Aims. Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy. Methods. Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high-fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analysis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq). Results. Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA-Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation. Conclusion. These findings suggest that elevated or prolonged inflammation contributes to delayed fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients. Cite this article: Bone Joint Res 2023;12(10):657–666

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

Aims. This study examined whether systemic administration of melatonin would have different effects on osseointegration in ovariectomized (OVX) rats, depending on whether this was administered during the day or night. Methods. In this study, a titanium rod was implanted in the medullary cavity of one femoral metaphysis in OVX rats, and then the rats were randomly divided into four groups: Sham group (Sham, n = 10), OVX rat group (OVX, n = 10), melatonin day treatment group (OVX + MD, n = 10), and melatonin night treatment group (OVX + MN, n = 10). The OVX + MD and OVX + MN rats were treated with 30 mg/kg/day melatonin at 9 am and 9 pm, respectively, for 12 weeks. At the end of the research, the rats were killed to obtain bilateral femora and blood samples for evaluation. Results. Micro-CT and histological evaluation showed that the bone microscopic parameters of femoral metaphysis trabecular bone and bone tissue around the titanium rod in the OVX + MD group demonstrated higher bone mineral density, bone volume fraction, trabecular number, connective density, trabecular thickness, and lower trabecular speculation (p = 0.004) than the OVX + MN group. Moreover, the biomechanical parameters of the OVX + MD group showed higher pull-out test and three-point bending test values, including fixation strength, interface stiffness, energy to failure, energy at break, ultimate load, and elastic modulus (p = 0.012) than the OVX + MN group. In addition, the bone metabolism index and oxidative stress indicators of the OVX + MD group show lower values of Type I collagen cross-linked C-telopeptide, procollagen type 1 N propeptide, and malondialdehyde (p = 0.013), and higher values of TAC and SOD (p = 0.002) compared with the OVX + MN group. Conclusion. The results of our study suggest that systemic administration with melatonin at 9 am may improve the initial osseointegration of titanium rods under osteoporotic conditions more effectively than administration at 9 pm. Cite this article: Bone Joint Res 2022;11(11):751–762

Aims. Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages. Methods. Rat-derived chondrocytes were isolated, and DCs induced with interleukin (IL)-1β were used for exo isolation. Rats with OA (n = 36) or macrophages were treated with DC-exo or phosphate-buffered saline (PBS). Macrophage polarization and autophagy, and degradation and chondrocyte activity of cartilage tissues, were examined. RNA sequencing was used to detect genes differentially expressed in DC-exo, followed by RNA pull-down and ribonucleoprotein immunoprecipitation (RIP). Long non-coding RNA osteoarthritis non-coding transcript (OANCT) and phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) were depleted in DC-exo-treated macrophages and OA rats, in order to observe macrophage polarization and cartilage degradation. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway activity in cells and tissues was measured using western blot. Results. DC-exo inhibited macrophage autophagy (p = 0.002) and promoted M1 macrophage polarization (p = 0.002). DC-exo at 20 μg/ml induced collagen degradation (p < 0.001) and inflammatory cell infiltration (p = 0.023) in rats. OANCT was elevated in DC (p < 0.001) and in cartilage tissues of OA patients (p < 0.001), and positively correlated with patients’ Kellgren-Lawrence grade (p < 0.001). PIK3R5 was increased in DC-exo-treated cartilage tissues (p < 0.001), and OANCT bound to fat mass and obesity-associated protein (FTO) (p < 0.001). FTO bound to PIK3R5 (p < 0.001) to inhibit the stability of PIK3R5 messenger RNA (mRNA) (p < 0.001) and disrupt the PI3K/AKT/mTOR pathway (p < 0.001). Conclusion. Exosomal OANCT from DC could bind to FTO protein, thereby maintaining the mRNA stability of PIK3R5, further activating the PI3K/AKT/mTOR pathway to exacerbate OA. Cite this article: Bone Joint Res 2022;11(9):652–668

A novel EP4 selective agonist (KMN-159) was developed [1] and has been proven that it can act as an osteopromotive factor to repair critical-size femoral bone defects in rats at a dose-dependent manner [2]. Based on its osteopromotive properties, we hypothesized that KMN-159 could also aid in bone formation for spinal fusion. Therefore, the aim of this study was to investigate its spinal fusion effect in a dorsolateral spinal fusion model in rats. This study was performed on 192, 10-week-old male Wistar rats. The rats were randomized into 8 groups (n = 12 per group): 1) SHAM (negative control), 2) MCM (scaffold only), 3) MCM + 20 µg BMP-2 (positive control), 4-8) MCM + 0.2, 2, 20, 200 or 2000 µg KMN-159. A posterolateral intertransverse process spinal fusion at L4 to L5 was performed bilaterally by implanting group dependent scaffolds (see above) or left empty in the SHAM group (protocol no. 25-5131/474/38). Animals were euthanized after 3 weeks and 6 weeks for µCT and biomechanical testing analysis. The results showed that KMN-159 promoted new bone formation in a dose-dependent manner at 3 weeks and 6 weeks as verified by µCT. The biomechanical testing showed that the dose of 20, 200 and 2000 µg KMN-159 groups obtained comparable strength with BMP-2 group, which higher than SHAM, MCM and lower doses of 0.2 and 2 µg KMN-159 groups. In conclusion, KMN-159 could be a potential replacement of BMP-2 as a novel osteopromotive factor for spinal fusion. Acknowledgements: We are grateful to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology)

Objectives. This study aimed to assess the effect of age and osteoporosis on the proliferative and differentiating capacity of bone-marrow-derived mesenchymal stem cells (MSCs) in female rats. We also discuss the role of these factors on expression and migration of cells along the C-X-C chemokine receptor type 4 (CXCR-4) / stromal derived factor 1 (SDF-1) axis. Methods. Mesenchymal stem cells were harvested from the femora of young, adult, and osteopenic Wistar rats. Cluster of differentiation (CD) marker and CXCR-4 expression was measured using flow cytometry. Cellular proliferation was measured using Alamar Blue, osteogenic differentiation was measured using alkaline phosphatase expression and alizarin red production, and adipogenic differentiation was measured using Oil red O. Cells were incubated in Boyden chambers to quantify their migration towards SDF-1. Data was analyzed using a Student’s t-test, where p-values < 0.05 were considered significant. Results. CD marker expression and proliferation of the MSCs from the three groups was not significantly different. The young MSCs demonstrated significantly increased differentiation into bone and fat and superior migration towards SDF-1. The migration of SDF-1 doubled with young rats compared with the adult rats (p = 0.023) and it was four times higher when compared with cells isolated from ovariectomized (OVX) osteopenic rats (p = 0.013). Conclusion. Young rat MSCs are significantly more responsive to osteogenic differentiation, and, contrary to other studies, also demonstrated increased adipogenic differentiation compared with cells from adult and ostopenic rats. Young-rat-derived cells also showed superior migration towards SDF-1 compared with MSCs from OVX and adult control rats. Cite this article: A. Sanghani-Kerai, L. Osagie-Clouard, G. Blunn, M. Coathup. The influence of age and osteoporosis on bone marrow stem cells from rats. Bone Joint Res 2018;7:289–297. DOI: 10.1302/2046-3758.74.BJR-2017-0302.R1

This study aims to assess the fracture mechanics of type-2 diabetic (T2D) femoral bone using innovative site-specific tests, whilst also examining the cortical and trabecular bone microarchitecture from various regions using micro-computed tomography (CT) of the femur as the disease progresses. Male [Zucker Diabetic Fatty (ZDF: fa/fa) (T2D) and Zucker Lean (ZL: fa/+) (Control)] rats were euthanized at 12-weeks of age, thereafter, right and left femora were dissected (Right femora: n = 6, per age, per condition; Left femora: n=8-9, per age, per condition). Right femurs were notched in the posterior of the midshaft. Micro-CT was used to scan the proximal femur, notched and unnotched femoral midshaft (cortical) of the right femur and the distal metaphysis (trabecular) of the left femur to investigate microarchitecture and composition. Right femurs were fracture toughness tested to measure the stress intensity factor (Kic) followed by a sideways fall test using a custom-made rig to investigate femoral neck mechanical properties. There was no difference in trabecular and cortical tissue material density (TMD) between T2D and control rats. Cortical thickness was unchanged, but trabeculae were thinner (p<0.01) in T2D rats versus controls. However, T2D rats had a greater number of trabeculae (p<0.05) although trabecular spacing was not different to controls. T2D rats had a higher connectivity distribution (p<0.05) and degree of anisotropy (p<0.05) in comparison to controls. There was no difference in the mechanical properties between strains. At 12-weeks of age, rats are experiencing early-stage T2Ds and the disease impact is currently not very clear. Structural and material properties are unchanged between strains, but the trabecular morphology shows that T2D rats have more trabecular struts present in order to account for the thinner trabeculae

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)

Autologous cancellous bone graft is the gold standard in large bone defect repair. However, studies using autologous bone grafting in rats are rare and donor sites as well as harvesting techniques vary. The aim of this study was to determine the feasibility of autologous cancellous bone graft harvest from 5 different anatomical sites in rats and compare their suitability as donor sites for autologous bone graft. 13 freshly euthanised rats were used to describe the surgical approaches for autologous bone graft harvest from the humerus, iliac crest, femur, tibia and tail vertebrae (n=4), determine the cancellous bone volume and microstructure of those five donor sites using µCT (n=5), and compare their cancellous bone collected qualitatively by looking at cell outgrowth and osteogenic differentiation using an ALP assay and Alizarin Red S staining (n=4). It was feasible to harvest cancellous bone graft from all 5 anatomical sites with the humerus and tail being more surgically challenging. The microstructural analysis showed a significantly lower bone volume fraction, bone mineral density, and trabecular thickness of the humerus and iliac crest compared to the femur, tibia, and tail vertebrae. The harvested volume did not differ between the donor sites. All donor sites apart from the femur yielded primary osteogenic cells confirmed by the presence of ALP and Alizarin Red S stain. Bone samples from the iliac crest showed the most consistent outgrowth of osteoprogenitor cells. The tibia and iliac crest may be the most favourable donor sites considering the surgical approach. However, due to the differences in microstructure of the cancellous bone and the consistency of outgrowth of osteoprogenitor cells, the donor sites may have different healing properties, that need further investigation in an in vivo study

Aims. Post-traumatic osteoarthritis (PTOA) is a subset of osteoarthritis (OA). The gut microbiome is shown to be involved in OA. However, the effect of exercise on gut microbiome in PTOA remains elusive. Methods. A total of 18 eight-week Sprague-Dawley rats were assigned into three groups: Sham/sedentary (Sham/Sed), PTOA/sedentary (PTOA/Sed), and PTOA/treadmill-walking (PTOA/TW). PTOA model was induced by transection of the anterior cruciate ligament (ACLT) and the destabilization of the medial meniscus (DMM). Treadmill-walking (15 m/min, 30 min/d, five days/week for eight weeks) was employed in the PTOA/TW group. The response of cartilage, subchondral bone, serology, and gut microbiome and their correlations were assessed. Results. Eight-week treadmill-walking was effective at maintaining the integrity of cartilage-subchondral bone unit and reducing the elevated systematic inflammation factors and microbiome-derived metabolites. Furthermore, 16S ribosomal ribonucleic acid (rRNA) sequencing showed disease-relevant microbial shifts in PTOA animals, characterized by the decreased abundance of phylum TM7 and the increase of phylum Fusobacteria. At the genus level, the abundance of Lactobacillus, Turicibacter, Adlercreutzia, and Cetobacterium were increased in the PTOA animals, while the increase of Adlercreutzia and Cetobacterium was weakened as a response to exercise. The correlation analysis showed that genus Lactobacillus and Adlercreutzia were correlated to the structural OA phenotypes, while phylum Fusobacteria and genus Cetobacterium may contribute to the effects of exercise on the diminishment of serological inflammatory factors. Conclusion. Exercise is effective at maintaining the integrity of cartilage-subchondral bone unit, and the exercise-induced modification of disease-relevant microbial shifts is potentially involved in the mechanisms of exercise-induced amelioration of PTOA. Cite this article: Bone Joint Res 2022;11(4):214–225

Osteoarthritis (OA) and diabetis mellitus type 2 (DMT2) are pathogenetically linked. Complement dysregulation contributes to OA and could be involved in DMT2. The inflammatory anaphylatoxin C5a is released during complement activation. This study aims to understand the specific responses of chondrocytes isolated from diabetic and non-diabetic rats exposed to C5a and/or the proinflammatory cytokine TNFα in vitro dependent on the glucose supply. Articular chondrocytes of adult Zucker Diabetic Fatty (ZDF) rats (homozygous: fa/fa, diabetic, heterozygous: fa/+, lean controls) were exposed to 10 ng/mL TNFα and 25 ng/mL C5a alone or in combination, both, under normo- (NG, 1 g/L glucose) and hyperglycemic (HG, 4.5 g/L glucose) conditions (4 or 24 h). Chondrocyte survival, metabolic activity and gene expression of collagen type 2, suppressors of cytokine signaling (SOCS)1, −3 and anti-oxidative hemoxygenase-1 (HMOX1) were assessed. The complement regulatory protein CD46 and cell nuclei sizes were analyzed. Chondrocyte vitality remained unaffected by the treatment. Metabolic activity was impaired in chondrocytes of non-diabetic rats under HG conditions. Collagen type 2 transcription was suppressed by TNFα under HG condition in chondrocytes from nondiabetic donors and under both conditions in those of DMT2 rats (24 h). Except for DMT2 chondrocytes under HG (4 h), HMOX1 was generally induced by TNFα +/- C5a (NG, HG). C5a elevated HMOX1 only in chondrocytes of controls. The SOCS1/3 genes were increased by TNFα (NG, diabetic, non diabetic, 4 and 24 h). This could also be observed in chondrocytes of diabetic, but not of lean rats (24 h, HG). At 4 h, C5a induced SOCS1 only in non diabetic chondrocytes (NG, HG). Cytoprotective CD46 protein was suppressed by TNFα under NG condition. Nuclear volumes of chondrocyte were lower in chondrocytes from DMT2 rats compared to those from controls. The differential response suggests that chondrocytes are irreversibly compromised by DMT2. Achnowledgement: The authors are grateful for the support by the “Stiftung Edoprothetik (S 04/21)”

Objectives. Diabetes mellitus (DM) is known to impair fracture healing. Increasing evidence suggests that some microRNA (miRNA) is involved in the pathophysiology of diabetes and its complications. We hypothesized that the functions of miRNA and changes to their patterns of expression may be implicated in the pathogenesis of impaired fracture healing in DM. Methods. Closed transverse fractures were created in the femurs of 116 rats, with half assigned to the DM group and half assigned to the control group. Rats with DM were induced by a single intraperitoneal injection of streptozotocin. At post-fracture days five, seven, 11, 14, 21, and 28, miRNA was extracted from the newly generated tissue at the fracture site. Microarray analysis was performed with miRNA samples from each group on post-fracture days five and 11. For further analysis, real-time polymerase chain reaction (PCR) analysis was performed at each timepoint. Results. Microarray analysis showed that there were 14 miRNAs at day five and 17 miRNAs at day 11, with a greater than twofold change in the DM group compared with the control group. Among these types of miRNA, five were selected based on a comparative and extended literature review. Real-time PCR analysis revealed that five types of miRNA (miR-140-3p, miR-140-5p, miR-181a-1-3p, miR-210-3p, and miR-222-3p) were differentially expressed with changing patterns of expression during fracture healing in diabetic rats compared with controls. Conclusions. Our findings provide information to further understand the pathology of impaired fracture healing in a diabetic rat model. These results may allow the potential development of molecular therapy using miRNA for the treatment of impaired fracture healing in patients with DM. Cite this article: S. Takahara, S. Y. Lee, T. Iwakura, K. Oe, T. Fukui, E. Okumachi, T. Waki, M. Arakura, Y. Sakai, K. Nishida, R. Kuroda, T. Niikura. Altered expression of microRNA during fracture healing in diabetic rats. Bone Joint Res 2018;7:139–147. DOI: 10.1302/2046-3758.72.BJR-2017-0082.R1

