloaded with 1 N and subsequently stretched at a rate of 1 mm/s until complete failure was observed. Non-operated tendons were used as a control (n=4). After 28 postoperative days, MRI demonstrated that four scaffolds (material 1: n=2, material 2: n=2) were slightly dislocated in the proximal part of hind limb. In total five failures of reconstruction could be detected in the tendon repairs (material 1: n=3, material 2: n=2). Tendons augmented with the bovine material 1 showed a maximum tensile load of 57.9 ± 17.9 N and tendons with porcine scaffold material 2 of 63.1 ± 19.5 N. The native tendons demonstrated only slightly higher loads of 76.6 ± 11.6 N. Maximum failure load of the tendon-scaffold construct in both groups did not differ significantly (p < 0.05). Stiffness of the tendons treated with the bovine scaffold (9.9 ± 3.6 N/mm) and with the porcine scaffold (10.7 ± 2.7 N/mm) showed no differences. Stiffness of the native healthy tendon of the contralateral site was significantly higher (20.2 ± 6.6 N/mm, p < 0.05). No differences in the mechanical properties between samples of both scaffold groups could be detected, regardless of whether the repaired tendon defect has failed or the scaffold has been dislocated. The results show that MRI is important as an auxiliary tool to verify the biomechanical outcome of tendon repair in animal models

We have compared the rates of infection and resistance in an animal model of an orthopaedic procedure which was contaminated with a low-dose inoculum of Staphylococcus epidermidis. We randomised 44 Sprague-Dawley rats to have bone cement implanted subcutaneously containing either gentamicin or saline (control). The wound was inoculated with a dilute solution of gentamicin-sensitive Staphylococcus epidermidis. At two weeks the cement was retrieved and microbiologically tested. A lower overall rate of infection was seen in the gentamicin-loaded cement group, but there was a significantly higher rate of gentamicin-resistant infection in this group (Fisher’s exact test, p < 0.01). Antibiotic-impregnated cement has an optimum surface for colonisation and prolonged exposure to antibiotic allows mutational resistance to occur. Gentamicin-loaded cement may not be appropriate for revision surgery if it has been used already in previous surgery

Objectives. Osteoporosis and osteomalacia lead to increased fracture risk. Previous studies documented dysregulated osteoblast and osteoclast activity, leading to a high-turnover phenotype, reduced bone mass and low bone mineral content. Osteocytes, the most abundant bone cell type, are involved in bone metabolism by enabling cell to cell interaction. Osteocytes presence and viability are crucial for bone tissue homeostasis and mechanical integrity. Osseo-integration and implant degradation are the main problems in developing biomaterials for systemically diseased bone. This study examines osteocyte localisation, morphology and on the implant surface and at the implant bone interface. Furthermore, the study investigates ECM proteins regulation correlated to osteocytes and mechanical competence in an ovariectomised rat model with a critical size metaphyseal defect. Methodology. After induction of osteoporosis, 60 female Sprague-Dawley rats were randomised into five groups: SrCPC (n=15), CPC (n=15), ScB30 (n=15), ScB30Sr20 (n=15) and empty defect (n=15). The left femur of all animals underwent a 4mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with the above mentioned implants or left empty. After six weeks, histomorphometric analysis showed a statistically significant increase in bone formation at the tissue-implant interface in the SrCPC group compared to the other groups (p<0.01). Osteocyte morphology and networks were detected using silver and staining. ECM proteins were investigated through immunohistochemistry. Cellular populations were tested using enzyme histochemistry. Mineralisation was assessed using time of flight secondary ion mass spectrometry (TOF-SIMS). Statistical analysis was performed using Mann Whitney U test with Bonferroni correction. Results. In the SrCPC and compared to other test groups, osteocytes presence and morphology was enhanced. An increased osteocytic activity was also seen in ScB30Sr20 when compared to SCB30 alone. Local osteomalatic lesions characterised by the presence of excessive unmineralised osteoid as revealed by the VKVG staining in the intact bone was also seen. A regular pattern of osteocytes distribution reflecting a better bone maturation was also seen in case of the Sr