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

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)

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

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

Introduction. Diabetes mellitus type 2 (DMT2) patients often develop Achilles tendon (AS) degeneration. The ZDF rat model is often used to study DMT2. Hence, this study investigated whether tenocytes isolated from diabetic and non diabetic ZDF rats respond differentially to normo- (NG) and hyperglycemic (HG) conditions in the presence of tumor necrosis (TNF)α. Method. AS tenocytes isolated from adult diabetic (fa/fa) or lean (fa/+) Zucker Diabetic Fatty (ZDF) rats were treated with 10 ng/mL TNFα either under NG or HG conditions (1 g/L versus 4.5 g/L glucose). Tendons were characterized histopathologically using Movin score. Tenocyte survival, metabolic activity, gene and/or protein expression of the main tendon extracellular matrix (ECM) component collagen type 1, the myofibroblast marker alpha smooth muscle actin (αSMA, Acta2), complement regulatory factors, the antioxidant defense enzyme heme oxygenase-1 (Hmox1), suppressors of cytokine signaling (Socs)1 and Soc3 were analyzed. Result. Tendons of diabetic rats showed significantly higher Movin score values suggesting tendon degeneration. Tenocyte vitality remained high, but metabolic activity was impaired by HG conditions, irrespectively of tenocyte origin. Higher amounts of αSMA were visualized in tendons/cells of diabetic rats or those exposed to TNFα. Collagen type 1 protein and gene expression was suppressed by TNFα (NG), but only in cells of non diabetic animals. The anaphylatoxin receptor C3aR was higher expressed in tenocytes from diabetic animals. CD46 was suppressed by TNFα (NG) in cells of diabetic rats. Hmox1, Socs1 and Socs3 were induced by HG, but only in tenocytes of diabetic rats (4 h). Conclusion. The response of tenocytes to TNFα depends on glucose supply and cell origin suggesting their irreversible impairment in DMT2

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

Aims. Ultrasound-guided injection techniques are expected to enhance therapeutic efficacy for skeletal muscle injuries and disorders, but basic knowledge is lacking. The purpose of this study was to examine the diagnostic accuracy of ultrasound for abnormal skeletal muscle lesions, and to examine the distribution patterns of solution and cells injected into abnormal muscle lesions under ultrasound guidance. Methods. A cardiotoxin (CTX)-induced muscle injury model was used. Briefly, CTX was injected into tibialis anterior muscle in rats under ultrasound observation. First, the diagnostic accuracy of abnormal muscle lesions on ultrasound was examined by comparing ultrasound findings and histology. Next, Fast Green solution and green fluorescent protein (GFP)-labelled cells were simultaneously injected into the abnormal muscle lesions under ultrasound guidance, and their distribution was evaluated. Results. Evaluation of short-axis ultrasound images and cross-sectional histological staining showed a strong correlation (r = 0.927; p < 0.001) between the maximum muscle damage area in ultrasound and haematoxylin and eosin (H&E) staining evaluations. Histological analysis showed that ultrasound-guided injection could successfully deliver Fast Green solution around the myofibres at the site of injury. In contrast, the distribution of injected cells was very localized compared to the area stained with Fast Green. Conclusion. This experimental animal study demonstrated the potential of ultrasound to quantitatively visualize abnormalities of skeletal muscle. It also showed that ultrasound-guided injections allowed for highly accurate distribution of solution and cells in abnormal muscle tissue, but the patterns of solution and cell distribution were markedly different. Although future studies using a more clinically relevant model are necessary, these results are important findings when considering biological therapies for skeletal muscle injuries and disorders. Cite this article: Bone Joint Res 2025;14(1):33–41

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

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

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

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

Aims. 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

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

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

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