Joint surface restoration of deep osteochondral defects represents a significant unmet clinical need. Moreover, untreated lesions lead to a high rate of osteoarthritis. The current strategies to repair deep osteochondral defects such as osteochondral grafting or sandwich strategies combining bone autografts with ACI/MACI fail to generate long-lasting osteochondral interfaces. Herein, we investigated the capacity of juvenile Osteochondral Grafts (OCGs) to repair osteochondral defects in skeletally mature animals. With this regenerative model in view, we set up a new biological, bilayered, and scaffold-free Tissue Engineered (TE) construct for the repair of the osteochondral unit of the knee. Skeletally immature (5 weeks old) and mature (11 weeks old) Lewis rats were used. Cylindrical OCGs were excised from the intercondylar groove of the knee of skeletally immature rats and transplanted into osteochondral defects created in skeletally mature rats. To create bilayered TE constructs, micromasses of human periosteum-derived progenitor cells (hPDCs) and human articular chondrocytes (hACs) were produced in vitro using chemically defined medium formulations. These constructs were subsequently implanted orthotopically in vivo in nude rats. At 4 and 16 weeks after surgery, the knees were collected and processed for subsequent 3D imaging analysis and histological evaluation. Micro-computed tomography (µCT), H&E and Safranin O staining were used to evaluate the degree of tissue repair. Our results showed that the osteochondral unit of the knee in 5 weeks old rats exhibit an immature phenotype, displaying active subchondral bone formation through endochondral ossification, the absence of a tidemark, and articular chondrocytes oriented parallel to the articular surface. When transplanted into skeletally mature animals, the immature OCGs resumed their maturation process, i.e., formed new subchondral bone, partially established the tidemark, and maintained their Safranin O-positive hyaline cartilage at 16 weeks after transplantation. The bilayered TE constructs (hPDCs + hACs) could partially recapitulate the cascade of events as seen with the immature OCGs, i.e., the regeneration of the subchondral bone and the formation of the typical joint surface architecture, ranging from non-mineralized hyaline cartilage in the superficial layers to a progressively mineralized matrix at the interface with a new subchondral bone plate. Cell-based TE constructs displaying a hierarchically organized structure comprising of different tissue forming units seem an attractive new strategy to treat osteochondral defects of the knee

Aims. The aims of this study were to develop an in vivo model of periprosthetic joint infection (PJI) in cemented hip hemiarthroplasty, and to monitor infection and biofilm formation in real-time. Methods. Sprague-Dawley rats underwent cemented hip hemiarthroplasty via the posterior approach with pre- and postoperative gait assessments. Infection with Staphylococcus aureus Xen36 was monitored with in vivo photoluminescent imaging in real-time. Pre- and postoperative gait analyses were performed and compared. Postmortem micro (m) CT was used to assess implant integration; field emission scanning electron microscopy (FE-SEM) was used to assess biofilm formation on prosthetic surfaces. Results. All animals tolerated surgery well, with preservation of gait mechanics and weightbearing in control individuals. Postoperative in vivo imaging demonstrated predictable evolution of infection with logarithmic signal decay coinciding with abscess formation. Postmortem mCT qualitative volumetric analysis showed high contact area and both cement-bone and cement-implant interdigitation. FE-SEM revealed biofilm formation on the prosthetic head. Conclusion. This study demonstrates the utility of a new, high-fidelity model of in vivo PJI using cemented hip hemiarthroplasty in rats. Inoculation with bioluminescent bacteria allows for non-invasive, real-time monitoring of infection. Cite this article: Bone Joint J 2021;103-B(7 Supple B):9–16

Aims. To fully verify the reliability and reproducibility of an experimental method in generating standardized micromotion for the rat femur fracture model. Methods. A modularized experimental device has been developed that allows rat models to be used instead of large animal models, with the aim of reducing systematic errors and time and money constraints on grouping. The bench test was used to determine the difference between the measured and set values of the micromotion produced by this device under different simulated loading weights. The displacement of the fixator under different loading conditions was measured by compression tests, which was used to simulate the unexpected micromotion caused by the rat’s ambulation. In vivo preliminary experiments with a small sample size were used to test the feasibility and effectiveness of the whole experimental scheme and surgical scheme. Results. The bench test showed that a weight loading < 500 g did not affect the operation of experimental device. The compression test demonstrated that the stiffness of the device was sufficient to keep the uncontrollable motion between fracture ends, resulting from the rat’s daily activities, within 1% strain. In vivo results on 15 rats prove that the device works reliably, without overburdening the experimental animals, and provides standardized micromotion reproductively at the fracture site according to the set parameters. Conclusion. Our device was able to investigate the effect of micromotion parameters on fracture healing by generating standardized micromotion to small animal models. Cite this article: Bone Joint Res 2021;10(11):714–722

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

The use of antifibrinolytic drugs and many other agents have a critical importance in bleeding control. Tranexamic acid [4- (aminomethyl) cyclohexanecarboxylic acid] is a synthetic amino acid lysine derivative with antifibrinolytic activity in humans. There are many studies in the literature that show that it is effective and effective both systemically and locally in spinal surgery. However, all of these studies have investigated the effects of topical tranexamic acid on bleeding and its effect on fusion has not been investigated yet. Aim of this study is to investigate the effect of topical tranexamic acid on fusion using macroscopic, radiologic and microscopic techniques. After approve of ethics committee with the protocol number 19/2019 for 28 Wistar Albino rats underwent intertransvers fusion. All rats were randomized into four (4) groups, using sealed envelopes. Local tranexamic acid (Transamin® 100 mg/ml, Teva İlaç, İstanbul) doses was determined based on previously conducted studies; 1mg/kg (D1 group), 10mg/kg (D10 group), 100 mg/kg (D100 group) and no tranexamic acid (D0 group). At the end of 8. th. weeks all rats were evaluated with manuel palpation, mammography and histopathologic analysis. Radiographic examination was performed two times to evaluate the intra and inter observer differences. 2 rats in-group D0 died after the radiographic examination. Assessment of fusion with manual palpation revealed that use of local 1mg / kg tranexamic acid had no effect on fusion (p=0.32), however with increasing doses of tranexamic acid had negative effect on fusion (p=0.002). On radiologic examination, spearman's rho correlation coefficient was found to be moderate in the first evaluation (r=0.46) and high in second evaluation (r=0.61). Radiological examination revealed that the control group was the best in fusion (p=0.007), and that tranexamic acid affected fusion adversely, independent of dosage (p=0.27). Among the groups in histopathologic examination, no statistical difference was found (p=0.134). Local administration of tranexamic acid affects the intertransverse fusion adversely depending on the dosage macroscopically and it also affects fusion adversely independent of the dosage radiologically and histopathologically

The effects of Hypericum perforatum on nerve regeneration after sciatic nerve injury have not yet been evaluated in all its aspects yet. In this experimental study, the effect of Hypericum perforatum on injured nerve tissue was histologically and biochemically investigated. Motor functional healing was surveyed by gait analysis. Rats were divided into 3 groups: Group I (n=8) was intact control group and no intervention and treatment was applied to this group. Group II (n=16) was surgical control group and Group III (n=16) was Hypericum perforatum group. After the operation, while any treatment was performed on Group II, 30 mg/kg dose Hypericum perforatum extract was intraperitoneally administered to the Group III per day for 8 weeks from the 1. st. day of post-op. Gait analysis was made to all rats for functional evaluation at 2. nd. , 3. rd. , 4. th. , 6. th. and 8. th. weeks, and sciatic functional index (SFI) was evaluated. At the end of the eighth week, sciatic nerve tissue samples were taken from the sacrificed rats. Tissues were examined biochemically, histologically and immnohistochemically. Malondialdehyde (MDA) as an indicator of oxidative stress and main antioxidant enzyme [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)] levels were biochemically measured. The nerve degeneration and regeneration were histologically viewed, and also cell count was immnohistochemically done by having done anti-S100 staining. It was seen that measurement results of SFI were statistically significantly difference between groups (p<0,001). In the sciatic nerve tissue samples taken from the rats, it was not determined a statistically significant difference between MDA, SOD, GPx and CAT levels detected by ELISA method (p>0,05). In the histological evaluation, it was seen that Hypericum perforatum affected positively the regeneration and immunohistochemically, it was found a statistically significant difference between the anti-S-100 positive cell numbers. The obtained results in this study show that; Hypericum perforatum, which was intraperitoneally administered on rats subjected to nerve injury, has affected positively the nerve regeneration and it can also provide an insight to future studies

Aims. A growing number of fractures progress to delayed or nonunion, causing significant morbidity and socioeconomic impact. Localized delivery of stem cells and subcutaneous parathyroid hormone (PTH) has been shown individually to accelerate bony regeneration. This study aimed to combine the therapies with the aim of upregulating fracture healing. Methods. A 1.5 mm femoral osteotomy (delayed union model) was created in 48 female juvenile Wistar rats, aged six to nine months, and stabilized using an external fixator. At day 0, animals were treated with intrafracture injections of 1 × 10. 6. cells/kg bone marrow mesenchymal stem cells (MSCs) suspended in fibrin, daily subcutaneous injections of high (100 μg/kg) or low (25 μg/kg) dose PTH 1-34, or a combination of PTH and MSCs. A group with an empty gap served as a control. Five weeks post-surgery, the femur was excised for radiological, histomorphometric, micro-CT, and mechanical analysis. Results. Combination therapy treatment led to increased callus formation compared to controls. In the high-dose combination group there was significantly greater mineralized tissue volume and trabecular parameters compared to controls (p = 0.039). This translated to significantly improved stiffness (and ultimate load to failure (p = 0.049). The high-dose combination therapy group had the most significant improvement in mean modified Radiographic Union Score for Tibia fractures (RUST) compared to controls (13.8 (SD 1.3) vs 5.8 (SD 0.5)). All groups demonstrated significant increases in the radiological scores – RUST and Allen score – histologically compared to controls. Conclusion. We demonstrate the beneficial effect of localized MSC injections on fracture healing combined with low- or high-dose teriparatide, with efficacy dependent on PTH dose. Cite this article: Bone Joint Res 2021;10(10):659–667

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. Mesenchymal stem cells (MSCs) are of growing interest in terms of bone regeneration. Most preclinical trials utilize bone-marrow-derived mesenchymal stem cells (bMSCs), although this is not without isolation and expansion difficulties. The aim of this study was: to compare the characteristics of bMSCs and adipose-derived mesenchymal stem cells (AdMSCs) from juvenile, adult, and ovarectomized (OVX) rats; and to assess the effect of human parathyroid hormone (hPTH) 1-34 on their osteogenic potential and migration to stromal cell-derived factor-1 (SDF-1). Methods. Cells were isolated from the adipose and bone marrow of juvenile, adult, and previously OVX Wistar rats, and were characterized with flow cytometry, proliferation assays, osteogenic and adipogenic differentiation, and migration to SDF-1. Experiments were repeated with and without intermittent hPTH 1-34. Results. Juvenile and adult MSCs demonstrated significantly increased osteogenic and adipogenic differentiation and superior migration towards SDF-1 compared with OVX groups; this was the case for AdMSCs and bMSCs equally. Parathyroid hormone (PTH) increased parameters of osteogenic differentiation and migration to SDF-1. This was significant for all cell types, although it had the most significant effect on cells derived from OVX animals. bMSCs from all groups showed increased mineralization and migration to SDF-1 compared with AdMSCs. Conclusion. Juvenile MSCs showed significantly greater migration to SDF-1 and significantly greater osteogenic and adipogenic differentiation compared with cells from osteopenic rats; this was true for bMSCs and AdMSCs. The addition of PTH increased these characteristics, with the most significant effect on cells derived from OVX animals, further illustrating possible clinical application of both PTH and MSCs in bone regenerative therapies. Cite this article:L. Osagie-Clouard, A. Sanghani-Kerai, M. Coathup, R. Meeson, T. Briggs, G. Blunn. The influence of parathyroid hormone 1-34 on the osteogenic characteristics of adipose- and bone-marrow-derived mesenchymal stem cells from juvenile and ovarectomized rats. Bone Joint Res 2019;8:397–404. DOI: 10.1302/2046-3758.88.BJR-2019-0018.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

Due to well-known disadvantages of the autologous bone graft, many alternatives have been studied for a reliable spinal fusion. Herein, we aimed to investigate the effects of human recombinant epidermal growth factor (EGF) on posterolateral lumbar fusion in a rat model. 36 male SD rats underwent posterolateral fusion at L4-5 level. They were randomly assigned to 3 groups: Sham control group, Hydoxyapatite β-tricalcium phosphate (HA/β-TCP) group and HA/β-TCP + EGF group. Rats were euthanized at 8 weeks post-surgery. 6 rats from each group were selected for manual palpation examination, micro-computed tomography analysis and histologic analysis; and the rest was used for biomechanical analysis. Based on manual palpation, there was no fusion in the sham control group. Fusion rate was 33.3% in the HA/β-TCP group and 66.7% in the HA/β-TCP + EGF group (p=0.085). Micro-CT results revealed that new bone formation was higher in the HA/β-TCP + EGF group (BV/TV: 40% vs. 65%) (p=0.004). Histologically newly formed bone tissue was more pronounced in the EGF group and compacted and bridging bone spicules were observed. The median maximum bending moment values were 0.51 Nmm (0.42– 0.59), 0.73 Nmm (0.49– 0.88) and 0.91 Nmm (0.66– 1.03) in the sham control, HA/β-TCP and HA/β-TCP + EGF groups, respectively (p=0.013). The median stiffness values were 1.69 N/mm (1.12–2.18), 1.68 N/mm (1.13–2.74) and 3.10 N/mm (1.66–4.40) as in the previous order (p=0.087). This study demonstrates that EGF enhances posterolateral lumbar fusion in the rat model. EGF in combination with ceramic grafts increased the fusion rates

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. Methods. EMF (20 Gauss) was examined in rats (aged 20 weeks), which underwent an ovariectomy (OVX; n = 8) or sham-ovariectomy (sham-OVX; n = 8). As a putative positive control, all rats received bilateral fibular osteotomies to examine the effects on fracture healing. Treatment was applied to one proximal lower leg (three hours a day, five days a week); the lower leg was not treated and served as a control. Bone architectural changes of the proximal tibia and bone formation around the osteotomy were evaluated using in vivo microCT scans at start of treatment and after three and six weeks. Results. In both OVX and sham-OVX groups, EMF did not result in cancellous or cortical bone changes during follow-up. Moreover, EMF did not affect the amount of mineralised callus volume around the fibular osteotomy. Conclusions. In this study we were unable to reproduce the strong beneficial findings reported by others. This might indicate that EMF treatment is very sensitive to the specific set-up, which would be a serious hindrance for clinical use. No evidence was found that EMF treatment can influence bone mass for the benefit of osteoporotic patients. Cite this article: Bone Joint Res 2014;3:230–5