substituted cements. Whereas in case of the ScB30 degenerated osteocytes with a comparatively irregular arrangement were seen. Nonetheless, ECM proteins indicating discrepant bone turnover (RANKL, OPG, BMP2, OCN; ASMA) were noticed to increase within these regions and were accompanied by the presence of apoptotic osteocytes. Interestingly, osteocytes were also localised near the blood vessels within the newly formed woven bone. On the other hand, osteocytes allocation at implant bone interface and on the implant surface were qualitatively better in the Sr substituted groups when compared to the other test groups. Furthermore, this correlates with healing enhancement and implant retention results obtained from the histomorphometry (BV/TV and Osteoclasts count). The first qualitative results of the sclerostin visualisation showed a lower expression in the Sr supplemented biomaterials compared to the Sr free ones. Conclusion. Osteoblasts, osteoclast and osteocytes are the key players to bone metabolism through production and mineralisation of ECM or resorption. The current study indicates the importance in therapeutically targeting osteocytes to regulate bone metabolism in osteoporotic/osteomalatic bone. Sr inhibits osteoclast activity which is important for implant degradation. However, in osteoporotic bone osteoclasts inhibition is crucial to enhance the healing. Our data suggest that osteocytes allocation at the bone implant interface and on the implant surface is aiding in implant degradation through osteocytes dependent resorption. Currently, discrepancies in mechanosensitivity, proliferation and fibrotic tissue formation are being investigated together with several anchorage proteins to quench further effects of osteocyte presence at the implant bone interface

Introduction. Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy affecting approximately 3 million people in China (Stone R, 2009). The precise aetiology of KBD is not clear, but the lack of selenium and the pollution of mycotoxins in food are a suspected cause of KBD. In this pilot study, we use a rat model to investigate the effect of low selenium and T-2 toxin on articular cartilage metabolism. Methods. 140 male Sprague-Dawley rats were fed with selenium-deficient or normal diet for 4 weeks to produce a low selenium or normal nutrition status. The rats were then fed for a further 4 weeks with low selenium or normal diets with or without T-2 toxin (100ng per gram body weight per day). The rat knee joints were fixed and paraffin embedded and histological and immunohistochemical staining was performed to analyse the metabolism of articular cartilage. Results. There was increased cell cluster formation in the middle and/or deep zones in rats fed with both diets. However, an apparent cell loss was observed in the low selenium + T-2 toxin group with an apparent increase in caspase-3 staining, indicating the increased cell apoptosis. Moreover, toluidine blue staining was reduced in the low selenium + T-2 toxin group, suggesting a loss of sulphated glycosaminoglycans. Similarly, there was reduced 2B6 and 6C3 staining in the territorial matrix of chondrocytes, indicating a reduced synthesis in 4-sulhated and native CS motifs. In contrast, increased 1B5 staining was observed in the articular cartilage from the low selenium + T-2 toxin group, suggesting a lack of CS sulphatransferase activity. Interestingly, there was increased 7D4 staining in the superficial zone of articular cartilage from low selenium + T-2 toxin group, suggesting an initiation of an osteoarthritis-like lesion. Discussion. These results indicated that low selenium nutrition and T-2 toxin could promote cell apoptosis and disrupt CS-GAG metabolism in ECM of rat articular cartilage in this animal model, which is similar to that observed in KBD patients. Collectively, our results support the hypothesis that low selenium and T-2 toxin are the cause of KBD

