Effect of GDF‐7 deficiency on tail tendon phenotype in mice

The subfamily of growth/differentiation factors (GDFs) known as GDFs 5, 6, and 7 appears to be involved in tendon maintenance and repair, although the precise nature of this role has yet to be elucidated. The aim of the present study was to examine the role of GDF‐7 in tendon maintenance by studying tail tendon fascicle gene expression, composition, and material property strain rate dependency in 16‐week‐old male and female GDF‐7 deficient mice. GDF‐7 deficiency did not affect the biochemical composition of tail tendon fascicles, nor did it significantly affect the tensile material properties obtained at either slow (5%/s) or fast (50%/s) strain rates. Further, no difference was found between genotypes in the strain rate sensitivity of any tensile material property. Consistent with the compositional analyses, QRT‐PCR data did not reveal any differences of twofold or greater in the gene expression levels of collagens I, III, V, nor in the proteoglycans decorin, fibromodulin, lumican, biglycan, versican, or aggrecan. Gdf5 expression was upregulated twofold in GDF‐7 deficient tail tendons, and Bmp7 expression was downregulated twofold. No notable differences in expression levels for Bmp1‐6 or Gdf6 were detected. GDF‐5 protein levels were 50% higher in GDF‐7 deficient tail tendon compared to wild type tail tendon. The results of this study support the intriguing possibility that compensation by Gdf‐5 may be at least in part responsible for the absence of a strong phenotype in GDF‐7 deficient mice. © 2008 Orthopaedic Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:834–839, 2008     

Sex matters in the establishment of murine tendon composition and material properties during growth

The existence of sex‐based differences in tendon and ligament injury rates has led investigators to test the hypothesis that sex plays a significant role in modulating tendon and ligament composition and material properties. To date, no studies have attempted to characterize how such differences develop during the course of normal tissue maturation and growth. Thus, the primary aim of the present study was to use a murine model to test the hypothesis that sex‐based differences in the normal age‐related development of tendon composition and material properties exist by assessing these parameters in the Achilles and tail tendons from 4‐, 6‐, 9‐, 12‐, and 15‐week‐old male and female C57Bl/6J mice. Despite significantly lower levels of total collagen content in females subsequent to sexual maturity (p < 0.0001), as well as a significant effect of sex on glycosaminoglycan content (p < 0.0001), Achilles tendon elastic modulus was not compromised in females. Female Achilles tendons did exhibit a significantly higher failure strain (p = 0.0201) and strain energy density (p = 0.0004) than did males, as well as a trend toward higher ultimate strength (p = 0.0556). In contrast to the high load‐bearing environment of the Achilles tendon site, sex did not have a statistically significant effect on any compositional or material property in the low load‐bearing tendon fascicles of the tail. These data support recent studies by others, which suggest that male and female tendons have a differential adaptational response to their local mechanical loading environment. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:631–638, 2010     

Accelerated hypertrophic chondrocyte kinetics in GDF‐7 deficient murine tibial growth plates

The Growth/Differentiation Factors (GDFs) are a subgroup of the Bone Morphogenetic Proteins (BMPs) well known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling molecules, GDF‐5, have recently been shown to exhibit a decreased rate of endochondral bone growth in the proximal tibia due to a significantly longer hypertrophic phase duration. GDF‐7 is a related family member, which exhibits a high degree of sequence identity with GDF‐5. The purpose of the present study was to determine whether GDF‐7 deficiency also alters the endochondral bone growth rate in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5‐week‐old female GDF‐7 ?/? mice and wild type control littermates were examined. GDF‐7 deficiency resulted in a statistically significant increase in growth rate (+26%; p = 0.0084) and rate of cell loss at the chondrosseous junction (+25%; p = 0.0217). Cells from GDF‐7 deficient mice also exhibited a significantly shorter hypertrophic phase duration compared to wild type controls (?27%; p = 0.0326). These data demonstrate that, in the absence of GDF‐7, the rate of endochondral bone growth is affected through the modulation of hypertrophic phase duration in growth plate chondrocytes. These findings further support a growing body of evidence implicating the GDFs in the formation, maturation, and maintenance of healthy cartilage. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:986–990, 2008     

Variability in tendon and knee joint biomechanics among inbred mouse strains.