Objectives. Insufficient protein ingestion may affect muscle and bone mass, increasing the risk of osteoporotic fractures in the elderly, and especially in postmenopausal women. We evaluated how a low-protein diet affects bone parameters under gonadal hormone deficiency and the improvement led by hormone replacement therapy (HRT) with 17β-oestradiol. Methods. Female Wistar rats were divided into control (C), ovariectomized (OVX), and 17β-oestradiol-treated ovariectomized (OVX-HRT) groups, which were fed a control or an isocaloric low-protein diet (LP; 6.6% protein; seven animals per group). Morphometric, serum, and body composition parameters were assessed, as well as bone parameters, mechanical resistance, and mineralogy. Results. The results showed that protein restriction negatively affected body chemical composition and bone metabolism by the sex hormone deficiency condition in the OVX group. The association between undernutrition and hormone deficiency led to bone and muscle mass loss and increased the fragility of the bone (as well as decreasing relative femoral weight, bone mineral density, femoral elasticity, peak stress, and stress at offset yield). Although protein restriction induced more severe adverse effects compared with the controls, the combination with HRT showed an improvement in minimizing these damaging effects, as it was seen that HRT had some efficacy in maintaining muscle and bone mass, preserving the bone resistance and minimizing some deleterious processes during the menopause. Conclusion. Protein restriction has adverse effects on metabolism, leading to more severe menopausal symptoms, and HRT could minimize these effects. Therefore, special attention should be given to a balanced diet during menopause and HRT. Cite this article: Bone Joint Res 2019;8:573–581

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

In the last years there is an increase in the interest in the study of growth factors that take part in the process of consolidation of the fracture to be used as treatment. The different types of fixations modify the natural process of the fracture healing and the production of growth factors could also be affected. There is not evidence in the literature of the effect that the intramedullary reaming has on the osteogenesis. We did a study to analyse the effect of intramedullary reaming on the production of growth factors during the process of fracture healing in the femur of rats. We did a pospective study in San Carlos Clinical Hospital from Madrid in which was made a fracture on the femur of 64 adults rats type Sprague-Dawley. The rats were divided in two main groups; each group received one different treatment: 30 rats with intramedullary nail and 34 rats did not receive any treatment. The rats of each group were sacrificed in 4 different moments: at the 24th hour, 4th, 7th and 15th days after the fracture was done, and we measured the amount of growth factors that appeared in the callus fracture, by anatomopathology study. The group in which was done the intramedullar nailing recovered normal walk after surgery. In this group were found more production of BMP and PDGF compared to the control group but did not reveal any significant difference between the groups (p> 0,05). Differences about other growth factors as TGF were not found. We conclude that in the results we have taken, the increase on BMP and PDGF could be produced by the intramedullary reaming by the surgery technique but we would need more studies

Aims. This study aimed to investigate whether human umbilical cord mesenchymal stem cells (UC-MSCs) can prevent articular cartilage degradation and explore the underlying mechanisms in a rat osteoarthritis (OA) model induced by monosodium iodoacetate (MIA). Methods. Human UC-MSCs were characterized by their phenotype and multilineage differentiation potential. Two weeks after MIA induction in rats, human UC-MSCs were intra-articularly injected once a week for three weeks. The therapeutic effect of human UC-MSCs was evaluated by haematoxylin and eosin, toluidine blue, Safranin-O/Fast green staining, and Mankin scores. Markers of joint cartilage injury and pro- and anti-inflammatory markers were detected by immunohistochemistry. Results. Histopathological analysis showed that intra-articular injection of human UC-MSCs significantly inhibited the progression of OA, as demonstrated by reduced cartilage degradation, increased Safranin-O staining, and lower Mankin scores. Immunohistochemistry showed that human UC-MSC treatment down-regulated the expression of matrix metalloproteinase-13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), and enhanced the expression of type II collagen and ki67 in the articular cartilage. Furthermore, human UC-MSCs significantly decreased the expression of interleukin (IL)-1β and tumour necrosis factor-α (TNF-α), while increasing TNF-α-induced protein 6 and IL-1 receptor antagonist. Conclusion. Our results demonstrated that human UC-MSCs ameliorate MIA-induced OA by preventing cartilage degradation, restoring the proliferation of chondrocytes, and inhibiting the inflammatory response, which implies that human UC-MSCs may be a promising strategy for the treatment of OA. Cite this article: Bone Joint Res 2021;10(3):226–236

Introduction. The fracture healing outcome is often evaluated via ex vivo testing of the fracture callus. However, there is only a small time window, where the callus stiffness is significantly different, i.e. a delayed fracture healing might be undetected if the time point of sacrifice is improper. The aim of this study was to develop an in vivo monitoring concept, which allows determining the fracture callus stiffness in vivo over the whole healing time in rats. Hypothesis. The fracture callus stiffness can be monitored by measuring the deformation of the external fixation device during gait analysis at several healing time points. Materials & Methods. The right femurs of sixteen wistar rats were osteotomized and stabilized with an external fixation device (stiffness 119 N/mm or 32 N/mm). The fixator body was instrumented with a stain gauge to measure the deformation. Gait analysis was performed once per week in a gait wheel equipped with a ground reaction force measuring device. Results. The deformation of the fixation devices decreased over the healing time indicating an increase of the callus stiffness. The flexible fixated group showed a later increase of the callus stiffness indicating a delay in fracture healing. Discussion & Conclusion. Measuring the deformation of the fixator and gait analysis provides a powerful tool to monitor the fracture healing process in rats. With this, it is possible to detect a delayed fracture healing process more reliable than with ex vivo analyses

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

Background. The effect of corticosteroids on tendon properties is poorly understood, and current data are insufficient and conflicting. The objective of this study was to evaluate the effects of corticosteroids injection on intact and injured rotator cuff (RC) through biomechanical and radiographic analyses in a rat model. Methods. 70 rats were assigned to seven groups:1)control - saline injection;2) no tear + single methylprednisolone acetate (MTA) injection; 3) no tear + triple MTA injection; 4) tear + single saline injection; 5) tear + single MTA injection; 6) tear+ triple saline injections; 7) tear+ triple MTA injections. Triple injections were repeated once a week. Following unilateral supraspinatus (SSP) injuries, MTA was injected subacromialy. Rats were sacrificed 1 week after last injection. Shoulders were harvested, grossly inspected, SSP was evaluated biomechanically. Bone density at the tendon insertion site on the greater tuberosity (GT) were assessed with micro-computed tomography (CT). Results. Exposure of the intact RC to the triple MTA injection resulted in significant decrease in maximal load and stiffness as compared to control group (p<0.05). In the injured tendons, at three week, steroids treated group presented with a significantly lower maximal load compared to the saline treated rats (p<0.01). Stiffness was slightly lower in the steroids treated group at three weeks (p=0.1). Micro-CT analysis showed significantly lower GT volume fraction and connectivity density in undamaged rats following triple MTA injection. Conclusions. Repeated dose of corticosteroids significantly weakens rat RC. Repeated MTA injections negatively affect bone quality and may deteriorate tendon to bone insertion site. However, data retrieved from animals must be scrupulously analysed prior to extrapolation to humans. Despite the limitations, our results clearly show a significant detrimental effect of corticosteroid exposure on the injured rat RC tendon biomechanics. These effects should be well thought-out against any potential benefit prior to administering a subacromial corticosteroid injection

Introduction The present experiment is undertaken to determine if a single dose addition of OP-1 device (rhBMP-7 and TCP-CMC) will enhance posterolateral spinal fusion in an osteoporotic rat mode (estrogen deficiency). Posterolateral intertransverse process spinal fusion using recombinant human osteogenic protein (rhBMP-7) was performed in ovariectomised female rats. OP-1 can be manipulated to enhance fusion rates and fracture healing with or without osteoporosis. Osteoporosis is characterised by low bone mass and micro-architectural deterioration of bone structure, resulting in bone fragility and an increase in susceptibility to fracture. Ovariectomised rats have been used as an osteoporotic model for posterolateral intertransverse process fusion in BMP experimental studies. Many studies have shown rhBMP-7 promotes spinal fusions in posterolateral fusion animal models. Not only is OP-1 able to promote spinal fusion in a standard animal model, but also it has been shown to overcome the inhibitory effects of nicotine in a rabbit posterolateral spinal fusion model. OP-1 Putty (Stryker) is an osteoinductive and osteoconductive bone graft material which consists of the recombinant human Osteogenic Protein (rhBMP-7), and TCP putty containing carboxymethylcellulose sodium (CMC) and tricalcium phosphate. This standard OP-1 device is somewhat different from the one Moazzaz et al used (. 1. ). The implication of OP-1 in osteoporotic model will open a new therapeutic window for osteoporotic or osteopaenial patients for the requirements of spinal fusion. Methods In present study, a total of 42 ovariectomised Sprague-Dawley female rats were randomly assigned to groups receiving 30 μg lactose + 400mg TCP-CMC, 90 μg lactose + 400 mg TCP-CMC, 30 μg rhBMP-7 + 400 mg TCP-CMC and 90 μg rhBMP-7 + 400 mg TCP-CMC. There was a group of rats receiving 400 mg TCP-CMC alone. Spinal fusion was evaluated by manual motion testing at each lumbar segment, Faxitron digital X-ray evaluation using the Lenke grading system, CT scans, DEXA scans and histology. Results Ovariectomized rats receiving 30 μg lactose + 400mg TCP-CMC, 90 μg lactose + 400 mg TCP-CMC, and 400 mg TCP-CMC alone did not show spinal fusion. OVX rats receiving 90 μg rhBMP-7 + 400 mg TCP-CMC showed significantly higher fusion rates than other groups (P < 0.0001). However, the rats receiving 30 μg rhBMP-7 + 400 mg TCP-CMC did not show solid fusion either radiologically and histologically. Discussion Therefore rhBMP-7, in dose of 90 μg, is able to overcome the inhibitory effects of estrogen deficiency on posterolateral spinal fusion and generate a relatively robust fusion. The effect of the OP-1 on osteoporotic spine is dose-dependent with/without carrier-dependent

Purpose: To investigate the effect of amifostine and dexrazoxane on bone mass of the vertebrae and femurs of doxorubicin treated male rats. Methods: Amifostine, Doxorubicin and Dexrazoxane were purchased from SMBD-Jewish General Hospital Pharmacy. Lactating Sprague Dawley dams with 14 male pups were purchased from Charles River Canada. At neonate day 10, rat pups were randomly divided into 4 groups of n=5. Pups were injected once intraperitoneally with either Phosphate Saline Buffer 1X (saline), or drugs, AMF (50 mg/kg), AMF + DOX (50 mg/kg +3 mg/ kg), or with AMF + DXR + DOX (50 mg/kg + 60 mg + 3 mg/kg, 20:1 DXR to DOX ratio). AMF and DXR were injected 30 minutes prior to the DOX injection. After injection, rat pups were returned to their mothers until weaning on neonate day 22. Rats were then sacrificed at day 38 (28 Post-Injection, PI). Bone mineral density (BMD) and micro computed tomography were analyzed. Results: Dissection of male pups days 1, 5 or 9 post-injection did not reveal any intestinal or organ damage. AMF treatment alone led to a slight but not significant increase in the right femoral, left femoral and lumbar vertebral BMDs. Similarly, AMF + DOX or AMF + DXR + DOX treated rats had no significant change in either femoral and vertebrae BMD. Conclusions: We recently showed that a single injection of DOX in young female rats is associated with low bone turnover resulting in vertebrae and femur bone growth deficits. However, no such a difference was detected when similarly treated males were examined. The role of sex steroid hormone at this age is unclear as sex hormones level are very low in neonates at the time of injection and the rats, male and female, were sacrificed prior to puberty. To define the role of sex hormone in the observed gender-specific drug susceptibility we plan on comparing the response of intact to ovariectomized female rats to the drug regimen. Funding: Other Education Grant. Funding Parties: CIHR

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

It has been shown that mesenchymal stem cells (MSCs) and BMP are involved in bone formation. The aim of the study was to evaluate the osteogenic potential of human bone marrow (hBM), human expanded MSC (hexp-MSC), BMP-7, and hexp-MSC plus BMP-7, to treat a rat femoral segmental defect. Sprague-Dawley (SD) and athymic rats (Nu) were used. SD rats where used in order to define surgical technique. Nu rats groups consisted of: G1-autoclaved bone and human bone marrow (hMNC); G2-bone and hexp-MSC; G3-bone with BMP-7 only; and G4-bone and hexp-MSC with BMP-7. A plate was attached to the femoral diaphysis with two cerclage wires. Then a 6-mm femoral gap was made and filled with a different graft. At regular intervals, the femoral defect was evaluated with radiographs, using a modified six-grade Cook classification. At 8 weeks G1 showed non-visible new bone formation; G2 minimal new disorganised bone; G3 disorganised new bone bridging the graft to host at both ends; and G4 significant new bone and graft remodelling. Histological analysis confirmed these results. Our results showed that although the osteogenic activity may be improved by hMSC (G2) as well as by BMP-7 (G3), the association hexp-MSC plus BMP-7(G4) produced graft osteointegration at 8 weeks after surgery. This may have a remarkable impact on future orthopaedics surgery strategies

Introduction: In avascular necrosis [AVN] of the femoral head the dead bone undergoes osteoclastic osteolysis and is replaced by newly synthesized, immature, weak bone, which cannot withstand the daily loads. The articular surface might caves in because of these changes, and osteoarthritic joint changes can develop. Alendronate interferes with the osteoclastic activities, it can slow-down the bone turnover of the necrotic bone and can differ these changes. The aim of this study is to delay the speedy renewal of living epiphyses by alendronate medication in order to describe the effects of it on the fate of the necrotic femoral heads in rats. Methods: Sixty female sprague-dawley rats, 6-month old weighing about 400–500 grams, underwent surgical AVN of the right femoral heads. Forty-four rats, the treated group, were treated with alendronate 200 μgm/kg/day. Sixteen rats, the control group, were treated with saline. Both groups were daily injected subcutaneously for six weeks and sacrificed. Both femoral heads were harvested and were evaluated microscopically and stained by H& E. Results: The necrotic femoral heads of the control group, which were not treated by alendronate, were severely distorted with osteoarthrosis features as; collapse of the epiphysis, pannus formation, filling of spaces by chronically and mildly inflamed densely textured fibrous tissue which was polluted by numerous tiny particles of necrotic bone. Additionally, large chunks of necrotic articular cartilage were haphazardly scattered in the fibrous tissue. All hematopoietic and fat cells of the intertrabecular spaces of the epiphysis were replaced by fibrous tissue. More often than not, the cartilage of the physis was focally or entirely absent such that osseous trabeculae of the epiphysis and metaphysis linked with each other, forming so-called epiphyseal-metaphyseal bridges. The above described alterations were encountered in all animals, yet their severity varied. The decisive difference between the necrotic femoral heads of otherwise untreated in opposition to the alendronate-medicated rats was the preservation of a hemispherical configuration of the femoral heads. There was no distortion of the femoral heads in the alendronate-treated animals and the femoral heads preserved their roundness. All femoral heads of the non-operated left hips were microscopically normal. Discussion: It has become clear that the degree of architectural distortion of the femoral epiphyses depends on the extent of bone turnover leading to resorption of all debris and its replacement by living osseous and soft tissues. The more rapidly and more extensively the reconstruction of living epiphyses progresses, the smaller is the prospect of reshaping a hemispherical or near-hemispherical femoral head. The recently rebuilt epiphyses cannot carry daily transarticular loads without caving in. The revascularization-related reconstitution of weak bony trabeculae is blamed for the collapse of the femoral heads. If this indeed is the case, the remodeling of the necrotic femoral heads should be delayed, rather than sped-up. Alendronate interferes with the osteoclastic activities and hence, slowing-down the bone turnover. The osteoclastic activity is detrimental for the conservation of a hemispherical femoral head because of the rapidly occurring replacement of the necrotic bone by living tissues. Halting the activities of the osteoclasts by a biphosphonate would stop the hasty osteoneogenesis, which is responsible for the early femoral capital disfigurement and might delay the regeneration of osteo-arthiritic changes of the joint later on