Fractures repair by two mechanisms; direct fracture healing and indirect fracture healing via callus formation. Research concerning the effects of bisphosphonate on fracture repair has solely assessed indirect fracture healing. Patients with osteoporosis on bisphosphonates continue to sustain fragility fractures. A proportion of osteoporotic fractures require plate fixation. Bisphosphonates impair osteoclast activity and therefore, may adversely affect direct fracture healing that predominates with plate fixation. Five skeletally mature Sprague-Dawley rats received daily subcutaneous injections of 1mg/kg Ibandronate (IBAN). Similarly, five control rats received saline (CONTROL). Three weeks following commencement of injections a tibial osteotomy was rigidly fixed with compression plating similar to that seen in routine clinical practice. Fracture healing was monitored with radiographs. Six weeks post plate fixation, animals were sacrificed. Radiographs were performed of the extricated tibiae following plate removal. The visibility of the osteotomy site was scored as totally visible, partially visible or absent as previously described. Mechanical testing was conducted on the healing osteotomies via 4-point bending. Fractures healed without visible external callus. In the IBAN group three animals had totally visible osteotomy lines and two had partially visible osteotomy lines. The CONTROL group had three animals with absent osteotomy lines and two with partially visible osteotomy lines. The mean (±SD) stress at failure for the healing tibial osteotomies at 6 weeks was 28.8 (±23.97)MPa in the IBAN group and 37.4(±29.20) MPa in the CONTROL group (p=0.62). Our results indicate that Ibandronate adversely affected direct fracture repair as demonstrated by the radiographic density of the fracture line. The strength of the repair was reduced but this did not reach statistical significance. Our results suggest that a sample size of 220 animals is required to detect a 15% difference (alpha 0.05, beta 0.2) which suggests the effect of bisphosphonates on direct fracture repair may be small

Summary Statement. The present study demonstrates the beneficial effects of strontium (Sr) modified calcium phosphate cement to improve new bone formation in a metaphyseal osteoporotic fracture defects in rats compared to calcium phosphate cement and empty defects. Keywords: strontium, fracture, calcium phosphate, bone formation. Introduction. Impaired fracture healing with subsequent implant failure is a dramatic problem in osteoporotic fractures. Biomaterials are of interest to stimulate fracture healing in osteoporotic defects and the objective of the current study is to investigate the effects of Strontium modified calcium phosphate cement (SrCPC) in a critical-size metaphyseal fracture defect of osteoporotic rats compared to calcium phosphate (CPC) and empty defect control group. Methods. 45 female Sprague-Dawley rats were randomized into 3 groups: SrCPC, CPC and empty defect (n=15 for each). A combinatorial approach of multi-deficiency diet for 3 months after bilateral ovariectomy was used for induction of osteoporosis. Left femur of all animals underwent a 4mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with CPC or SrCPC and internally stabilised with a T shaped mini-plate. Empty defect served as a control. After 6 weeks femora were harvested followed by histological, histomorphometrical, immunohistochemical (bone-morphogenic protein 2, osteocalcin and osteoprotegerin), and molecular biology analysis (alkaline phosphatase, collagen10a1 and osteocalcin) to demonstrate the effects of the biomaterials on new bone formation. Time of flight secondary ion mass spectrometry (TOF-SIMS) technology was used to assess the distribution of released strontium ions and calcium appearance of newly formed bone. Results. Histomorphometric analysis showed a statistically significant increase in the bone formation at the tissue-implant interface in the SrCPC group (p<0.001). A statistically significantly more cartilage and unmineralised bone formation was also seen in the SrCPC group in comparision to the CPC group alone (p<0.05) and also to the empty defect (p<0.05) in the former fracture defect zone. These data were confirmed by the immunohistochemistry results which revealed an increase in bone-morphogenic protein 2, osteocalcin and osteoprotegerin and an increase in expression of genes responsible for bone formation viz. alkaline phosphatase, collagen10a1 and osteocalcin. TOF-SIMs analysis showed a higher release of Sr from the SrCPC into the interface region and related to a higher calcium content in this area compared to CPC. Discussion/Conclusion. SrCPC treatment showed enhanced new bone formation in a metaphyseal osteoporotic fracture defect of rats after 6 weeks compared to CPC-filled and empty defects in histomorphometry, immunochemistry and gene expression analysis. Strontium ranelate is a well-known anti-osteoporotic drug increasing bone formation and reducing bone resorption. As revealed by TOF-SIMS release of Sr out of the the SrCPC cement is most likely attributable for new bone formation. Therefore, Sr seems to be a good candidate not only for systemic treatment in osteoporosis but also in Sr-modification of biomaterials for local stimulation of new bone formation in osteoporotic fracture defects

