Estrogen prevents osteoporotic bone loss by attenuating bone resorption; however, the molecular basis for this is unknown. Here, we report a critical role for the osteoclastic estrogen receptor alpha (ERalpha) in mediating estrogen-dependent bone maintenance in female mice. We selectively ablated ERalpha in differentiated osteoclasts (ERalpha(DeltaOc/DeltaOc)) and found that ERalpha(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Further, we show that estrogen induced apoptosis and upregulation of Fas ligand (FasL) expression in osteoclasts of the trabecular bones of WT but not ERalpha(DeltaOc/DeltaOc) mice. The expression of ERalpha was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. Our results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/FasL system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs.

Based on the fact that aging is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow and that osteoblasts and adipocytes share a common progenitor, this study investigated the role of PPARγ γ, a key regulator of adipocyte differentiation, in bone metabolism. Homozygous PPARγ γ-deficient ES cells failed to differentiate into adipocytes, but spontaneously differentiated into osteoblasts, and these were restored by reintroduction of the PPARγ γ gene. Heterozygous PPARγ γ-deficient mice exhibited high bone mass with increased osteoblastogenesis, but normal osteoblast and osteoclast functions, and this effect was not mediated by insulin or leptin. The osteogenic effect of PPARγ γ haploinsufficiency became prominent with aging but was not changed upon ovariectomy. The PPARγ γ haploinsufficiency was confirmed to enhance osteoblastogenesis in the bone marrow cell culture but did not affect the cultures of differentiated osteoblasts or osteoclast-lineage cells. This study demonstrates a PPARγ γ-dependent regulation of bone metabolism in vivo, in that PPARγ γ insufficiency increases bone mass by stimulating osteoblastogenesis from bone marrow progenitors.Nonstandard abbreviations used: alkaline phosphatase (ALP); bone morphogenetic protein-2 (BMP-2); bone volume (BV); CCAAT enhancer-binding proteins (C/EBPs); computed tomography (CT); LDL receptor-related protein 5 (LRP5); leukemia inhibitory factor (LIF); M-CSF-dependent bone marrow macrophage (M-BMMφ); receptor activator of nuclear factor κB ligand (RANKL); ovariectomy (OVX); PPAR responsive element (PPRE); tartrate-resistant acid phosphatase (TRAP); tissue volume (TV); type I collagen α1 chain (COL1A1).

Based on the fact that aging is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow and that osteoblasts and adipocytes share a common progenitor, this study investigated the role of PPARγ γ, a key regulator of adipocyte differentiation, in bone metabolism. Homozygous PPARγ γ-deficient ES cells failed to differentiate into adipocytes, but spontaneously differentiated into osteoblasts, and these were restored by reintroduction of the PPARγ γ gene. Heterozygous PPARγ γ-deficient mice exhibited high bone mass with increased osteoblastogenesis, but normal osteoblast and osteoclast functions, and this effect was not mediated by insulin or leptin. The osteogenic effect of PPARγ γ haploinsufficiency became prominent with aging but was not changed upon ovariectomy. The PPARγ γ haploinsufficiency was confirmed to enhance osteoblastogenesis in the bone marrow cell culture but did not affect the cultures of differentiated osteoblasts or osteoclast-lineage cells. This study demonstrates a PPARγ γ-dependent regulation of bone metabolism in vivo, in that PPARγ γ insufficiency increases bone mass by stimulating osteoblastogenesis from bone marrow progenitors.Nonstandard abbreviations used: alkaline phosphatase (ALP); bone morphogenetic protein-2 (BMP-2); bone volume (BV); CCAAT enhancer-binding proteins (C/EBPs); computed tomography (CT); LDL receptor-related protein 5 (LRP5); leukemia inhibitory factor (LIF); M-CSF-dependent bone marrow macrophage (M-BMMφ); receptor activator of nuclear factor κB ligand (RANKL); ovariectomy (OVX); PPAR responsive element (PPRE); tartrate-resistant acid phosphatase (TRAP); tissue volume (TV); type I collagen α1 chain (COL1A1).

Low-intensity pulsed ultrasound (LIPUS) has been shown to accelerate fracture healing in both animal models and clinical trials, but the mechanism of action remains unclear. In fracture healing, various consecutive cellular reactions occurred until repair. We investigated whether the advanced effects of LIPUS depended on the duration and timing of LIPUS treatment in a rat closed femoral fracture model to determine the target of LIPUS in the healing process. Sixty-nine Long-Evans male rats that have bilateral closed femoral fractures were used. The right femur was exposed to LIPUS (30 mW/cm 2 spatial and temporal average [SATA], for 20 minutes/day), and the left femur was used as a control. Rats were divided into four groups according to timing and duration of treatment (Ph-1, days 1-8; Ph-2, days 9 -16; Ph-3, days 17-24; throughout [T], days 1-24 after the fracture). Animals were killed on day 25. After radiographs and microfocus X-ray computed tomography (CT) tomograms were taken, the hard callus area (HCA), bone mineral content (BMC) at the fracture site, and mechanical torsion properties were measured, and histological analysis was conducted. Interestingly, the maximum torque of the LIPUS-treated femur was significantly greater than that of the controls in all groups without any changes in HCA and BMC. The multiviewing of three-dimensional (3D) CT reconstructions and histology supported our findings that the partial LIPUS treatment time was able to accelerate healing, but longer treatment was more effective. These results suggest that LIPUS acts on some cellular reactions involved in each phase of the healing process such as inflammatory reaction, angiogenesis, chondrogenesis, intramembranous ossification, endochondral ossification, and bone remodeling. (J Bone Miner Res 2001;16:671-680)