Bone strength is influenced by bone quality besides its density. This study aimed to evaluate the effects of teriparatide on changes of bone strength as well as trabecular and cortical bone microstructures at femoral neck in female ovariectomized (OVX) rats. Eighteen female Wister rats were divided into three groups: the sham control, OVX and treatment (Tx) groups. All of them were sacrificed after 3-month intermittent teriparatide intervention in Tx group. All left femurs were removed and scanned using micro-CT and followed by mechanical test for each femoral neck. Regarding micro-CT, four trabecular parameters including bone volume fraction (BV/TV), trabecular thickness (TbTh), trabecular separation (TbSp), and trabecular number (TbN) and three cortical parameters including volumetric bone mineral density (vBMD), cortical cross-sectional area (CtAr) and cortical thickness (CtTh) were measured at femoral neck region. All data were analyzed and was presented as median ± SEM. The mean bone strength of femoral neck significantly decreased in OVX group when compared to the control group (p < 0.05) and was significantly restored in Tx group (p < 0.01). Regarding the trabecular parameters, the BV/TV and TbTh significantly decreased in OVX group while compare to Tx group. However, no significant difference was observed in TbSp and TbN between the groups. Regarding the cortical parameters, CtTh was significantly greater in Tx group than that in OVX group (p<0.01). As our findings, intermittent teriparatide can improve the deteriorated bone strength of femoral neck due to ovarian deficiency via changing both trabecular microarchitecture and cortical morphology

Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-a (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics

The purpose of this study was to evaluate whether epidural fibrosis formation around the spinal cord was affected by endogenous oestrogen deficient state after lumbar laminectomy in the rats. Thirty-six 12-month-old adult female Sprague-Dawley rats were used in this study. Bilaterally ooferectomy were done in 18 rats. Rats were divided into two groups: oophrectomised (oestrogen deficient) group and sham operated (oestrogen maintained) group. Three weeks after the ooferectomy each rat underwent complete bilaterally laminectomy at the L2 and L3 vertebral levels (two levels per rat). The rats were randomly divided into three equal groups (12 rats in each group). The rats were sacrificed at four, eight, and twelve weeks postoperatively and the lumbar spine excised en bloc, fixed and decalcified. Section stained with hematoxylin and eosin and Masson’s trichrome were used to evaluate epidural fibrosis, acute inflammatory cells, chronic inflammatory cells and vascular proliferation. Sections were analyzed by investigator blinded to the study and graded on a five-point grading system. Statistic were performed using Mann-Whitney U test when compare two variable and Kruskal-Wallis test when compare more than two variables. Compared with the oopherectomised group, the sham operated group showed decreased rate of epidural fibrosis and higher acute and chronic inflammatory cells response at four and eight weeks but this was no statistically significant (p> 0.05). The results of this study revealed that endogenous oestrogen may decrease epidural fibrosis formation after lumbar laminectomy in the rats

Surgical site infection related to orthopaedic implants is one of the serious complications. In the previous works, we developed a novel thermal spraying technology combined silver with hydroxyapatite (HA) in order to resolve such problems, and reported the property and antibacterial effect of them in vitro. However, no previous reports have investigated in vivo. Therefore, we monitored serum silver level in rats to clarify in vivo kinetics of silver released from the coating. HA loaded with 3 wt % of silver oxide (HA-Ag) and plain HA powder were sprayed on surface of titanium disks (20 mm diameter × 1 mm thick) by the flame spraying, which is a kind of thermal spraying method with acetylene torch. All these test pieces were obtained from Japan Medical Materials Corporation (JMM, Osaka, Japan). Both samples were implanted singly into the back subcutaneous pockets of male Sprague-Dawley rats (150–200 g). Rats were housed individually and given ad libitum access to food and water. After 24 h, 48 h, 7 d, 14 d and 28 d, the rats were sacrificed, and then the blood was drawn from common iliac vein. All procedures were operated under anesthesia. These blood samples were spun down and serum silver levels were measured by an inductively coupled plasma mass spectrometry. The average serum silver level in HA-Ag group had increased to more than 40 ppb until 48 h after implantation, and then decreased rapidly to normal level. There were significant differences (p < 0.05) between HA-Ag and HA group, at each measurement period. This is the first report to elucidate the serum silver level in rats implanted HA-Ag coatings. To date, reported coating technologies have included direct-loading antibacterial agents or heavy metals including silver with prosthesis base. The combine technology HA with silver would be effective in not only antibacterial but also osteoconductive respect. Our experimental results highlight the following 2 features: the serum silver levels peaked relatively early, and the levels reduced immediately to normal level after the peak. Therefore, we speculate that the released silver would not be accumulated generally, which not contribute long-term toxicity, and the coating would be suitable for prevention of early surgical site infections. This study provides novel and important information on in vivo release- property for HA-Ag coating, and suggests this coating is effective against not late but rather early infection related to orthopaedic implants

Purpose of this study is to create an experimental model of electrophysologic evaluation of the supraspinatus muscle on rats, after traumatic rupture of its tendon. The population of this study consisted of 10 male Sprague Dawley rats weighting 300–400g. Under general anaesthesia we proceeded with traumatic rupture of the supraspinatus tendon and exposure of the muscle. The scapula was immobilized, and the supraspinatus tendon was attached to a force transducer using a 3–0 silk thread. A dissection was performed in order to identify the suprascapular nerve, which was then stimulated with a silver electrode. Stimulations were produced by a stimulator (Digitimer Stimulator DS9A) and were controlled by a programmer (Digitimer D4030). Fiber length was adjusted until a single stimulus pulse elicited maximum force during a twitch under isometric conditions. Rectangular pulses of 0.5 ms duration were applied to elicit twitch contractions. During the recordings, muscles were rinsed with Krebs solution of approximately 37 8C (pH 7.2–7.4) and aerated with a mixture of 95% O2 and 5% CO2. The output from the transducer was amplified and recorded on a digital interface (CED). The following parameters were measured at room temperature (20–21 8C): single twitch tension; time to peak; half relaxation time; tetanic tensions at 10, 20, 40, 80 and 100 Hz; and fatigue index, which was evaluated using a protocol of low frequency (40 Hz) tetanic contraction, during 250 ms in a cycle of 1 s, for a total time of 180 s. The fatigue index value was then calculated by the formula [fatigue index=(initial tetanic tension − end tetanic tension) ∗ 100/(initial tetanic tension)]. In the end, the transducer was calibrated with standard weights and tensions were converted to grams. The mean single twitch was 8.2, the time to peak 0.034 msec and the half relaxation time 0.028 msec. The strength of titanic muscle contractures was 5.7 msec at 10Hz and 17.7 at 100Hz. Finally, the fatigue index was calculated at 48.4. We believe that electrophysiologic evaluation of the supraspinatus muscle in rats will help us understanding the pathology of muscle atrophy after rotator cuff tears and possibly the functional restoration after cuff repair

The pineal hormone melatonin was recently shown to have a free radical scavenging ability which reduces lipid peroxidation and has also been shown to scavenge the hydroxyl radical and other reactive oxygen species. Melatonin stimulates several antioxidative enzymes, which increases its efficiency as an antioxidant. Also, melatonin stimulates osteoblast differentiation and mineralization of matrix and it may regulate osteoclastic activity via superoxide dismutase in vitro. Therefore, the effect of melatonin in fracture healing depends in part on the free radical scavenging and osteoblastic-osteoclastic regulatory properties of melatonin. In this study the effect of melatonin (MEL) on fracture healing in rat tibia model was studied by using biochemical, radiological and histopathologic methods. Male Sprague-Dawley rats (n=80) were randomized into control and melatonin groups with 8 rats per group according to the day of sacrifice (Days 1, 3, 7, 14, 28). Group B (melatonin group) received 30 mg/kg melatonin intraperitoneally (i.p.) for the duration of the experiment. Malondialdehyde (MDA) levels in MEL group decreased at days 3, 7, 14, and 28 compared to control values (p< 0.05). Superoxide dismutase (SOD) activity in MEL group decreased at days 3, 7, and 14, and returned to the first day value after 28 days. Myeloperoxidase (MPO) values in MEL group decreased at days 1, 3, and 7 (p< 0.001). Both radiologically and histopathologically fracture healing was significantly more advanced in the MEL group (p< 0.05, p< 0.05 respectively). We conclude that exogenously administering a pharmacological dose of melatonin has a positive effect on fracture healing and may be beneficial as a supportive agent in fracture cases

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

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

Purpose: The study was conducted to evaluate the effect of salmon calcitonin (sCT) on fracture healing in normal and hypogonadal male rats. Material and Method: Fifty six male Wistar rats, aged 3 months, were undertaken hemiosteotomy of the distal femur shaft, of standard length and width. Half of the animals had been orchiectomised at the age of 2 months. The animals were divided in 8 groups, 7 rats each, as follow: A, a (normal, no sCT), B, b (normal+Sct), C,c(orchiectomised) and D,d (orchiectomised+Sct). Salmon calcitonin was administered immediately after the hemiosteotomy in a dose of 5IU/day subcutaneously. Groups A, a, C and c were given placebo. The animals of the groups a b, c, and d were killed at 2 weeks, while the animals of the groups A, B, C, and D were killed at 4 weeks. After the euthanasia, total bone density and cortical bone density of the callus was estimated by peripheral quantitative computed tomography (pQCT). Histological and histomorfometric parameters of the callus were estimated as well. Results: The mean cortical bone density was 1221.93±13.82 (g/cm. 3. ±SE) for the group a, 1281.3±13.57 for b, 1221.41±18.24 for c, 1245±17.12 for d, 1173.45±34.14 for A, 1298.9±11 for B, 1280.78±13.68 for C, and 1279.4U19.2 for D. The mean total bone density was 843.95±13.69 (g/cm. 3. ±SE) for the group a, 859.84±26.46 for b, 892.27±25.3 fore, 861.37±10.88 for d, 818.97±32.5 for A, 926.39±19.6 for B, 888.31±24.19 for C, and 912.75±28.13 for D. Values of cortical bone density in group b and B were significantly greater than a and A, respectively (b> a, p=0.01 and B> A, p=0.002). Total bone density of the callus was statistically greater in group B than A (B> A, p=0.01). According to the histological and histomorphometric results, sCT increased the amount of cartilage (p=0.014) and the amount of woven bone (p=0.015) in group b compared to a, while osteoblasts number showed no difference between the two groups. Comparing groups c and d, sCT increased the amount of cartilage (p=0.036) and the amount of woven bone (p=0.0014) in group d compared to c, while decreased osteoblasts number in group d (p=0.03). In four weeks the amount of cartilage is significant greater in group D versus C (p=0.006), as well as the amount of woven bone (p=0.0004). The size of the callus is significant greater in group D compared to C as well (p=0.052). Conclusion: It appears that salmon calcitonin administration improves significantly the parameters of callus bone density in normal rats and increases the amount of cartilaginous callus and woven bone both in normal and orchiectomised rats

Purpose: Bony metastases in vertebrae secondary to breast cancer can result in osteolysis and an increase in skeletal related events. Bisphosphonates (BP) are the current standard of care for breast cancer patients with skeletal disease. Photodynamic therapy (PDT) is a non-radiative treatment, which has been successfully applied to various malignancies and shown to successfully ablate vertebral human breast cancer (MT1) metastases in a murine model. Previous in-vitro study has shown that pre-treatment of MT-1 cells with the BP zoledronic acid (Zometa. ®. ) renders them more susceptible to PDT. The aim of this study was to evaluate the influence of pre-treatment with BPs on the effect of PDT treatment on tumour ablation in metastatically involved vertebrae in vivo. Method: Metastases were induced in fourteen 5–6 weeks old female athymic rats (Hsd:RH-Foxn1rnu) by intra-cardiac injection of 2x10^6 MT-1 cells. Four groups were formed:. control, no treatment;. BP only;. PDT only;. BP and PDT combined. Seven days after MT-1 injection 60 μg/kg of zoledronic acid was injected. PDT treatment was administered on day 14 using the photosensitizer BPD-MA (1.0 mg/kg; Visudyne). Fifteen minutes later, laser-light (690nm; 75J) was administered to the lumbar vertebrae. The rats were euthanized 7 days after PDT treatment. A total of 45 vertebrae were evaluated using a histomorphometric program (GENIE™, Aperio) to assess tumour burden. Statistical analyses were performed using a one-way ANOVA with a Tukey post hoc test. A p-value p< .05 was considered to be statistically significant. Results: The total The total tumour burden within vertebrae of rats pre-treated with BP and/or PDT was significantly lower compared to the control rats (p< .001). In addition, the PDT alone treated group demonstrated significantly less tumour burden than the combined BP+PDT group. In the control and BP-only groups, large tumours were found to include regions of necrosis. The PDT treatment groups (PDT and BP+PDT) exhibited areas of necrosis throughout the entire vertebral bodies with adjacent formation of granulation tissue. Conclusion: BP, PDT and combined BP+PDT treatments resulted in a lower overall tumour burden at day 21 post MT-1 cell injection compared to control rats. A surprising increased level of tumour burden was found in comparing the combined treatment group to the PDT-only group. These findings are in contrast to previous in-vitro results, where the pre-treatment with BPs made the cells more susceptible to PDT. Pre-treatment with BP affects both the bone and tumour cells, and as such may induce different cellular pathways in response to PDT treatment. However, the ability of PDT applied at day 14 to cause a similar reduction in tumour burden compared to BP treatment at day 7, suggests its ability to rapidly and effectively ablate the tumour within the bone, even in the presence of BP

Introduction and Aims: There is good preliminary evidence that Bone Morphogenic Protein 7 (BMP-7) plays an integral role in fracture healing and metabolism of bone. It is not known, however, whether the implantation of an OP-1 device will enhance the rate of fracture healing in the presence of osteoporosis. The object of this study was to determine the effects of OP-1 on osteoporotic fracture healing in rats. Method: An open fracture of the mid-shaft of the femur was created in 60, three months post-surgical ovarectomised female Sprague Dawley rats. Thirty rats had OP-1 device with CMC putty implanted into the fracture site and 30 rats had CMC putty implanted without OP-1. The fracture was stabilised with a 1.4mm K-wire. Muscle and skin closed. Ten rats from each group were sacrificed at three time points – 12, 20 and 31 days post-surgery, and bilateral femurs harvested. The fractured femurs were analysed by DEXA scanning, high-resolution radiography, cross-sectional area, biomechanical assessment and histology. Results: There was a statistically significant acceleration of fracture healing with the use of OP-1 in DEXA, radiological, cross-sectional area and biomechanical analysis and a qualitative enhancement by histological analysis. Conclusion: The results show that an OP-1 device can enhance fracture healing in the presence of osteoporosis in a rat

Purpose of this study is to create an experimental model on rats for EMG evaluation of the supraspinatus muscle after traumatic rupture of its tendon. The population of this study consisted of 5 male rats of 300–400g. Under general anaesthesia we proceeded with traumatic rupture of the supraspinatus tendon and exposure of the muscle. The electrode of a stimulator was placed under suprascapular nerve and the supraspinatus tendon was sutured on a transducer for digital record of the produced signal. Initially we found the resting length and the electric intensity for higher muscle contracture. The parameters that were evaluated after single contracture (single twitch) were strength, time to peak, half relaxation time. Furthermore, it was evaluated the strength of tetanic contractures at 10,20,40,80,100 Hz (Stimulation for 350msec each time).Finally it was evaluated the muscle fatigue with stimulation at 40Hz for 250msec and total duration of 3 minutes. Fatigue index was calculated according to the decrease of titanic muscle contracture (Initial value-Final Value/Initial Value x 100). Our results are presented in mean ± sd. The single twitch was 8.2(5.1),the time to peak 0.034(0.02) msec, the half relaxation time 0.028(0.008)msec. The strength of titanic muscle contractures was 5.7msec at 10Hz and 17.7 at 100Hz. Finally the fatigue index was calculated at 48.4. We believe that EMG evaluation of the supraspinatus muscle in rats will help us understanding the pathology of muscle atrophy after rotator cuff tears and possibly the functional restoration after cuff repair