Hereditary factors are thought to be responsible for impaired tendon function and joint laxity. The present study investigated the genotypic variability of knee laxity and stiffness and tendon mechanical and geometric properties among 16-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mice. In one group of mice, knee mechanics were quantified using a custom loading apparatus enabling translation of the tibia against a stationary femur. In a second group, flexor digitorum longus and Achilles tendons from the left hind limb underwent biomechanical testing, while those of the contralateral limb were analyzed histologically for determination of cross-sectional area. Our results demonstrate that tendon and joint mechanics varied significantly among the inbred mouse strains, indicating that biomechanical properties are genetically determined. A/J mouse knees exhibited greater laxity (p < 0.001) and lower stiffness (p < 0.001) compared to those of the B6 and C3H mice. The genotypic differences in whole joint properties were similar to those of the tendons' structural biomechanical traits. Although body mass did not differ (p > 0.2) among the three strains, significant genotypic differences were found at the whole tendon, material quality, and morphological levels of the tissue hierarchy. Furthermore, genetic regulation of tendon mechanical properties varied with anatomic site. Patterns of genotypic differences in tendon size were not consistent with those of biomechanical properties, suggesting that unique combinations of structural and compositional factors contribute to tendon growth, adaptation, and development. Therefore, the three inbred strains constitute a useful experimental model to elucidate genetic control of structure-function relationships in normal and healing tendons and ligaments.     

Hypochlorhydria‐induced calcium malabsorption does not affect fracture healing but increases post‐traumatic bone loss in the intact skeleton

Efficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post‐traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr?/? mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr?/? and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium‐enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro‐computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme‐linked immunosorbent assay. Fracture healing was unaffected in Cckbr?/? mice. However, Cckbr?/? mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr?/? mice. Therefore, under conditions of hypochlorhydria‐induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914–1921, 2016.

     

Thrombospondin‐1 Regulates Bone Homeostasis Through Effects on Bone Matrix Integrity and Nitric Oxide Signaling in Osteoclasts

Thrombospondin‐1 (TSP1), an endogenous antiangiogenic, is a widely expressed secreted ligand with roles in migration, adhesion, and proliferation and is a target for new therapeutics. While TSP1 is present in the bone matrix and several TSP1 receptors play roles in bone biology, the role of TSP1 in bone remodeling has not been fully elucidated. Bone turnover is characterized by coordinated activity of bone‐forming osteoblasts (OB) and bone‐resorbing osteoclasts (OC). TSP1?/? mice had increased bone mass and increased cortical bone size and thickness compared to wild type (WT). However, despite increased size, TSP1?/? femurs showed less resistance to bending than expected, indicative of diminished bone quality and a bone material defect. Additionally, we found that TSP1 deficiency resulted in decreased OC activity in vivo and reduced OC differentiation. TSP1 was critical during early osteoclastogenesis, and TSP1 deficiency resulted in a substantial overexpression of inducible nitric oxide synthase (iNOS). Importantly, administration of a NOS inhibitor rescued the OC function defects of TSP1?/? mice in vivo. To investigate the role of bone‐derived TSP1 in osteoclastogenesis, we found that WT pre‐OCs had defective iNOS expression when cultured on TSP1?/? bone compared to WT bone, suggesting that TSP1 in bone plays a critical role in iNOS signaling during OC development. These data implicate a new role for TSP1 in bone homeostasis with roles in maintaining bone matrix integrity and regulating OC formation. It will be critical to monitor bone health of patients administered TSP1‐pathway directed therapeutics in clinical use and under development. © 2014 American Society for Bone and Mineral Research.     

A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal

Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six‐generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild‐type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG‐mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain‐of‐function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development. © 2015 American Society for Bone and Mineral Research.     

Sex‐specific regulation of body size and bone slenderness by the acid labile subunit

Insulin‐like growth factor 1 (IGF‐1) is a crucial mediator of body size and bone mass during growth and development. In serum, IGF‐1 is stabilized by several IGF‐1‐binding proteins (IGFBPs) and the acid labile subunit (ALS). Previous research using ALS knockout (ALSKO) mice indicated a growth retardation phenotype, and clinical reports of humans have indicated short stature and low bone mineral density (BMD) in patients with ALS deficiency. To determine the temporal and sex‐specific effects of ALS deficiency on body size and skeletal development during growth, we characterized control and ALSKO mice from 4 to 16 weeks of age. We found that female ALSKO mice had an earlier‐onset reduction in body size (4 weeks) but that both female and male ALSKO mice were consistently smaller than control mice. Interestingly, skeletal analyses at multiple ages showed increased slenderness of ALSKO femurs that was more severe in females than in males. Both male and female ALSKO mice appeared to compensate for their more slender bones through increased bone formation on their endosteal surfaces during growth, but ALSKO females had increased endosteal bone formation compared with ALSKO males. This study revealed age‐ and sex‐specific dependencies of body size and bone size on the ALS. These findings may explain the heterogeneity in growth and BMD measurements reported in human ALS‐deficient patients. © 2010 American Society for Bone and Mineral Research.     