Summary Statement. Tendon-bone interface becomes matured with the perforating fiber and the cells striding over the bone area. We suggest that both “perforating fiber” and “cell stride” could play a crucial role in regeneration after rotator cuff repair. Introduction. To obtain a successful outcome after rotator cuff repair, repaired tendon requires to be anchored biologically to the bone. However, it is well known that the histological structure of the repaired tendon-bone insertion is totally different from the normal insertion. This morphological alteration may contribute to biological instability after surgical repair. To address these issues, it is fundamental to clarify the difference of the structure between the normal and the repaired insertion in detail. Surprisingly, few studies on the tendon-bone insertion using electron microscopy has been performed so far, since the insertion area is solid (bone/cartilage) and extremely limited for the analysis. Recently, a new scanning electron microscopical method (FIB/SEM tomography) has been developed, making it possible to analyze the wider area with the higher resolution and reconstruct 3D ultrastructures. The purpose of this study was to analyze the ultrastructure of the repaired supraspinatus tendon-bone insertion in rat using FIB/SEM tomography. Materials and Methods. Adult Sprague-Dawley rats underwent complete cuff tear and subsequent repair of the supraspinatus tendon. The repaired supraspinatus tendon-bone interface was evaluated at 2 and 4 weeks after surgery. At each time point, 6 shoulders were used for biomechanical testing (ultimate load-to-failure and linear stiffness), 3 shoulders for conventional histological analysis and 3 shoulders for the ultrastructural analysis. The supraspinatus tendon insertion of the age-matched adult SD rats was used as normal control. For statistical analysis, the Wilcoxon's rank sum test was used to compare load-to-failure and linear stiffness. Differences of P<0.05 were considered significant. Results. <Biomechanical testing> All shoulders failed at the tendon-bone interface. The ultimate load-to-failure and the linear stiffness were significantly greater at 8 weeks than at 4 weeks (p<0.05). Normal tendon-bone insertion: The normal supraspinatus insertion consists of four-layered structure: tendon, fibrocartilage, mineralised fibrocartilage and bone. Repaired tendon-bone interface. At week 2, the fibro-vascular tissue was intervened between the tendon and bone at the repaired site. At week 4, the fibro-vascular tissue became organised, and perforating fibers were partially observed. <Ultrastructure using FIB/SEM tomography> Normal tendon-bone insertion: The ultrastructure of the normal supraspinatus insertion was very smooth. The cells were located between collagen bundles and arranged with their cell processes parallel to the bundles. Repaired tendon-bone interface: At week 2, the cells in the fibro-vascular tissue were arranged irregularly. At week 4, a part of the cells became arranged regularly and participated in linkage between the fibro-vascular tissue and bone, striding their processes across the bone side. Apparent boundary separating the fibro-vascular tissue from bone was observed throughout the periods. Conclusion. At 4 weeks after surgery, the repaired supraspinatus insertion remains to be immature and biologically weak. At 8 weeks after the surgery, it becomes matured with the perforating fiber and the cells striding over the bone area. We therefore suggest that both “perforating fiber” and “cell stride” could play a crucial role in regeneration of the tendon-bone interface after rotator cuff repair

Summary Statement. The mechanism of spinal cord injury varies across the human population and this may be important for the development of effective therapies. Therefore, detailed understanding of how variables such as impact velocity and depth affect cord tissue damage is important. Introduction. Studies have shown an independent effect of impact velocity and depth on injury severity, thereby suggesting importance of the interaction between the two for spinal cord injury. This work examines both the individual and interactive effects of impact velocity and impact depth on demyelination, tissue sparing, and behavioural outcomes in the rat cervical spinal cord. It also aims to understand the contribution of the