The growth plates of rapidly growing animals have been studied extensively. Nevertheless, several questions remain unanswered, partly because many events happen simultaneously, especially at the vascular front. Terminal chondrocytes are thought to undergo programmed cell death, but the fate of the cell remnants remains unclear. Are the dying cells released into the vascular space and phagocytosed by macrophages, as one would expect for apoptosis? Or are the cells eliminated prior to opening of the lacunae, leaving empty lacunae? Do all terminal chondrocytes die or do some become bone-forming cells? Rodents maintain a growth plate into old age, long after longitudinal growth has ceased. These stationary growth plates have several features not found in the growth plates of rapidly growing animals and closer study of these features may provide answers to the above questions. Femurs and tibiae from 4–16 week-old and 62–80 week-old rats were decalcified, processed into paraffin, and the morphological changes were documented. Between 4–16 weeks, the heights of the growth plates decreased due to loss of the large hypertrophic chondrocytes, but the various zones were still present. In the aged rats, the growth plates were identifiable as a narrow cartilaginous band with some short columns of inactive cells. The vascular front was irregular, the narrow spicules of primary spongiosa were absent and the much thicker spicules, which are normally seen in secondary spongiosa, directly abutted to the cartilage. Horizontal apposition of bone matrix onto the cartilage edge was frequently present. In addition, the following features were noted. 1) Acellular areas: Nearly all growth plates contained regions of cartilage from which all cells and their lacunae had disappeared. In some cases, these acellular regions stretched from the reserve zone to the vascular front and even persisted as a relatively wide core within the spicules of spongiosa, indicating increased resistance of acellular cartilage to resorption. The absence of cells or cell debris was consistent with an autophagic mode of cell death and subsequent collapse of the lacunae. 2) Remodelling within the growth plate; in some growth plates, large regions of growth plate cartilage had been resorbed and new bone had been laid down in a pattern similar to the remodelling of cortical bone. This suggested that the normal resistance of cartilage to vascular invasion had been lost locally, but was maintained in adjacent non-remodelled regions. 3) Trans-differentiation of chondrocytes to bone-forming cells; extensive new medullary bone formation was noted in the diaphysis of approximately 30% of the aged rats, suggesting that they had received an (unknown) osteogenic stimulus. In these rats, bone matrix was identifiable inside chondrocytic lacunae, and spreading beyond the confines of the lacunae, thus directly replacing growth plate cartilage with bone matrix. The results suggest that i) chondrocytes are capable of self-elimination, perhaps by a mechanism similar to the autophagic cell death that occurs during insect metamorphosis; ii) resorption of cartilage and vascular invasion requires the presence of the viable chondrocytes; and iii) chondrocytes have the capacity to transdifferentiate to bone-forming cells, but only do so when receiving an increased osteogenic stimulus

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

Introduction: The existence of peripheral blood (PB) derived mesenchynal stem cells (PBMSCs) have been documented in several species including human. The circulating skeletal stem cells may provide a new source of stem cells that may be used for skeletal and other tissue engineering applications. The objective of this study is to further investigate and compare the biological characteristics of the PBMSCs with bone marrow derived MSCs in the GFP rats. Methods: The peripheral blood (PB) from the GFP rats was harvested by cardiac puncture using syringes containing sodium heparin. Mononuclear cells were isolated by density gradient centrifugation method and plated at a density of 1–3~105/cm2 in flasks with D-MEM medium containing 15% FCS. The bone marrow (BM) was also collected for obtaining BMMSCs, the bone chips for osteoblastic cells, and the skin for skin fibroblasts. The phenotypes of the cells were characterized by immunocytochemistry (ICC), and flow cytometry methods. Gene expression profiles of 3-paired PBMSCs and BMMSCs cDNA samples were examined by Affymetrix gene chips microarray analysis. The multipotent differentiation potentials of PBMSCs into osteoblasts, chondrocytes, and adipocytes were examined under specific inductive conditions and checked with lineage specific markers. Finally, the osteogenic potential of the PBMSCs was examined by an in vivo implantation model in which the PBMSCs were seeded with HA-TCP powder complexes, and implanted subcutaneously in the severe compromised immunodeficiency (SCID) mice for 12 weeks, whereas the bone-derived osteoblasts and skin fibroblasts were used as controls. Results: Compared with the BMMSCs, the PBMSCs shared some but not all common surface markers as demonstrated by (ICC) and flow cytometry examinations. The osteogenic differentiation of PBMSCs was defined with positive staining of type I collagen and osteocalcin; positive staining for alkaline phosphatase and Von Kossa staining for mineralized bone nodules. Adipogenic differentiation was evidenced by positive Oil red-O staining for accumulated lipids, and chondrogenic differentiation by positive type II collagen and Saferinin O positive staining. For gene expression profiles, in the Affymetrix chip general analysis, 83 genes were up regulated and 84 genes down regulated in the PBMSCs (vs BMMSCs, > 2 fold, E-B/B-E> 100, p< 0.05). Most of which genes are related to cell proliferation, differentiation, cytoskeleton, and calcium/iron homeostasis. After 12 weeks implantation in SCID mice, newly formed lamellar bone was clearly evident in the groups with PBMSCs implants, so as in the groups with osteoblasts implants, but only fibrous tissue was found in the group implanted with skin fibroblasts. Discussion: This study demonstrated that the multi-potent PBMSCs in the GFP rats resemble BMMSCs in many aspects, but they are distinguishable from the BMMSCs in some biological characteristics and gene profiles. Our study has confirmed that these PBMSCs possess osteogenic potential in vitro and in vivo, suggesting that these circulating stem cells could serve as an alternative source as bone marrow derived MSCs for tissue engineering purposes

Aim. The purpose of this study was to examine the symmetry of the mechanical and tomographic properties of the rat femur and tibia, frequently used in biomedical research. Materials-Methods. For this purpose both femora and tibiae of 43 Wistar rats underwent tomographic (pQCT) evaluation while both femora and tibiae of 40 further Wistar rats were subjected to destructive three-point bending testing. The age of the animals ranged between 3 and 12 months and all were males. Results. While there was significant variation between the structural and the biomechanical properties between different animals the differences between the femora and the tibiae of the same animal were not statistically significantly different in the majority of animals. Conclusion. We conclude that in healthy, sexually mature Wistar rats, in the first year of their life, there is no significant diversity in terms of tomographic and biomechanical properties of their opposing posterior limb long bones

1. Into osseous defects cut in the pelvis of rats, Kiel bone grafts were implanted after impregnation with the animals' own fresh bone marrow, obtained by femoral puncture. Unimpregnated Kiel bone grafts and Kiel bone grafts impregnated with an antibiotic solution were implanted as controls. 2. Histological examination of the implant area showed that in the marrow-impregnated grafts new bone formation could be observed after twelve days, and that during an observation period of 135 days after implantation bone formation occurred in thirteen out of nineteen rats. In four of these cases a continuous bony bridge developed over the defect. 3. In the unimpregnated grafts no more than a small amount of new bone was seen in only one of seven rats. In the antibiotic-impregnated grafts no bone formation was found in six rats during the same period of observation

Purpose: This study was designed to determine whether indomethacin, a potent anti-inflammatory agent, reduces muscle damage secondary to elevated ICP. Method: 16 adult Wistar rats were randomized to 4 groups. In group 1 (control), no intervention occurred. Group 2 (indo) rats were administered indomethacin (12mg/kg) with no elevation of ICP. Group 3 (CS) rats had elevated ICP (30–40mmHg × 45 minutes) using saline injection. Group 4 rats (CS/indo) had elevated ICP and indomethacin administration. After 45 minutes, hindlimb fasciotomy was performed. The extensor digitorum longus muscle was reflected onto an intravital microscope. Capillary perfusion was measured by comparing the number of continuously perfused capillaries to intermittent and non perfused capillaries. Inflammation was determined using the number of activated (rolling and adherent) white blood cells. Muscle cell damage was measured using differential fluorescent staining. Perfusion, inflammation, and muscle damage were compared in all 4 groups using a one-way ANOVA (p< 0.05). Results: Perfusion: Indomethacin treatment (CS/indo) increased the proportion of intermittently perfused capillaries (39.1 ± 2.2 vs 30.3 ± 2.7) and decreased nonperfused capillaries (38.4 ± 1.8 vs 50.1 ±2.5) compared to CS (p=0.0002). Control and indo groups demonstrated more continuously perfused capillaries compared to CS or CS/indo groups (p0.05). Conclusion: Treatment of elevated ICP with indomethacin improved microvascular perfusion and reduced cell damage. The protective mechanism of indomethacin is unknown, but may be related to an anti-oxidative and vasodilatory effect. Treatment of elevated intracompartmental pressure with indomethacin dramatically reduces muscle damage and may have important future clinical benefit. Further research is required to determine the mechanism of action

Purpose: The aim of this study was to investigate the results of end-to-side neurorraphy of the common peroneal nerve (CPN) to the tibial nerve (TN) in rats, after administration of bFGF or NGF. Materials: Five (5) groups of adult male Wistar rats, each comprising 25 animals, were studied:. End-to-side neurorraphy (4 groups) Group A bFGF (20ng) Group B NGF (25ng) Group C (normal saline) Group X [bFGF (20ng) + NGF (25ng)]. Negative control group (G) Animal keeping was conform to standard conditions set by the NIH (appropriate cages for housing; standard rat chow and water ad libitum; 12h – light/darkness exposure). All experimental procedures were performed under the supervision of a veterinarian and were prospectively approved by the Animal Experimental Ethics Committee. Methods: In groups A, B and X, the CPN was sharply divided at a distance of 7mm distal to its origin from the rat sciatic nerve; the proximal CPN stump was then sutured into the thigh muscles, whereas the distal CPN stump was sutured terminolaterally to the ipsilateral TN. Sub sequently, a total volume of fifty microliters (50μl) of the corresponding solution of growth factor(s) was administered in each case beneath the epineurium, proximal to the CPN/TN coaptation site, with the aid of a microsyringe. The same surgical procedure was carried out in group C (positive control group), as well, but an equal volume (i.e. 50μl) of normal saline was administered instead. Finally, in each of the animals of the remaining group G, both the proximal and the distal stump of the CPN were carefully sutured into the neighbouring muscles; hence, the latter would constitute a negative control group, thanks to the resulting atrophy of the CPN – innervated musculature. All surgical procedures took place with the animals under dissociative anaesthesia and were performed under sterile conditions, using the operating microscope and applying microsurgical techniques. In each case, the right CPN was operated upon; the contralateral (left) CPN remained intact, thus serving as control. Euthanasia was achieved by means of intracardiac administration of high – dose sodium pentobarbital. Results: The evaluation of the outcome four (4) months postoperatively was based on clinical examination, walking-track analysis, electromyographic and histomorphometric studies. Our data indicate that the administration of the growth factors under investigation has a favorable effect on the outcome of CPN repair; the administration of bFGF, in particular, seems to improve the results of terminolateral neurorraphy in the time span studied. Conclusions: In rats, CPN repair via end-to-side neurorraphy to the TN can be enhanced by the administration of bFGF or NGF

Apart from preliminary notices of present work, previous reports of experimental and clinical trials of the effects of a high-peak pulsed electromagnetic field (PEMF) on degeneration and regeneration of peripheral nerves lacked statistical analysis. Therefore, we designed experiments with standardised operative, histological, cytological and morphometric techniques to assess the effect of PEMF on lesions of the common peroneal nerves in paired male rats matched for age, environmental conditions and level and type of lesion. One of two types of lesion was induced in the left common peroneal nerve: in 12 pairs of rats the nerve was crushed just above the knee and in the remaining 12 pairs the nerve was cut and immediately sutured at the same level. The right common peroneal nerve of each rat served as a control. Animals received 15 minutes of PEMF produced by a Diapulse machine or sham treatment daily for periods ranging from three and a half days to eight weeks after injury. Healthy nerves were unaffected, but after damage there were statistically significant differences between PEMF treated and sham treated rats. PEMF accelerated the recovery of injured limbs and the degeneration, regeneration and maturation of myelinated axons; epineural, perineural and intraneural fibrosis was reduced; and the luminal cross-sectional area of intraneural vessels increased after both types of lesion. Findings are discussed and the need for clinical trials is stressed

The aim of our study was to evaluate if PTH is able to increase the trabecular density of osteoporotic bone at the site of an implant and whether the anabolic effect of PTH at this side is stronger then the effect of an osteoclast inhibitor like alendronate. 48 cement rod was inserted in the tibia of 48 female rats, of which 36 had been ovariectomized. The cement rods, which served as implants, were made of Palacos R bone cement. After implantation, the 36 ovariectomized rats were divided in 3 groups. One was injected subcutaneusly with PTH (1–34) at a dose of 60 g/kg BW. The second was injected with alendronate at a dose of 205 g/kg BW. The third with vehicle only. The remaining 12 sham operated rats were also injected with vehicle only. All injections were given three times a week and the rats were killed 2 weeks after implantation. The tibial segments around the hole of the rods were prepared histologically. Thus the surfaces which had been in contact with the rod appeared as straight lines and could be analyzed histomorphometricly. The trabecular density of the bone closest to the implant was measured. One femur of all animals was used for measurement by DEXA. There was a substantial increase in the trabecular density close to the rods with PTH treatment (Anova p=0.002). PTH lead to a trabecular density of 89%, where as the ovariectomized animals revealed a trabecular density of 58% and the sham operated control of 68%. No significant increase of implant related trabecular density could be found in the alendronate treated group. In this group a density of 72% was established. DEXA showed the expected differences in bone mineral content (Anova p=0.001). In this study, intermittent PTH treatment increased implant-related trabecular density in osteoporotic bone after 2 weeks. No such positive effect could be found with alendronate treatment at such a short period of time. We think the reason for this phenomenon could be the early onset of the anabolic PTH effect on regenerating bone, whereas alendronate is thought to only inhibit bone resorption, which might lead to a later effect. The early onset of PTH effects even in osteoporotic bone suggests that intermittent PTH treatment might lead to an increased micro-interlock between implant and bone and might therefore be considered as a possible drug to enhance incorporation of orthopedic implants

In vitro experiments have shown, that stabilisation of the fibula in complete fractures of the lower leg give more stability compared to a single stabilisation of the tibia. However it is not known how this biomechanical conditions influence the bone healing process. To investigate the effect of fibula stability in tibia fracture healing tibial osteotomies in rats with and without fibula fractures were compared. Male wistar rats (n=18) were operated by a transverse osteotomy of the proximal tibia of the left leg. Fracture was stabilised by intramedullary nailing. In 8 cases an additional closed fibula fracture was performed. The healing period was 21 days. Each whole leg was examined by x-ray. After explantation of the tibia and removing of the nail and the fibula, the tibia was examined by CT-Scan, three-point-bending and histological evaluation. Animals, who had a fibula fracture along with the tibia fracture presented with delayed healing. Density in CT-scan was 30% lower (p=0,0002) in animals with a fibula fracture (405mg/ccm, SD:64) compared to those without a fibula fracture (mean=577mg/ccm, SD:17). In three point bending the bending stiffness was 79% lower (p=0,0006) in animals with a fibula fracture (mean=252Nmm/mm, SD:118) compared to animals without a fibula fracture (mean=1219Nmm/mm, SD:478). The breaking force was 59% lower (p=0,0004) in animals with a fibula fracture (mean=17,5N, SD:6) compared to animals without a fibula fracture (mean=42,4N, SD:14). Complete fractures of the lower leg healed considerably worse than solitary fractures of the tibia. We conclude that the missing of rotational stability of our k-wire fixation of the tibia with a unfixed fibula fracture is one of the reasons for the delay in fracture repair. The results support the in vitro findings of the biomechanical importance of the fibula for the stability of tibia fractures