TNF is required for the induction but not the maintenance of compression‐induced BME signals in murine tail vertebrae: Limitations of anti‐TNF therapy for degenerative disc disease

While bone marrow edema (BME) is diagnostic of spondyloarthropathy, its nature remains poorly understood. In contrast, BME in ankylosing spondylitis is caused by tumor necrosis factor (TNF)‐induced vascular and cellular changes. To investigate the relationship between chronic compression and TNF signaling in compression‐induced BME we utilized a tail vertebrae compression model with WT, TNF‐Tg, and TNFR1&2?/? mice to evaluate: (i) healing following release of chronic compression, (ii) induction of BME in the absence of TNFR, and (iii) efficacy of anti‐TNF therapy. Compression‐induced normalized marrow contrast enhancement (NMCE) in WT was significantly decreased threefold (p < 0.01) within 2 weeks of release, while the NMCE values in TNF‐Tg vertebrae remained elevated, but had a significant decrease (p < 0.05) by 6 weeks after the release of compression. TNFR1&2?/? mice were resistant to compression‐induced BME. Anti‐TNF therapy did not affect NMCE versus placebo. Histological examination revealed that NMCE values significantly correlated with marrow vascularity and cellularity (p < 0.05), which account for 76% of the variability of NMCE. Collectively, these data demonstrate a critical role for TNF in the induction of chronic compression‐induced BME, but not in its maintenance. Amelioration of BME is achieved through biomechanical stability, but is not affected by anti‐TNF therapy. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1367–1374, 2011     

Aging does not alter tendon mechanical properties during homeostasis,but does impair flexor tendon healing

Aging is an important factor in disrupted homeostasis of many tissues. While an increased incidence of tendinopathy and tendon rupture are observed with aging, it is unclear whether this is due to progressive changes in tendon cell function and mechanics over time, or an impaired repair reaction from aged tendons in response to insult or injury. In the present study, we examined changes in the mechanical properties of Flexor Digitorum Longus (FDL), Flexor Carpi Ulnaris (FCU), and tail fascicles in both male and female C57Bl/6 mice between 3 and 27 months of age to better understand the effects of sex and age on tendon homeostasis. No change in max load at failure was observed in any group over the course of aging, although there were significant decreases in toe and linear stiffness in female mice from 3 to 15 months, and 3 to 27 months. No changes in cell proliferation were observed with aging, although an observable decrease in cellularity occurred in 31‐month old tendons. Given that aging did not dramatically alter tendon mechanical homeostasis we hypothesized that a disruption in tendon homeostasis, via acute injury would result in an impaired healing response. Significant decreases in max load, stiffness, and yield load were observed in repairs of 22‐month old mice, relative to 4‐month old mice. No changes in cell proliferation were observed between young and aged, however, a dramatic loss of bridging collagen extracellular matrix was observed in aged repairs suggest that matrix production, but not cell proliferation leads to impaired tendon healing with aging. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2716–2724, 2017.

     

Gender and estrogen manipulation do not affect traumatic brain injury in mice

As epidemiological data have suggested that female patients may have improved clinical prognoses following traumatic brain injury (TBI) compared to males, we designed experiments to determine the role of gender and estrogen in TBI-induced brain injury and inflammation in rodents. To this end, male and female C57Bl/6 mice were separated into the following four groups: intact males, intact females with vehicle supplementation, ovariectomized females with vehicle supplementation, and ovariectomized females with estrogen supplementation. All mice were subjected to a controlled cortical impact model of TBI, and cortical injury, hippocampal degeneration, microglial activation, and brain cytokine expression were analyzed after injury. Additionally, the spleens were harvested and cytokine release from cultured splenic cells was measured in response to specific stimuli. Data indicate that TBI-induced cortical and hippocampal injury, as well as injury-related microglial activation were not significantly affected by gender or estrogen manipulation. Conversely, brain levels of MCP-1 and IL-6 were significantly increased in males and intact females following TBI, but not in female mice that had been ovariectomized and supplemented with either estrogen or vehicle. Evaluation of splenic responses showed that the spleen was only moderately affected by TBI, and furthermore that spleens isolated from mice that had been given estrogen supplementation showed significantly higher release of the anti-inflammatory cytokine IL-4, regardless of the presence of absence of TBI. Overall, these data indicate that while estrogen can modulate immune responses, and indeed can predispose splenic responses towards and anti-inflammatory phenotype, these effects do not translate to decreased brain injury or inflammation following TBI in mice.     