energy applied during impact, not only the impact factors. Decoupling the effects of these two impact parameters will help to describe the injury mechanism. Maximum principal strain has also been shown to be useful as a predictor for neural tissue damage in vivo and in finite element (FE) models. A better understanding of this relationship with experimental results may help to elucidate the mechanics of spinal cord injury. Methods. In this study, 54 male Sprague-Dawley rats were given a contusion spinal cord injury at impact speeds of 8 mm/s, 80 mm/s, or 800 mm/s with depths of 0.9 mm or 1.5 mm. Animals recovered for 7 days followed by behavioural assessment and examination of the spinal cord tissue for demyelination and tissue sparing at 1 mm intervals, ±3 mm rostrocaudally to the epicentre. In parallel, a previously developed finite element model of the rat spinal cord was used to examine the resulting maximum principal strains in the spinal cord for correlations with histological and mechanical impact data. Results and discussion. Impact depth was a consistent factor in predicting axonal damage, tissue sparing, and the resulting behavioural deficit. Increased impact velocity resulted in significantly higher impact energies and measureable tissue damage at the 1.5 mm impact depth, but not at the 0.9 mm impact depth and is best displayed by the percentage of axon damage at the injury epicentre. Linear correlation analysis with FEA strain showed significant (p≪0.001) correlations with axonal damage in the ventral (R2=0.86) and lateral (R2=0.74) regions of the spinal cord and with white matter (R2=0.90) and grey matter (R2=0.76) sparing. Discussion and Conclusion. The difference in injury severity to velocity at different impact depths identifies the existence of threshold interactions between the two impact factors. Beyond this point incremental increases in either velocity or depth are more likely to result in significantly increased impact energy and thus tissue damage and functional impairment. The relationship between the impact depth and velocity of injury demonstrated a more rate sensitive response to spinal cord tissue damage at the deep (1.5 mm) impact depth than at the shallow (0.9 mm) impact depth. Impact velocity also became quickly less significant than impact depth in determining tissue damage further from the epicentre. Furthermore, the results shown by this work extend the research identifying significant correlations between maximum principal strain and neurological tissue damage

Summary. Coagulase-negative staphylococci, including S. epidermidis, have emerged as the leading pathogens of hospital-acquired biomaterial-related infections. These infections can be clinically indolent and challenging also for diagnostic imaging. In the current model of catheter-related infections, . 68. Ga-labeled Siglec-9 PET/CT imaging was able to detect peri-implant S. epidermidis bone infections. Introduction. Coagulase-negative staphylococci, including S. epidermidis, have emerged as the leading pathogen of nosocomial (hospital-acquired) biomaterial-related infections, including periprosthetic infections and intravascular catheter-related bloodstream infections. Pathogenic S. epidermidis strains exhibit robust attachment to implant surfaces and subsequent biofilm formation. By nature, the clinical picture of periprosthetic S. epidermidis infections can be indolent with vague signs of infection. These infections are also highly challenging for diagnostic imaging and microbiologic studies. Our recent experimental study of . 18. F-FDG-PET/CT confirmed that subacute peri-implant S. epidermidis infections, reflecting limited inflammatory reaction, are characterised by low . 18. F-FDG uptake. Vascular adhesion protein-1 (VAP-1) is an inflammation inducible endothelial protein, which controls leukocyte migration to sites of inflammation and infection. Siglec-9 is a leukocyte ligand of VAP-1. We hypothesised that . 68. Ga-labeled Siglec-9, developed for PET imaging of inflammation and cancer, could be a novel tracer also for early defection of S. epidermidis peri-implant bone infections. Material & Methods. Thirty adult male Sprague-Dawley rats were randomised into three groups (n=10/group). A clinical intravenous polymer catheter was introduced into the medullary cavity of the left tibia followed by injections of a clinical isolate of S. epidermidis (T-54580, 3 × 10. 8. CFU/mL) and an adjunct sodium morrhuate. In the positive control group, a clinical isolate of S. aureus (52/52A/80, 3 × 10. 