Introduction/Background: This study was designed to determine the osteoinductive capacity of rhBMP-2 in a non-critical size femoral defect in normal rats and rats with diabetes mellitus (DM). It was hypothesized that DM would result in impaired bone regeneration in the femoral non-critical size defects due to reduced bone formation and local delivery of rhBMP-2 would accelerate non-DM healing and normalize impaired bone healing in DM rats to the levels of Non-DM bone healing. Materials/Methods: A total of 80 BB Wistar rats were used in the project. A 3mm defect was created during surgery and stabilized with a polyimide plate and either a 0.05cc/11μg dose of rhBMP-2 or buffer in a collagen sponge was implanted into the defect. Microradiographs were taken on the day of sacrifice and processing of samples for PECAM-1 immunohistochemistry, histomorphometry, and mechanical testing was performed. Results: Both Non-DM and DM groups treated with rhBMP-2 demonstrated significantly higher radiographic scoring, total new bone formation, BV quantification, and mechanical testing parameters compared to those treated with buffer at all timepoints with no significance noted between Non-DM and DM groups treated with rhBMP-2. Discussion/Conclusions: This study reveals decreased amount of new bone formation in DM animals compared to Non-DM animals, showing the detrimental effects of DM upon bone healing. A single application of rhBMP-2 resulted in new bone formation in DM animals similar to Non-DM animals, suggesting a critical role for rhBMP-2 in ameliorating the deleterious effects of DM on bone regeneration and formation. Besides these groups 15 more DM rats were used for PECAM-1 staining for angiogenesis (7 with 1 loss at a 3 week time point) and mechanical testing (8 at a 9 week time point)

Introduction. Nowadays, autologous platelet-rich plasma is used commonly in wound treatment. However, platelet gel, which was derived from allogeneic platelet-rich plasma (PRP) [1,2], has never been studied about efficacy in vivo or animal models. We aimed to determine efficacy of allogeneic platelet-gel on wound healing in rats by comparing with untreated, antibiotic-gel (Mupirocin 2%) treated and gel (sodium carboxymethylcellulose(NaCMC))-treated control. Methods. Fresh frozen plasma was centrifuged at 1200-G for 15 minutes to extract PRP which would be freeze-dried at −70°c, sterilized with gamma ray of Cobalt source 25 kGy and stored at −70°c. Then, processed freeze-dried PRP was mixed with gel base (NaCMC) as in form of allogeneic platelet-gel concentrated 30 mg/1g by sterilization process (table 1). Full-thickness of 6-mm-diameter skin punch biopsies were performed on 18 female Wistar rats which each rat had four wounds at back. Each wound was applied with untreated care, antibiotic-gel, NaCMC-gel and platelet-gel, respectively. Wound healing was studied from day 0–12. Animals were sacrificed with wound tissues removal on day 3, 7, 12 post-biopsy. Digital planimetric measurement device (VISITRAK, Smith and Nephew) was used in evaluation of total wound area on day 0, 3, 7, 12 post-biopsy. Histopathological changes of wound healing were studied, using 4-μm thickness section with haematoxylin-eosin (H&E) and Masson's trichrome-stain, under light microscope. Results. Platelet-gel reduced wound size more rapidly on day 3, 7 than other groups with statistical significance (p<0.05), although no statistically significant difference compared to antibiotic-treated wounds. Histological study confirmed earlier granulation forming and more collagen fibers in platelet-gel treated group when compared with others. discussion & Conclusions. Allogeneic platelet gel produced the satisfactory efficacy on acute wound healing in rat. This platelet gel needs further study in human for efficacy and safety that might be developed for using in acute wound treatment in the future

Purpose: Intraarticular use of anaesthetic agents is common for postoperative pain relief after arthroscopic knee surgery. In this study, we have evaluated and compared the effects of Bupivacaine, Levobupivacaine and Tramadol both invivo and invitro experimental rat models on articular cartilage and chondrocytes. Materials and Methods: Invivo Experiment: 1. Injections: Thirty mature Sprague-Dawley rats weighing 230 – 300 g were randomized into 3 groups. Bupivacaine (Group 1), Levobupivacaine (Group 2) and Tramadol (Group 3) were injected into the right knee joints and physiological 0.9% saline into the left. 2. Histopathologic Analysis: The specimens were fixed, decalcified and stained with Hematoxylen and Eosin (H& E) and Toluidin Blue. All slides were examined by the same pathologist, who was blinded to the injectate used in each joint. All samples were evaluated histopathologically according to the recommendation of International Cartilage Repair Society’s osteoarthritis and cartilage histopathology grading and staging system. Invitro Experiment: Articular cartilage cells of the rats were cultured and seeded. Cartilage cell seeded samples (104 cells/mL) were incubated in three different anesthetic agents (0,5%); Bupivacaine, Levobupivacaine, and Tramadol respectively. Cell Titer 96TM Nonradioactivity Cell Proliferation (MTS) assay was used to determine the cell density on the samples. Results:. Invivo: There were pathologic changes like cartilage hypertrophy, active chronic inflammation with abscess formation, cellular proliferation, focal vertical fissures and focal discontunity on cartilage matrix at superficial zone in all three groups on the drug injected sides. Although those histopathologic findings were not found statistically significant when compared the OARSI grade, OA stage and OA score with the control groups (p> 0.05), statistically significant higher OARSI grade, OA stage and OA scores were detected when compared the Levobupivacaine injected group after 10 days with the Levobupivacaine injected group after 48 hours (p< 0.01 [ p=0.008]). Invitro: MTS results show that 0.5% Tramadol is cytotoxic to rat chondrocyte in vitro after 30 min of exposure. Also the cell number in both Bupivacaine and Levobupivacaine treated wells showed decrease throughout 15, 30 and 60 min exposures. Conclusion: No report has been appointed comparing the effects of the mentioned three drugs both invivo and invitro. Although chondrotoxicity of Bupivacaine was less harmful than Levobupivacaine and Tramadol, these findings suggest that local anesthetics negatively affect articular cartilage and chondrocytes. Given that chondrocyte loss has been implicated in the development of chondrosis and osteoarthritis, orthopaedic surgeons should be careful in their preference for pain control with intraarticular drug injections after arthroscopic procedures

1. In growing rats oestrogen, cortisone and thyroxine in high doses suppress bone formation, and this effect is probably part of a general suppression of body growth. 2. Growth hormone and thyroxine in small doses stimulate both body growth and bone formation. 3. Testosterone has no effect on bone formation. 4. Oestrogen and cortisone suppress bone resorption. The effect of cortisone may be modified in conditions of calcium depletion. 5. Thyroxine appears on the other hand to increase bone resorption. 6. Testosterone has no effect on bone resorption

1. Experimental arthritis was induced in rats by the intradermal injection of modified Freund's adjuvant. 2. The granulation tissue occurring in and around the joints was examined with the electron microscope. 3. Intracellular collagen was demonstrated in many of the cells. 4. Collagen formation by these cells was studied by autoradiographic techniques using tritiated proline as a label. 5. The proline turnover was rapid, as most of the labelled proline had become extracellular one hour after its injection. 6. It was concluded that the collagen was present within the cells as a result of phagocytosis despite the fact that the cells had the electron microscopic features of fibroblasts

Introduction: The purpose of this study was to determine whether regional blood flow (microspheres) in the femur is diminished with aging, and whether a reduction in flow is associated with impaired endothelium-dependent vasodilation. Materials and Methods: Blood flow and PNA endothelium-dependent vasodilation was measured in young (4–6 months old) and aged (24–26 months old) male Fischer-344 rats. Results: Blood flow in the aged rats was ~25% lower in femoral bone and 45% lower in diaphyseal marrow. Endothelium-dependent vasodilation was lower with old age (young: 83 ± 6% maximal relaxation; aged: 62 ± 5% maximal relaxation) and was mediated through impairment of the NOS signaling pathway, which resulted in a lower nitric oxide bioavailability (young: 168 ± 56 nM nitric oxide; aged: 50 ± 7 nM nitric oxide). Discussion: Such age-related changes in bone perfusion and nitric oxide signaling could impact clinical bone loss, increase risk of fracture, and impair fracture healing in the elderly

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

1. The distribution of isotope following a single injection of either Ca. 45. or C. 14. -proline has been studied in young rats in which one tibia had previously been removed, killed and reimplanted. 2. The dead tibia took up about 25 per cent as much Ca. 45. or C. 14. as did the living tibia and the possible processes by which this occurred are discussed. 3. Determination of the "accretion rate " by kinetic analysis of the Ca. 45. data showed that this was much too high unless the physico-chemical process of uptake of isotope by bone was taken into account. 4. Under the conditions of the experiment it was not possible to estimate the rate of bone matrix formation using C. 14. -proline as a tracer

The aetiology of idiopathic scoliosis, despite of long-lasting efforts to disclose it, remains unknown. The purpose of the study was to evaluate the spine development after pinealectomy or cortical sensory motor area damage in the growing rats. Method: The authors operated 69 Wistar albino rats (aged three to four weeks) in antraperitoneal anaesthesia. In the first group (22 rats) pinealectomy – PIN was performed, in the second one (25) the sensory motor cortical area 2x1x1 mm bellow the coronal suture was removed – SMCA. The sham operation consisted of craniotomy – CRA (11 rats) and craniotomy with durotomy – CRDU (11 rats). All surgeries were performed from the left side. Radiography was made three months after surgery. Scoliosis, C2-T7 lordosis, T7-S1 kyphosis were measured. Results have been processed by software Statistica 7.1. StatSoft, Inc. (2005). We used ANOVA test for evaluation of potential difference between groups, in the case of approving the difference between groups, we tested difference between each two groups by two-sample t-test. Those tests were realised on 0,05 significant level. Results: In the PIN group scoliosis 9–14 degrees (mean value 10,8) developed in five animals, in SMCA group scoliosis 10 – 24 degrees (mean value 15,9) was observed in eight animals. These statistically significant differences were found: higher surgery weight in PIN, longer surgery time in PIN and SMCA, lower lordosis in PIN and higher in CRDU, differences of all groups in kyphosis and in an end weight. Conclusion: Our results indicate the importance of cortical area damage, together with craniotomy and durotomy in the development of growing rat spine. We cannot exclude the influence of peri-operative bleeding, brain hypoxia or metabolic effect of anaesthetics. These damages could cause a disorder of balance between smaller inhibitory and greater facilitating area of CNS, controlling the muscular tone and resulting in the development of lordosis and scoliosis due to muscle imbalance

The parameters of cellular proliferation and growth in the growth plates of immature rats were measured after unilateral tibial osteotomy and used to calculate growth rates. Distal osteotomy of one tibia was followed by a bilateral increase in the calculated growth rate of the distal growth plates. However, the ipsilateral distal growth plate grew faster than the contralateral between 12 and 18 days after operation, which appeared to be related to increased cell proliferation and height. Proximal osteotomy led to an increase in growth rates proximally which was more marked on the contralateral side. The lesser response of the ipsilateral growth plate may have been due to local impairment of blood supply, or to greater local release of metabolites after bony damage. Distal tibial osteotomy gave similar results to circumferential release of the distal tibial periosteum. Proximal osteotomy, however, produced a relative impairment of growth on the operated side. This may be of importance in the correction of childhood deformities associated with inequality of leg length

AO Spine Reference Centre & Institute of Health & Biomedical Innovation, Queensland University of Technology, Brisbane, Australia. Traumatic spinal cord injury (SCI) is a devastating condition with no curative therapy. Pro-inflammatory therapy has been suggested recently to try and reduce the inhibitory glial scar and promote neural regeneration and healing. The aim of this study is to investigate the potential of sustained delivery of angiogenic/pro-inflammatory growth factors to reduce the secondary degeneration after spinal cord injury. Adult male Wistar Kyoto rats (200-300g; 12-16weeks old) were subjected to cord hemisections via a T10 laminectomy. Animals were randomised to treatment or control groups after the spinal cord injury had been induced. Treatment consisted of implantation of a mini-osmotic pump capable of delivering 5 micrograms vascular endothelial growth factor (VEGF) and 5 micrograms platelet-derived growth factor (PDGF), via a catheter, to the site of the lesion, over 7 days(n=6). Control animals were subjected to either cord lesion only (n=6) or lesion plus mini-pump delivering PBS (phosphate-buffered saline) solution (n=6). Rats were sacrificed at one month and the spinal cords were harvested and examined by immunohistology, using anti-neurofilament-200 and anti-Glial Acidic Fibrillary Acidic Protein (GFAP) antibodies. RESULTS: Active treatment spinal cords showed a higher level with aboration of the axonal filament through the defect and more dense neurofilament-200 staining at the lesion site compared to both control groups. The treatment also showed the elevated presence of activated microglia in the lesion, whilst distal to the lesion the microglia and astrocytes retained an unreactive phenotype. Pro-inflammatory therapy in the rat spinal cord-injury model showed favourable histological findings after sustained delivery of PDGF and VEGF

The right sciatic nerve of 50 one-month-old male rats was cut under general anaesthesia. Groups of animals were sacrificed at intervals of up to 12 weeks after operation and the length of the femora, tibiae and first and fifth metatarsals were measured with a caliper accurate to 0.05 mm. From the first week, both metatarsals were between 3% and 5% shorter on the denervated side, but there was no further increase of the discrepancy. The femora were less than 1% longer in the denervated limb at the second and eighth week. No difference was found between the lengths of the tibiae. The various factors which could possibly be responsible for these findings are discussed

1. Dislocation and subluxation of the hip has been produced in young rats by application of splints reaching from the hip to the foot, bringing the hip into extension. 2. Progressive acetabular dysplasia and anatomical abnormalities of the head and neck of the femur occurred. 3. Results of the experiments suggest that post-natal extension of the hip is of importance in the pathogenesis of congenital dislocation of the hip in man

1. A method of constricting sciatic nerves of rats was devised which produced lesions resembling macroscopically and electromyographically those of carpal tunnel and related syndromes. 2. The nerves became swollen and hyperaemic proximal and distal to the constriction. The swellings were largely caused by an accumulation of fluid between the axons, but the axons themselves were also increased in size. 3. This accumulation of fluid was an oedema secondary to a partial obstruction of the vasa nervorum