Ribosomal protein L29/HIP deficiency delays osteogenesis and increases fragility of adult bone in mice

Mice lacking HIP/RPL29, a ribosomal modulator of protein synthesis rate, display a short stature phenotype. To understand the contribution of HIP/RPL29 to bone formation and adult whole bone mechanical properties, we examined both developing and adult bone in our knockout mice. Results indicated that bone shortening in HIP/RPL29‐null mice is due to delayed entry of chondro‐osteoprogenitors into the cell cycle. Structural properties of adult null bones were analyzed by micro‐computed tomography. Interestingly, partial preservation of cortical thickness was observed in null males indicating a gender‐specific effect of the genotype on cortical bone parameters. Null males, and to a lower extent null females, displayed increased bone material toughness to counteract decreased bone size. This elevation in a bone material property was associated with increased bone mineral density only in null males. Neither male nor female null animals could withstand the same maximum load as gender‐matched controls in three‐point bending tests, and smaller post‐yield displacements (and thus increased bone brittleness) were found for null animals. These results suggest that HIP/RPL29‐deficient mice exhibit increased bone fragility due to altered matrix protein synthesis rates as a consequence of ribosomal insufficiency. Thus, sub‐efficient protein translation increased fracture risk in HIP/RPL29‐null animals. Taken together, these studies provide strong genetic evidence that the ability to regulate and amplify protein synthesis rates, including those proteins that regulate the cell cycle entry during skeletal development, are important determinants for establishment of normal bone mass and quality. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:28–35, 2009     

An immunosufficient murine model for the study of human islets

For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti‐CD45RB. To assess whether anti‐CD45RB‐induced transplant tolerance requires B cells, B6 recipients received additional anti‐CD20 or B6μMT?/? mice were used. For some anti‐CD45RB‐treated B6μMT?/? mice, additional anti‐CD25 mAb was applied at the early or late stage post‐transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti‐CD45RB/anti‐CD20‐treated Foxp3‐GFP B6 mice. The results showed that anti‐CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti‐CD20, and 88.9% of xenografts accepted in anti‐CD45RB‐treated μMT?/? mice. These μMT?/? mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti‐CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post‐transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.     

Treadmill running exercise results in the presence of numerous myofibroblasts in mouse patellar tendons

Mechanical loading is known to alter tendon structure, but its cellular mechanisms are unclear. This study aimed to determine the effect of mechanical loading on tendon cells in vivo. C57BL/6J female mice were used in a treadmill running study. The treadmill running protocol consisted of treadmill training for 1 week, followed by sustained moderate running at 13 m/min for 50 min/day, 5 days/week, for 3 weeks. Immunohistochemical staining of tendon sections of mice after treadmill running revealed that numerous cells in the tendon section expressed α‐SMA, whereas in the tendon sections of control mice, only a few cells exhibited weak α‐SMA signals. Furthermore, mouse patellar tendon cells (MPTCs) derived from treadmill running mice were generally larger in culture, proliferated faster, expressed a higher level of α‐SMA, and formed more abundant stress fibers compared to MPTCs from control mice. In addition, MPTCs from treadmill running mice generated larger traction forces (169 ± 66.1 Pa) than those from control mice (102 ± 34.2 Pa). Finally, cells from treadmill running mice produced higher levels of total collagen (516.4 ± 92.7 µg/10,000 cells) than their counterparts (303.9 ± 34.8 µg/10,000 cells). Thus, mechanical loading via treadmill running increased the presence of myofibroblasts in mouse patellar tendons. As myofibroblasts are activated fibroblasts, their presence in the tendon following treadmill running indicates that they actively repair and remodel tendon tissue under strenuous mechanical loading, leading to known changes in tendon structure. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1373–1378, 2009     

Changes of MicroRNA Profile and MicroRNA‐mRNA Regulatory Network in Bones of Ovariectomized Mice