5. CFU/mL) with sodium morrhuate was injected. In the negative control group, equal amount of sterile saline was injected via the catheter. The catheter, cut at the level of tibial tuberosity, was left in situ to serve as the implant. Two weeks after surgery, PET imaging with . 68. Ga-DOTA-Siglec-9 was performed with quantitative analysis of the standardised uptake value (SUV) in the region of interests both in vivo and ex vivo. SUV ratio between the operated and contralateral intact tibia was calculated. The presence of infections and the absence of contamination in the negative control group were verified by separate microbiological analyses of bone samples and retrieved implants. The presence of microbial biofilms on catheters was verified ex vivo with fluorescence microscope. Histologic inflammatory reaction was graded using a scoring system. Intergroup differences were tested by means of ANOVA with a post-hoc test. Results. Both staphylococcal strains caused histologically acute osteomyelitic changes. In . 68. Ga-DOTA-Siglec-9 PET/CT imaging of the negative control group, there was a significant difference (29.5%, p<0.001) in the SUV ratio of the operated and contralateral tibia, demonstrating aseptic inflammatory reaction to catheter implantation. The corresponding SUV ratio values were 58.1% in the S. epidermidis group and 41.7% in the S. aureus group. The uptake in the S. epidermidis group was significantly (p=0.009) higher than in the negative control group. Discussion/Conclusion. The animal model was reproducible in creation of culture-positive biomaterial-related infections. . 68. Ga-labeled Siglec-9 PET/CT imaging was able to demonstrate aseptic inflammation in the negative control group and the tracer also detected peri-implant bone infections caused by S. epidermidis

Summary Statement. The stable inhibition of miR-214 in the aged osteoporotic rats induced by OVX could be achieved by periodic administration of AntagomiR-214 at a dosage of 4 mg/kg and at an interval of 7 days, which will provide a potential bone anabolic strategy for treatment of osteoprosis. Introduction. MiR-214 has a crucial role in suppressing bone formation and miR-214 inhibition in osteogenic cells may be a potential anabolic strategy for ameliorating osteoporosis (Wang X, et al. 2013). An aged ovariectomised rat has been regarded as a golden model to test bone anabolic agents for reversing established osteoporosis in aged postmenopausal women (Li X, et al. 2009). However, there is still lack of evidence to demonstrate bone anabolic potential of therapeutic inhibition of miR-214 within osteogenic cells in the golden model. So, it should be necessary to establish RNAi-based administration protocol toward stable inhibition of miR-214 at a low level in the golden model. A targeted delivery system specifically facilitating Antagomir-214 approaching osteogenic cells, i.e. (Asp-Ser-Ser). 6. -liposome (Zhang G, et al 2012), was employed in this study. Objectives. This study was to investigate optimal dosage and duration for therapeutic inhibition of miR-214 within osteogenic cells in the aged osteoporotic rats induced by ovariectomy. Materials and Methods. Six-month-old female Sprague-Dawley rats were ovariectomised (OVX) and left untreated for 12 months to establish aged osteoporosis. To determine the optimal dosage for therapeutic inhibition of miR-214, the OVX rats were injected intravenously with the AntagomiR-214 at a dosage of 0.5mg/kg, 1mg/kg, 2mg/kg, 4mg/kg, 6mg/kg and 8mg/kg (n=6 for each dosage group) delivered by (Asp-Ser-Ser). 6. -liposome, respectively. Thereafter, miR-214 expression level in osteogenic cells from bilateral femur was quantified at day 2 post injection by real-time PCR analysis in combination with laser captured dissection (LCM). To determine the optimal duration of miR-214, the OVX rats were intravenously injected with the AntagomiR-214 (AntagomiR-214 group) or non-sense AntagomiR-214 (NC group) delivered by (Asp-Ser-Ser). 6. -liposome at the optimal dosage or (Asp-Ser-Ser). 