Introduction: Patients who are prescribed bisphosphonates are still at risk to endure a fracture from weak and brittle bones. The question is what pharmacologic strategy should be taken to accelerate fracture healing when the patient is currently taking a bisphosphonate. Ibandronate, was tested in an osteoporotic rat model to determine how it modified the callus healing and resistance to torsion after a transverse fracture was produced in a femur. Materials and Methods: 36 female rats were divided into 3 groups; ovariectomized (OVX) placebo control, non-OVX control and Ibandronate. Prior to the osteotomy, the Ibandronate treatment group was injected with the drug over 21 days healing. Each sample was scanned by the SCANCO uCT 80 to measure volume of the callus and quality of the trabeculae in the proximal femur. Instron testing recorded the modulus of rigidity and torque until failure. Yield point and toughness were also calculated. Results: uCT images taken over the fracture gap showed that the Ibandronate rats had greater bone volume fraction of woven callus by ANOVA compared to control groups (p< 0.05). Significant in total callus volume for Ibandronate, were shown to be 32% larger than the non-OVX control group and 45% larger than the placebo group. Ibandronate also increased BMD of woven bone in the callus by 14%. Ibandronate showed the highest polar moment of inertia as well. The torsion testing in Ibandronate had 51% greater toughness than placebo and 69% greater than the non-OVX group. Ibandronate increased trabecular number significantly over the placebo and was not significantly different from the non-OVX group. Trabecular separation was less in Ibandronate compared to the placebo group. Volume in the trabecular neck increased by 35% for the Ibandronate over the placebo. Discussion: Ibandronate had an anabolic effect to produce more callus tissue at the fracture site, most likely by suppressing osteoclast remodelling activity. A large callus with more bone would increase fracture stability and reduce risk of non union. This is supported by a larger polar moment of inertia. Ibandronate had greater resistance to torsion, which could indicate better healing. However increased rigidity would not entirely benefit the healing unless the bone could handle load plastically. The toughness results showed that Ibandronate can absorb more energy than the control groups before refracturing. Continued treatment with this drug after a fracture could form a larger callus with greater mechanical toughness while also treating the disease of osteoporosis in other fracture risk sites of the body

Sclerostin is a negative regulator of osteoblast differentiation and bone formation. Expressed by osteocytes, it acts through antagonising the Wnt/â-catenin pathway and/or BMP activity. Distraction osteogenesis, used for limb lengthening and reconstruction, can be complicated by disuse osteopenia and poor healing response, both of which would benefit from pro anabolic therapy. We examined the effects of Sclerostin Antibody (Scl-AbIII, Amgen Inc.,) in a rat model of distraction osteogenesis. A femoral osteotomy was stabilized with an external fixator in male Sprague Dawley rats. After a week of latency, the gap was distracted twice daily for 14 days to a total of 7 mm. Saline or Scl-Ab was administered twice weekly throughout the distraction period and up to 4, 6 or 8 weeks post commencement of distraction. Three groups were examined: Saline, Continuous Scl-Ab throughout the study (C Scl-Ab), and Delayed Scl-Ab with commencement of Scl-Ab after distraction (D Scl-Ab). Regenerate bone mineral content (BMC), determined by DEXA, was increased 36% at 4 weeks and 86% at 6 weeks with C Scl-Ab, resulting in a 65% increase in bone mineral density (BMD) at 6 weeks, compared with Saline (p<0.01). D Scl-Ab treatment showed a 41% increase in BMC and a 31% increase in BMD compared with Saline at 6 weeks (p<0.05). At 8 weeks, C Scl-Ab remained significantly increased over Saline (72% in BMC; 60% in BMD). Micro-CT scans of the regenerate revealed increases in bone volume of 88% with C Scl Ab and 65% with D Scl-Ab compared with Saline at 6 weeks (p<0.05). By 8 weeks, these increases were 36% for C Scl-Ab (p<0.05) and 37% for D Scl-Ab compared with Saline (p<0.01). Importantly, mean moment of inertia was increased over two-fold in both Scl-Ab groups at 6 weeks compared with Saline (p<0.05). Histology at 6 weeks confirmed micro-CT data with 85–88% increases in bone volume/tissue volume (BV/TV) in the regenerate with both C Scl-Ab and D Scl-Ab compared with Saline (p<0.05). Analysis of bone formation at 6 weeks revealed increases in mineral apposition rate of 56% in C Scl-Ab and 52% in D Scl-Ab compared with Saline (p<0.05). Scl-Ab treatment increased bone formation in this model of distraction osteogenesis, resulting in a larger regenerate callus (increased BMC and BV/TV). We expect further studies to reveal increases in mechanical strength. Scl-Ab may hold promise as a therapeutic to accelerate regenerate formation and consolidation in distraction osteogenesis for limb reconstruction

An orientated substratum has been implicated in the development and regeneration of axons and synapses. We prepared a basement membrane matrix from autogenous striated muscle, used it to repair the sciatic nerve in rats, then investigated the results by histology and electrophysiology. When treated grafts were coaxially aligned with the nerve fascicles functional recovery appeared within 30 days, with good growth of axons into the distal nerve. Grafts with myotubes at right angles to the nerve fascicles supported nerve regeneration but at a slower rate. Grafts of coaxially aligned but untreated muscle allowed axon penetration only through naturally degenerated muscle fibres, with minimal axon penetration of the distal nerve. It is concluded that in the rat a treated graft with correctly orientated empty myotubes can facilitate and guide the regeneration of peripheral nerve after injury and so lead to recolonisation of the distal stump with functional recovery

Introduction: The aim of this study was to investigate if steroids enhance the vasoconstrictive effect of nor-adrenaline on femoral arteries, which may result in femoral head blood flow reduction. Materials and Methods: Ten male Wistar rats 62 to 88 days of age, 254 to 318 g of body weight, were used. Twenty femoral artery segments were harvested. These arterial segments were mounted as ring preparations on a small vessel myograph for isometric force measurements. The arteries were stimulated cumulatively with noradrenaline before and after incubation with methylprednisolone (5 μg/ml). Isometric wall tension was plotted and quantified by the EC50, the vasoconstrictor concentration resulting in halfmaximal contraction. Results: The noradrenaline dose-response curve displayed a shift to the left for the steroid group in relation to the controls. This was reflected by a significantly lower EC50 of 9.5*10. −7. M ± 5.1*10. −7. M for the steroid vessels compared to 2.5*10. −6. M ± 1.1*10. −6. M for the control vessels (mean ± SD; p< 0.005). Discussion: This study showed that incubation with methylprednisolone enhanced noradrenaline-mediated contraction of femoral arteries. Enhanced contraction of femoral arteries can diminish blood flow within the vascular bed supplying the femoral head. This may be a relevant cofactor in the early pathogenesis of steroid-associated femoral head necrosis

After the simultaneous administration of radiocalcium and radiophosphorus to young rats the rate of deposition of calcium and of phosphorus in various skeletal parts was computed. Agreement was found between the two sets of data. No difference was thus found in the metabolism of the calcium and of the phosphorus of the bone salt

1. An experimental study of the healing mechanism in circumscribed defects in femora of albino rats of the Wistar strain is described. 2. Only the outer one-fifth of the defect is repaired by subperiosteal bony callus, the rest of the defect being repaired by endosteal callus. 3. Subperiosteal callus does not bridge the defect until endosteal callus is developed fully. 4. As peripheral callus matures the greater part of the endosteal callus is resorbed, with the exception of trabeculae attached to the margin of the defect. 5. The resorbed area in the medullary part of the defect is gradually obliterated by deposition of inner circumferential lamellae. 6. There appear to be differences between the mechanism responsible for repair of fractures of a long bone and that which heals circumscribed bone defects

1. Some physical properties of living and dead bone have been studied in rats; most of these are interrelated and ultimately depend upon the composition of the tissue. 2. Dead bone, remaining within the body, does not take up measurable amounts of mineral from the tissue fluid but retains its original physical properties of radiographic density, specific gravity, strength and composition. 3. The altered radiographic density of avascular bone seen in clinical practice is almost certainly relative unless there has been concomitant appositional new bone formation. 4. Some other explanation must be sought for the finding that dead bone takes up significant amounts of bone-seeking isotopes in radioactive tracer studies

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

We examined the cellular responses to various particles injected into the knees and the intramedullary femoral cavities of rats in the presence of polymethyl-methacrylate (PMMA) plugs. The intra-articular particles were mainly ingested by synovial fibroblasts. Increased numbers of macrophages were not detected and there was only a slight increase in synovial thickness. Cellular responses in the intramedullary space were similarly mild and bone resorption around the PMMA plug did not occur. Bone formation was inhibited only by polyethylene particles. In contrast to current views, our study shows that wear particles per se do not initiate bone resorption

The purpose of this study was to understand the effects of terminal sterilisation and residual calcium on human demineralised bone matrix (DBM) in ectopic bone formation in nude rat. The intramuscular implantation of human DBM prepared by the Queensland Bone Bank (QBB) from four donors into eight male athymic rats was used to assess osteoinductivity. The DBM contained different levels of residual calcium and treated with or without gamma-irradiation at 11kGy. At 6 weeks post-implantation, calcium deposition was assessed by manual palpitation and radiological imaging. Tissue morphology and cellular interactions was analysed using various histological staining methods whilst protein expression of anabolic and catabolic biomarkers were examined through immunohistochemistry. All results were then analysed in qualitative, semi-quantitative and quantitative manners and tested for statistical significance. Bone formation was observed in all specimens at the gross level. This was confirmed by histology which revealed bony capsules surrounded by soft tissue in the muscle pockets and differences in tissue components. On a cellular level, variations in osteoclast expression were found between the two groups as well as amongst individual donors through statistical analysis which resulted in an imbalance of the expression of anabolic and catabolic markers. Furthermore, a positive relationship between residual calcium and new bone formation in gamma irradiated DBM samples was found. To date, no studies have compared the effect of calcium in gamma irradiated DBM. Our results suggest that gamma irradiation even at low doses and residual calcium may affect new bone formation. Taken together, this study stresses the importance of selecting ideal conditions for graft processing and the need to identify an optimal level of irradiation and remaining calcium levels that confers a balance between osteoinductivity and sterility

Continuous strontium administration first induces typical "rickets" in young rats receiving adequate calcium phosphorus and vitamin D but later the widened cartilage spontaneously calcifies intermittently leaving transverse bands consisting largely of osteoid tissue in the metaphysis; in addition to intermittent calcification bone changes indicate that skeletal growth is not uniformly progressive. Subsequently areas of the epiphysial cartilage fail to calcify and localised defects develop; among these are wedge-shaped metaphysial osteoid tissue masses, "invagination" of the epiphysial plate to form multiple nodules of cartilage with proliferating cells in the middle and hypertrophic ones at the periphery, perforation and fragmentation of the epiphysial plate with formation of large cartilage nodules. Multiple cartilage nodules of different sizes appear in the epiphysis, metaphysis and bone shaft. Most bone margins are lined by osteoid seams which only slowly calcify and concomitantly resorption is decreased so that the rate of remodelling of the skeleton is diminished. This type of process may help to explain the results of treatment of osteoporosis by strontium administration

We have studied the effect of hydroxyapatite (HA) coating in 15 ovariectomised and 15 normal rats which had had a sham procedure. Twenty-four weeks after operation, HA-coated implants were inserted into the intramedullary canal of the right femur and uncoated implants into the left femur. The prostheses were removed four weeks after implantation. Twelve specimens in each group had mechanical push-out tests. Sagittal sections of the other three were evaluated by SEM. The bone mineral density (BMD) of the dissected left tibia was measured by dual-energy x-ray absorptiometry. The difference in BMD between the control and ovariectomised tibiae was 35.01 mg/cm. 2. (95% CI, 26.60 to 43.42). The push-out strength of the HA-coated implants was higher than that of the uncoated implants in both groups (p < 0.0001), but the HA-coated implants of the ovariectomised group had a reduction in push-out strength of 40.3% compared with the control group (p < 0.0001). Our findings suggest that HA-coated implants may improve the fixation of a cementless total hip prosthesis but that the presence of osteoporosis may limit the magnitude of this benefit

Introduction. In degenerative disorders of the spine such as disc herniation, intervertebral discs can affect neural tissue, which may result in pain as demonstrated in both basic science and clinical investigations. Previous in vitro and in vivo studies have shown that notochordal cells and chondrocyte-like cells in nucleus pulposus affect nervous tissue differently. The aim of the present study was to evaluate the morphology of spinal neural tissue in an in vivo rat model following application of cells derived from nucleus pulposus. Material and method. A disc herniation model in rats (n=58) was used. The L4 nerve root was exposed to a) nucleus pulposus (3mg), b) notochordal cells (25,000 cells) or c) chondrocyte-like cells (25,000 cells). Four control groups were included: 1) application of nucleus pulposus (3 mg) and mechanical displacement of the spinal nerve complex, 2) sham operated animals, 3) application of cell diluent (50 μl) and 4) naïve animals. Seven days after surgery the L4 nerve roots with their dorsal root ganglion were harvested and prepared for blinded neuropathological examinations using light microscopy. Results. Damage and loss of myelinated nerve fibers as well as epineural granulation tissue were most pronounced in the group that had been subjected to nerve root displacement and application of nucleus pulposus. There was significantly less nerve fiber damage in all other groups. The number of myelinated nerve fibers with enlarged outer Schwann cell compartment was significantly higher in all experimental groups as compared to naïve animals, except for animals in which the nerve root complex had been exposed to cell diluent, notochordal cells and chondrocyte-like cells. Discussion and Conclusion. This is the first examination nerve root and dorsal root ganglion morphology after exposure to notochordal cells and chondrocyte-like cells in an in vivo model. The results indicate that application of notochordal cells and chondrocyte-like cells, per se, do not structurally affect the myelinated nerve fibers compared to naïve animals. However, one cannot exclude that there may be physiological effects of notochordal cells and chondrocyte-like cells on nerve tissue in vivo although no morphological differences were observed with the present method. The findings in the present study support previous observations that mechanical nerve tissue displacement and application of nucleus pulposus can induce pronounced morphological nerve tissue changes. However, the combination of mechanical nerve tissue displacement and application of notochordal cells and/or chondrocyte-like cells was not tested. In conclusion, the present study suggests that mechanical nerve tissue displacement is a prerequisite for the induction of morphological changes following application of disc tissue and its components on neural tissue. Summary. The effects of notochordal cells and chondrocyte-like cells on spinal nerve tissue might be dependent on concurrent mechanical nerve tissue deformation

The main findings in this experimental work on rats fed on lathyrus odoratus (sweet-pea) meal are as follows:. 1. Growth is retarded. 2. The growth plate is disorganised and normal ossification at the metaphysis is interfered with. 3. The small blood vessels are seriously affected and probably contribute quite largely to the disorganisation and lack of calcification. 4. Alkaline phosphatase activity is increased. 5. Raising of the periosteum and laying down of new bone result in exostoses. The possible underlying etiology and the role of cement substance, endocrine factors and the blood vessels are discussed

Introduction: Since Albright first proposed the concept of diabetic osteopenia, many studies have investigated the levels of mineral bone density (BMD) and risk of osteoporosis. In this study we investigate the effect of exercise, alfacalcidol and parathyroid hormone (1–34) on bone marker, BMD and bone mechanical properties in spontaneously diabetic GK/Jcl rats. Methods: 18 week-old male GK/Jcl rats were divided into 4 groups; no treatment (NT), exercise (Ex), alfacalcidol (ALF), and parathyroid hormone (PTH). The bone mineral density (BMD) of the lumbar vertebrae (L2-L4) and the left femur was measured by dual energy X-ray absorptiometry (DXA). Serum calcium (Ca), inorganic phosphorus (Pi) and osteocalcin (OC) were measured. Urinary Ca, Po, and creatinine (Cre) were measured. Urinary deoxypyridinoline (D-Pyr) was measured and the data were corrected for urinary Cre concentration. Mechanical strength of L5 was measured by the compression test. The mechanical strength of the right femur was measured by the three-point bending test. Results: The serum Oc levels in Ex and ALF group slightly increased (mean 5%). The serum Oc in PTH group increased significantly compared with that in the NT group (mean 70%). The urinary D-Pyr/Cre in the Ex group decreased compared with that in the NT group (mean 9 %). The urinary D-Pyr/Cre in the groups treated with ALF for 3 months were significantly decreased compared with that in the NT group (mean 20%). The urinary D-Pyr/Cre in the PTH group significantly increased compared with that in the NT group (mean 10%). The BMD of the L2–L4 in ALF group increased compared with NT group (mean 12%). The BMD of the L2–L4 in PTH group significantly increased compared with NT group (mean 10%). In the ALF group, however, the mechanical strength of the lumber vertebra was significantly higher (mean 25%) than that in the NT group. In the PTH group, the compressive load of the lumber vertebra (mean 70%) and breaking strength of the femur (mean 9%) was significantly higher than that in the NT group. Discussion: Treatment of osteoporosis has so far mainly utilized anti-resorptive agents such as estrogen, calcitonin and bisphosphonate, and bone anabolic agents stimulating bone resorption would be useful especially in low-turnover type of osteoporosis such as diabetic osteopenia. ALF treatment suppressed osteoclastic bone resorption while maintaining or even stimulating bone formation, and consequently increased bone mass with a parallel improvement in the mechanical strength of bone. PTH (1–34) had strong effects for improve the mechanical strength of the spine. In conclusion, it was demonstrated that ALF and PTH differed in their potency for improving the strength of the spine. Our results of biochemical parameter analysis demonstrated that ALF caused a significant suppression of bone resorption and maintained formation. The other hand, PTH had a strong effect on stimulating the bone turnover and bone strength, whereas it could affect the bone quality and reduce the risk of the spine fracture. These results provide important clues in understanding the action mechanisms of these agents on bone metabolism in the treatment of diabetic osteopenia