Growing evidence shows the possibility of a role of microRNAs (miRNA) in regulating bone mass. We investigated the change of miRNAs and mRNA expression profiles in bone tissue in an ovariectomized mice model and evaluated the regulatory mechanism of bone mass mediated by miRNAs in an estrogen‐deficiency state. Eight‐week‐old female C3H/HeJ mice underwent ovariectomy (OVX) or sham operation (Sham‐op), and their femur and tibia were harvested to extract total bone RNAs after 4 weeks for microarray analysis. Eight miRNAs (miR‐127, ‐133a, ‐133a*, ‐133b, ‐136, ‐206, ‐378, ‐378*) were identified to be upregulated after OVX, whereas one miRNA (miR‐204) was downregulated. Concomitant analysis of mRNA microarray revealed that 658 genes were differentially expressed between OVX and Sham‐op mice. Target prediction of differentially expressed miRNAs identified potential targets, and integrative analysis using the mRNA microarray results showed that PPARγ and CREB pathways are activated in skeletal tissues after ovariectomy. Among the potential candidates of miRNA, we further studied the role of miR‐127 in vitro, which exhibited the greatest changes after OVX. We also studied the effects of miR‐136, which has not been studied in the context of bone mass regulation. Transfection of miR‐127 inhibitor has enhanced osteoblastic differentiation in UAMS‐32 cells as measured by alkaline phosphatase activities and mRNA expression of osteoblast‐specific genes, whereas miR‐136 precursor has inhibited osteoblastic differentiation. Furthermore, transfection of both miR‐127 and miR‐136 inhibitors enhanced the osteocyte‐like morphological changes and survival in MLO‐Y4 cells, whereas precursors of miR‐127 and ‐136 have aggravated dexamethasone‐induced cell death. Both of the precursors enhanced osteoclastic differentiation in bone marrow macrophages, indicating that both miR‐127 and ‐136 are negatively regulating bone mass. Taken together, these results suggest a novel insight into the association between distinct miRNAs expression and their possible role through regulatory network with mRNAs in the pathogenesis of estrogen deficiency–induced osteoporosis. © 2014 American Society for Bone and Mineral Research.     

Histological and molecular analysis of the biceps tendon long head post‐tenotomy

Tendinopathy is a vexing clinical problem as its onset and development is often asymptomatic and unrecognized until tendon rupture. While extensively studied in the rotator cuff, Achilles, and patellar tendons, no study to date has examined the histological and molecular characteristics of the tendinopathic biceps long‐head (LHB). The anatomy of the LHB is unique in that it comprises intra‐ and extra‐articular portions, each exposed to differing loading patterns. Eleven LHBs post‐tenotomy were sectioned, fixed in formalin, and stained (H&E; Alcian Blue), and gross structural organization of collagen measured using polarized light microscopy. Protein expression of intra‐ and extra‐articular portions of the tenotomized biceps for IGF‐I, collagen III, and MMP‐1, ‐2, ‐3, and ‐13 was determined with Western blot analyses. The intra‐articular LHB exhibited significantly greater histological evidence of tendinopathy inclusive of increased proteoglycan (p < 0.05) and decreased organization as measured by polarized light microscopy (p < 0.01). The intra‐articular LHB also had significantly increased expression of collagen type III (p < 0.01) and of MMP‐1 and 3 (p < 0.01, p < 0.05 respectively). No significant differences were found for IGF‐I or for MMP‐2 and ‐13. The intra‐articular LHB exhibited histological characteristics of tendinopathy. Protein expression of the intra‐articular LHB did not universally display signs of tendinopathy in comparison to the extra‐articular portion of the tendon. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1379–1385, 2009     

Loss of Sex-Specific Difference in Femoral Bone Parameters in Male Leptin Knockout Mice

Sex-dependent differences were identified in the femoral bone parameters of male and female ob/ob (leptin knockout) mice compared with their C57BL/6 wild-type background strain. Total fat, lean weight and body weight were not different between adult male and female leptin knockout mice. However, leptin knockout males exhibited lower lean weights than C57BL/6 males. Peripheral quantitative computerized tomographic measurements at the femoral midshaft revealed that the normal differences in the periosteal circumference, endosteal circumference, total bone mineral content, and polar moment of inertia normally observed between adult male and female wild-type mice were lost between adult male and female ob/ob mice. Significant reductions in these bone parameters were seen in male ob/ob mice compared to male wild-type mice but not in female ob/ob mice compared to female wild-type mice. In prepubertal mice, there were no differences in phenotype and femoral bone parameters between males and females within any strain, suggesting sex hormone functions. Serum free testosterone levels were 5.6-fold higher in adult male ob/ob mice than in adult male C57BL/6 wild-type mice, and serum estradiol levels were 1.8- and 1.3-fold greater in adult male and female ob/ob mice, respectively, than in their wild-type counterparts. Androgen receptor gene expression was not different in femur-derived bone cells of male ob/ob mice compared with wild-type mice. The loss of sex-related differences in these bone parameters in adult male ob/ob mice might result from deficient signaling in the androgen signaling pathway and the fact that leptin functions are permissive for androgen effects on bone development. Xiaoguang Wang, Charles H. Rundle contributed equally to this work.