6. -liposome alone (Vehicle group). Then, the miR-214 level in osteogenic cells from bilateral femur was quantified at 1, 3, 5, 7, 9, 12, 14, 16, 21 day after the single dosing (n=6 for each time-point) by real-time PCR analysis in combination with LCM, respectively. To examine the long-term effect of the AntagomiR-214 after periodic pulsed dosing, the OVX rats were administrated with the AntagomiR-214 at the optimal dosage and duration for 5 repeated injections and then the miR-214 level in osteogenic cells from bilateral femur was quantified by real-time PCR analysis in combination with LCM. Results. The miR-214 level was efficiently decreased in a dose-dependent manner by the AntogomiR-214 and reached the level lower than 10% of the baseline at a dosage of 4 mg/kg at least in the aged osteoporotic rats. The effective duration for miR-214 at a level lower than 50% of the baseline lasted for 7 days in the osteoporotic rats after the single dosing. The miR-214 level was continuously lowered until 28 days and continuously maintained later at the level lower than 10% of the baseline by the 5 pulsed dosing of the AntagomiR-214 at an interval of 7 days and at a dosage of 4 mg/kg in the osteoporotic rats. Conclusions. The stable inhibition of miR-214 for bone anabolic strategy in the aged osteoporotic rats induced by OVX could be achieved by periodic administration of AntagomiR-214 at a dosage of 4 mg/kg and at an interval of 7 days

Summary Statement. Umbilical cord derived stem cell secretion could enhance the osteogenic differentiation of human bone marrow stem cells. It may promote bone, cartilage and tendon regeneration in rat models, but the effect was not significant up to now. Introduction. Mesenchymal stem cells (MSCs) are multipotent cells that have extensive proliferative capacity. MSCs synthesise various exosomes, growth factors and cytokines. Stem cell secretions were made from serum free conditioned medium of stem cells collected from different human tissues, such as adipose tissue and dental pulp. Our hypothesis is umbilical cord stem cell secretion could promote multiple proliferation and differentiation of MSCs, also enhance the regeneration of musculoskeletal tissues. Methods. In vitro: Human bone marrow mesenchymal stem cells (hBMSCs) were cultured in high glucose dulbecco's modified eagle medium with 10% serum. hBMSCs were treated by differential medium for osteogenic, tenogenic and chondrogenic differentiation. Alizarin red S staining, alcian blue staining and sirius red staining were used to test osteogenesis, chondrogenesis and tenogenesis of hBMSCs after treated by secretion. RNA expression level of hBMSCs were detected by real-time reverse transcriptase polymerase chain reaction. In vivo: 10 weeks male Sprague-Dawley rats were used in all the animal studies. Rat calvarial bone defect model, rat femoral closed fracture model with internal fixation, rat articular cartilage defect model and rat patella tendon window defect model were used in animal experiments. Radiography analysis, micro-computed tomography imaging analysis, mechanical test, ultrasound test and histology analysis were used to evaluate the regeneration of bone, cartilage and tendon. Results. Alizarin red S staining showed the minimal effective concentration of 20ug/ml umbilical cord stem cells secretion could promote strong osteogenesis of hBMSCs, with enhanced expression of osteogenic markers runx2 and ocn. 20ug/ml umbilical cord stem cells secretion could promote tenogenic differentiation. The bone defect healing study using rat calvarial defect model indicated no significant difference (p»0.05) between 0.5ug/1ug umbilical cord secretion treated group (agarose gel with secretion was implanted in defect) and control (PBS) in 4 weeks or 8 weeks time points. In the rat femoral closed fracture model, the difference of bone repair between 10ug umbilical cord secretion local injection group (injected 10ug in callus after surgery) and control (PBS injected) was not significant (p»0.05) in 4 weeks or 8 weeks. In the rat articular cartilage defect model, 1ug umbilical stem cell secretion with 20ul alginate gel group recovered better than alginate gel only group in 6 weeks(p<0.05), but the difference of cartilage healing was not significant (p»0.05) between other groups (alginate gel with BMSCs) in 6 weeks or 9 weeks. In the rat patella tendon window defect model, there were more compact collagen fibers in 1ug umbilical cord secretion group (secretion with fibrin glue), but the alignment of new tissue was not better than control (PBS with fibrin glue). Also the stress of defected area was not significantly different (p»0.05) between treated and control in 6 weeks and 9 weeks. Discussion/Conclusion. The umbilical cord stem cell secretion demonstrated osteogenic, and tenogenic effect in vitro, but the result in the healing of bone, cartilage and tendon was not significant. The optimal dosage and slow release method will be considered to improve the experiment. The mechanism of stem cell secretions will be studied in further research