It has been shown in experimental animals that the living cells in a bone autograft can make an important contribution to osteogenesis. However, some common clinical techniques, such as the topical use of antibiotic powders on grafts or on the graft bed, are likely to damage or kill the cells. In this experimental study in rats, bone isografts dusted with chloramphenicol or methicillin powder or with Polybactrin spray before subcutaneous implantation produced little or no new bone over a period of two weeks whereas untreated, control grafts showed abundant osteogenesis, as did grafts pretreated with solutions of antibiotics. The effect of short-term storage of the grafts for 3 to 24 hours in air, saline or culture medium before implantation was also examined. Grafts stored in culture medium generally did as well as, or better than, fresh control grafts whereas immersion in saline inhibited osteogenesis. The importance of these results for clinical bone grafting is discussed

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

Purpose: Severe fractures damage blood vessels and disrupt circulation at the fracture site resulting in an increased risk of poor fracture healing. Endothelial progenitor cells (EPCs) are bone-marrow derived cells with the ability to differentiate into endothelial cells and contribute to neovascularization and re-endothelialization after tissue injury and ischemia. We have previously reported that EPC therapy resulted in improved radiographic healing and histological blood vessel formation in a rat fracture model. The purpose of this study was to further quantify the effects of EPC therapy with microCT and biomechanical analyses. Method: Five-millimeter segmental defects were created and stabilized in the femora of 14 fisher 344 rats. The treatment group (n=7) received 1x106 EPCs within gelfoam locally at the area of the bone defect and control animals (n=7) received only saline-gelfoam with no cells. The formation and healing of bone after 10 weeks were asessed by radiographic, micro-CT and biomechanical analyses. Results: Radiographically all the animals in EPC-treated group healed with bridging callus formation, whereas control group animals demonstrated radiographic non-union. Micro-CT assessment demonstrated significantly improved parameters of bone volume (35.34 to 20.68 mm. 3. , p=0.000), bone volume density (0.24 to 0.13%, p=0.001), connectivity density (25.13 to 6.15%, p=0.030), trabecular number (1.14 to 0.51 1/mm, p=0.000), trabecular thickness (0.21 to 0.26 mm, p=0.011), trabecular spacing (0.71 to 1.88 mm, p=0.002), bone surface area (335.85 to 159.43mm, p=0.000), and bone surface to bone volume ratio (9.43 to 7.82 1/mm, p=0.013) in the defect site for the EPC group versus the control group respectively. Biomechanical testing showed that the EPC treatment group had a significantly higher torsional strength compared with the control group (EPC=164.6±27.9 Nmm, Control=29.5±3.8 Nmm; p value = 0.000). Similarly, the EPC treated fractures demonstrated significantly higher torsional stiffness versus controls (EPC=30.3±5.0 Nmm/ deg, Control=0.9±0.1 Nmm/deg; p value = 0.000). When biomechanically compared to contralateral intact limbs, the EPC treated limbs had similar torsional stiffness (p=0.996), but significantly lower torsional strength (p=0.000) and smaller angle of twist (p=0.002). Conclusion: These results suggest that local EPC therapy significantly enhances fracture healing in an animal model. The biomechanical results show that control animals develop a mechanically unstable non-union. In contrast, EPC therapy results in fracture healing that restores the biomechanical properties of the fractured bone closer to that of intact bone

Revascularisation of syngeneic and allogeneic intramuscular bone grafts have been studied using radioactive microspheres to measure the ingrowth of blood vessels. New bone formation and resorption were measured by 85strontium uptake and by graft weight reduction. Revascularisation, and mineralisation rate were significantly higher in syngeneic grafts than in allogeneic grafts at two, three and six weeks after implantation. The syngeneic grafts lost weight faster indicating that the allogeneic grafts resorbed more slowly. The ingrowth of new vessels is impaired in allogeneic bone, and this probably inhibits the rate of bone formation and resorption of the grafts.

We examined solvent-dried, gamma-irradiated (SD-R) allografts and fresh-frozen (FF) allografts mechanically and morphologically. Before transplantation, FF grafts were more than six times stronger than SD-R grafts. After four weeks, the tensile strength was about the same in both groups. At 24 weeks only collagen fibrils of small diameter were observed in the SD-R grafts while in FF grafts fibrils of small and intermediate diameter were seen. Clinically, we suggest that SD-R grafts could be used as a favourable alternative to FF grafts if care was taken regarding their initial mechanical weakness.

1. The radiographs of paired living and dead rat tibiae, obtained in an experiment previously reported, have been examined by densitometry.

2. The dead bone became progressively less dense than the living bone as the duration of the implantation increased.

3. The change in density was related to the quantity, but not to the quality, of the bone tissue examined.

1. The results of the present investigation indicate that in the foetal rat the juxta-epiphysial vascular bed consists of a dense irregular network of sinusoids in direct contact with the growth cartilage, supplied by end-arteries, and drained by a profusion of metaphysial sinusoids.

2. The circulation is a closed one–that is, the endothelium is unbroken in its continuity and microhaemorrhages do not occur against the cartilage.

3. It is possible that juxta-epiphysial endothelial cells or their derivatives are chondrolytic, and that they participate directly, together with other mesenchymal derivatives, in the removal of cartilage as a preparatory stage in enchondral bone formation.

One of the aims of this work was to find criteria by which the quality of bone as a supporting tissue might be judged. This inevitably involves discussion and, if possible, assessment, of the relative importance of the inorganic and organic material of the bone. It is relatively easy to measure the mineral content, and for that reason it has always received more than its due share of attention.

In the present experiment the composition of the ash of all bones was remarkably constant, with a Ca/P ratio of 2. Furthermore, X-ray crystallography showed that the structure of the inorganic material was the same in all cases. The great difficulty of measuring variations in the quality of the organic material which is, of course, protein in nature makes it impossible to say how much it influences bone strength. Since at least 40 per cent. of the bone is collagen, either a quantitative or a qualitative alteration might alter bone strength. X-ray crystallography revealed no qualitative differences in the collagen material of bones of the three groups; so that for the present it would seem safer to assume that alterations in the physical properties of the bones are due to variations in the relative proportions of organic and inorganic constituents (Dawson 1946, Bell et al. 1947).

These experiments show that the three diets produce highly significant differences in the percentage of ash, in S_B, and in E. It is possible that some variations in the percentage of ash are due to variations in the absolute collagen (weight of collagen in unit volume of bone substance); but the range of variation in the percentage of ash leaves no reasonable doubt that differences in percentage ash between the diet groups are due essentially to differences in absolute ash. Presumably the collagen contributes something to the strength of the bone; but the indications are that it plays a minor part and that the relative weakness and flexibility of rachitic bones is due to decrease in the absolute ash content. Within any one diet group, the relation between percentage ash and the other two variables, S_B and E, is masked by other sources of variation such as those associated with the many measurements involved; and thus the correlation between percentage ash and S_B, and also between percentage ash and E, is not significant.

At first sight, the scatter diagrams (Figs. 5 and 6) appear to indicate a correlation between ash and S_B, and between ash and E. Closer inspection shows, however, that the apparent trend is due largely to differences between the means of the diet groups, and that the points within any one group show no such obvious trend. Figure 7 shows that the position with regard to correlation between S_B and E is very different. Here there is an obvious trend within each diet group; the amount of scatter is very much less. Calculation shows that, even when the differences between the means of diet groups is excluded, there is still a significant correlation between S_B and E. The question of the correlation between the three variables is discussed more fully in the addendum to this paper.

Although the "goodness" of a bone is usually judged by its breaking stress, the experimental findings recorded above suggest that it may be assessed equally well on the basis of elastic properties as shown by Young's modulus. Normal bones, group S in these experiments, were elastic up to 79 per cent. of their breaking stress (Table II): the poorer bones of groups R and N were, however, only a little inferior in this respect. In some cases there was no apparent deviation of the load-deflexion curve from a straight line until the bone was about to break. Such a curve was published in the first paper of this series (Bell, Cuthbertson and Orr 1941), but in the light of further experience this curve is scarcely typical. The terminal falling over of the curve is illustrated in Figure 4 and is much more marked in the bones of group R.

While stress at the upper limit of elasticity varies over a wide range in the three groups (Table II and Fig. 4), the strain at this point is remarkably constant at about 1·5 per cent. This same percentage displacement must occur between the molecules of the bone material at the elastic limit—and it may be that, up to this amount of molecular displacement, the deformation is reversible; but that beyond it, plastic changes occur. We have no evidence as to whether the limiting displacement concerns mineral or protein constituents of the bone, or both.

We have already commented on the remarkable strength of bone material (Bell et al., 1941). The breaking stress of normal rat bone is about the same as that of cast iron, and about half that of mild steel. Young's modulus, however, is only one-tenth that of cast iron and one-twentieth that of steel. Thus bone, despite its lightness (specific gravity about 2·5 as compared with 7·9 for iron), is remarkably strong and at the same time more flexible than might be expected. Presumably the biological advantage is that greater flexibility helps to absorb sudden impacts. It is unusual in metallic substances to find the elastic modulus proportional to the strength; this is more characteristic of materials like concrete and timber. Another remarkable property of bone is that it remains elastic up to three-quarters of the breaking stress. Most metals show considerable ductility before reaching their breaking point.

While Young's modulus is of interest, both on its own account and as an index of the quality of the bone, its close association with breaking stress suggests that it might be used to predict the maximum load which a bone can carry safely. Since E, unlike S_B, can be measured without damage, useful information might be gained by measuring the elasticity of living human bones.

Experimentally produced fractures in long bones studied by light and electron microscopic histochemistry were found to heal by a process of enchondral calcification. There was intense proliferation in the cells of the cambium layer of the periosteum, with differentiation to chondroblasts and osteoblasts, suggesting that this layer was the primary tissue responsible for development of the callus. Cytoplasmic processes of the hypertrophic chondrocytes appeared to bud and produce matrix vesicles. Alkaline phosphatase activity was detected along the plasma membrane of the hypertrophic chondrocytes and around the matrix vesicles, before any signs of mineral deposition. Calcification took place by deposition of hydroxyapatite crystals in and around these matrix vesicles which frequently showed alkaline phosphatase activity. It is suggested that there is a close functional association between alkaline phosphatase activity and calcification in the process of fracture healing, which is another type of enchondral calcification mediated by matrix vesicles.

1. The process of repair after fracture of the humerus of the growing rat has been studied by histological, histochemical and biochemical methods.

2. Both periosteal and surrounding mesenchymal cells take part in the process of repair.

3. The primary framework of collagen bridging the gap is mainly formed by the mesenchymal cells, while calcification and ossification of the framework is largely a function of the periosteum.

4. The mucopolysaccharide content rises rapidly in the first week after injury, and is followed by a rise in the collagen content during the second week. The deposition of calcium phosphate during the third and fourth weeks causes an apparent fall in the collagen content during that period. The collagen content tends to return to normal during the phase of remodelling in the fifth and sixth weeks.

5. The tensile strength of the healing bone bears a close relation to its collagen content.

Gentamicin was described with negative effects on bone formation. Arginin-Glycin-Aspartat (RGD) sequences play a key role in the adhesion of osteoblasts and have proven to improve implant integration. We have already shown a significant reduction in infection rates by a combined gentamicin-hydroxyapatite (HA) and gentamicin-RGD-hydroxyapatite coating in a rabbit infection model for cementless joint prostheses.

The purpose of the study was to assess whether the gentamicin-HA coating had a negative effect on the implant integration and new bone formation, compared to pure HA coating, and whether this could be enhanced by additional gentamicin-RGD-HA coating.

There were 5 study groups (8 animals per group) with 5 different stainless steel K-wires: uncoated, HA coated, gentamicin-HA, RGD-coated, gentamicin-RGD-HA coated. A 2.0 mm K-wire with one type of coating was introduced into the intramedullary canal of the tibia. The tibiae were harvested after 12 weeks and standardised longitudinal and transverse sections were performed to study new bone formation around the implant and implant bone contact. New bone formation and osseointegration of the implant surface was assessed using histomorphometrical methods by computerised semi-quantitative analysis and histological methods.

There were no significant differences between the HA and the gentamicin-HA group although new bone formation and implant bone contact were always higher for the pure HA coating. Additional RGD coating on the gentamicin-RGD-HA coating did not show significant improvement of bone formation and implant integration compared to gentamicin-HA. There was a very similar histological appearance of new bone formation between all groups with very low frequency of giant cells, indicating good biocompatibility.

Gentamicin-HA coating did not have significant negative effects on bone formation and bone implant contact, compared to pure HA coating. In combination with the excellent ability to reduce infection rates, gentamicin-HA coating may have a high interest in cement-less arthroplasty.

Cartilage formation was provoked in the skull vault of the young rat by making multiple incisions, and scraping the periosteum to reduce the blood supply to the injured area. The hypothesis that ischaemia induces osteogenic cells to produce cartilage in the course of fracture repair thus receives experimental support.

1. It has been shown that in experimental rickets the well known changes in the epiphysial cartilage which so seriously affect growth are accompanied by severe interference with the progress of the metaphysial vessels into the growth cartilage.

2. Further evidence has been found that, by the repeated increase in their number, the cartilage cells occupying the more distal part of the proliferative segment become more and more affected by their remoteness from the epiphysial vessels, which supply the transudates to these cells. At a given distance these cells are affected and change, becoming hypertrophic, with increasingly large vacuolae, and are rich in glycogen and alkaline phosphatase.

3. The hypertrophic cells alter the nature of the intercellular substance they deposit and this becomes calcifiable. Provided that the metaphysial vessels are situated at an appropriate distance–about three cell capsules away–and that the blood has its necessary components, calcification occurs.

4. Calcification produces the advancing, rigid multitubular structure within which the progressing metaphysial vessels are protected.

5. The interruption of calcification by the withdrawal of fat-soluble vitamins breaks down the whole mechanism of growth and stops the vessels growing into their proper position. The administration of the required vitamins re-establishes the normal sequence of events and allows the vessels to play their decisive role in osteogenesis.

6. Any mechanism which causes the interruption of the vascular progression, whether from metaphysial ischaemia (Trueta and Amato 1960), from severe pressure (Trueta and Trias 1961) or from lack of calcification by withdrawing the fat-soluble vitamins, equally interrupts growth.