Although accumulated evidence has shown the bone anabolic effects of bone morphogenetic proteins (BMPs) that were exogenously applied in vitro and in vivo, the roles of endogenous BMPs during bone formation remain to be clarified. This study initially investigated expression patterns of BMPs in the mouse long bone and found that BMP2 and BMP6 were the main subtypes expressed in hypertrophic chondrocytes that induce endochondral bone formation. We then examined the involvement of the combination of these BMPs in bone formation in vivo by generating the compound-deficient mice (Bmp2+/-;Bmp6-/-). Under physiological conditions, these mice exhibited moderate growth retardation compared with the wild-type (WT) littermates during the observation period up to 52 weeks of age. Both the fetal and adult compound-deficient mice showed a reduction in the trabecular bone volume with suppressed bone formation, but normal bone resorption, whereas the single deficient mice (Bmp2+/- or Bmp6-/-) did not. When a fracture was created at the femoral midshaft and the bone healing was analyzed, the endochondral bone formation, but not intramembranous bone formation, was impaired by the compound deficiency. In the cultures of bone marrow cells, however, there was no difference in osteogenic differentiation between WT and compound-deficient cells in the presence or absence of the exogenous BMP2. We thus concluded that endogenous BMP2 and BMP6 cooperatively play pivotal roles in bone formation under both physiological and pathological conditions.

ObjectivesThe aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy.MethodsUsing tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined.ResultsIn tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased.ConclusionThis study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes.Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362–372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2

Triamcinolone acetonide (TA) injections are widely used to treat enthesopathy, but they may induce adverse effects such as tendon impairment and rupture. Platelet-rich plasma (PRP) is a blood fraction containing high platelet concentrations and various growth factors that play a role in tissue repair processes. The purpose of this study is to investigate whether TA has deleterious effects on human rotator cuff-derived cells, and if PRP can protect these cells from the effects of TA. Human rotator cuff-derived cells were cultured with and without TA and PRP, and the culture without any additive served as the control. Cell morphology was assessed at days 7 and 21. Cell viability was evaluated at days 1, 7, 14, and 21 by a water-soluble tetrazolium salt assay. Induction of apoptosis was measured by immunofluorescence staining and flow cytometry at day 7. Induction of cleaved caspase-3 was measured by immunofluorescence staining at day 7. The cells cultured with TA had a flattened and polygonal shape at day 7. The cells cultured with both TA and PRP were similar in appearance to control cells. Exposure to TA also significantly decreased cell viability, but cell viability did not decrease when PRP was added along with TA. The number of apoptotic cells increased with TA exposure, while addition of PRP prevented cell apoptosis. In conclusion, the deleterious effect of TA was prevented by PRP, which can be used as a protective agent for patients receiving local TA injections. Keywords: triamcinolone acetonide (TA); platelet-rich plasma (PRP); rotator cuff-derived cells; apoptosis; cell viability Enthesopathy caused by rotator cuff injury clinically impairs joint function. Rotator cuff disease represents a spectrum of pathological conditions ranging from tendinosis to full-thickness tears. Conservative therapy, including rest, physical therapy, non-steroidal anti-inflammatory medications, and steroid injections are the primary treatments for these conditions. 1,2The effectiveness of subacromial steroid injections for treatment of rotator cuff tendinosis has been equivocal. 3,4 Some clinical studies have demonstrated improvement in shoulder function and decrease in shoulder pain following steroid injection.3,5 However, other studies have shown that steroids afford no clinical benefits over those provided by injections of lidocaine alone 4 or physical therapy alone. 6 Triamcinolone acetonide (TA) injections are widely used for treatment of rotator cuff tendinosis, but they may cause tendon impairment and rupture; many cases of tendon rupture after TA injections have been reported. 7-11Despite previous studies on the deleterious histological and biomechanical changes in tendons after TA injections, 8,12 TA administration is still considered to be a conservative treatment option.Platelet-rich plasma (PRP) is an autologs concentration of platelets that contains many growth factors, including platelet-derived growth factor (PDGF), transforming growth factor b (TGF-b), fibroblastic growth factor (FGF), vascular endoth...

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