机械应力、骨细胞凋亡和骨重建

骨细胞是骨中数量最丰富的细胞,有诸多重要作用。其作为骨组织的机械应力感受细胞,将机械载荷转化为体内生化反应过程,如细胞凋亡,同时产生一些调节成骨细胞和破骨细胞活性的因子和激素,共同参与骨组织的重建。骨细胞凋亡是一些骨代谢疾病的发病机制,可导致骨组织缺失或障碍性。本文简述了外界机械应力通过对成骨细胞和破骨细胞活性的调节,并刺激机体产生激素调节骨细胞凋亡,从而影响骨塑型和骨重建。通过机械应力对骨细胞凋亡调节的研究,为预防、治疗、诊断骨代谢疾病提供新的理论依据。     

Quantifying the osteocyte network in the human skeleton

Osteocytes form an extensive cellular network throughout the hard tissue matrix of the skeleton, which is known to regulate skeletal structure. However due to limitations in imaging techniques, the magnitude and complexity of this network remain undefined.We have used data from recent papers obtained by new imaging techniques, in order to estimate absolute and relative quantities of the human osteocyte network and form a more complete understanding of the extent and nature of this network.We estimate that the total number of osteocytes within the average adult human skeleton is ~ 42 billion and that the total number of osteocyte dendritic projections from these cells is ~ 3.7 trillion. Based on prior measurements of canalicular density and a mathematical model of osteocyte dendritic process branching, we calculate that these cells form a total of 23 trillion connections with each other and with bone surface cells. We estimate the total length of all osteocytic processes connected end-to-end to be 175,000 km. Furthermore, we calculate that the total surface area of the lacuno-canalicular system is 215 m². However, the residing osteocytes leave only enough space for 24 mL of extracellular fluid. Calculations based on measurements in lactation-induced murine osteocytic osteolysis indicate a potential total loss of ~ 16,000 mm³ (16 mL) of bone by this process in the human skeleton. Finally, based on the average speed of remodelling in the adult, we calculate that 9.1 million osteocytes are replenished throughout the skeleton on a daily basis, indicating the dynamic nature of the osteocyte network.We conclude that the osteocyte network is a highly complex communication network, and is much more vast than commonly appreciated. It is at the same order of magnitude as current estimates of the size of the neural network in the brain, even though the formation of the branched network differs between neurons and osteocytes. Furthermore, continual replenishment of large numbers of osteocytes in the process of remodelling allows therapeutic changes to the continually renewed osteoblast population to be rapidly incorporated into the skeleton.     

Risedronate and alendronate suppress osteocyte apoptosis following cyclic fatigue loading

PURPOSE: The purpose of this study was to determine whether bisphosphonate treatment can prevent or delay osteocyte apoptosis in a cyclic fatigue animal model and if there are differences between two different bisphosphonates in their effects on osteocyte apoptosis. INTRODUCTION: Fatigue loading induces microdamage in long bones in rats and causes osteocyte apoptosis. In vitro data suggest that the bisphosphonates can prevent osteocyte apoptosis. MATERIALS AND METHODS: Six month old female Sprague-Dawley rats (n=72) were given a daily subcutaneous (sc) injection of saline vehicle, risedronate (RIS: 0.05 mug/kg per day) or alendronate (ALN: 0.1 mug/kg per day). On the 8th day of drug treatment, an axial compressive load was applied to the right ulna using a load-controlled electromagnetic device (17N, 6000 cycles, 2 Hz, 10% loss of stiffness approximately 1 h). Three, seven or ten days after loading, the animals were sacrificed. Immunohistochemistry for caspase-3 was performed to assess the extent of osteocyte apoptosis in loaded and non-loaded ulnas. RESULTS: Microdamage (Mdx) created by cyclic loading of the ulna induced a significant increase (p=0.03) in the number of apoptotic osteocytes compared to non-damaged regions of the same ulna, and compared to the contralateral non-loaded ulna. Risedronate and alendronate had an early effect (3 days after loading) on reducing load-induced osteocyte apoptosis. Risedronate significantly reduced the density of apoptotic osteocytes compared to vehicle-treated controls by approximately 50% in the Mdx area, whereas alendronate reduced it by approximately 40%. There were no differences among groups by seven days following loading. CONCLUSIONS: (1) Low doses of risedronate or alendronate suppressed osteocyte apoptosis induced by fatigue loading of the ulna in rats. (2) There was no difference between the effects of risedronate or alendronate on osteocyte apoptosis at these doses.     

Caspase-dependent cleavage of cadherins and catenins during osteoblast apoptosis.

As transmembrane, Ca2+-dependent cell-cell adhesion molecules, cadherins play a central role in tissue morphogenesis and homeostasis. Stable adhesion is dependent on interactions of the cytoplasmic domain of the cadherins with a group of intracellular proteins, the catenins. In the present study, we have detected the expression of alpha-, beta-, and gamma-catenins in human osteoblasts, which assemble with cadherins to form two distinct complexes containing cadherin and alpha-catenin, with either beta- or gamma-catenin. In osteoblasts undergoing apoptosis, proteolytic cleavage of N-cadherin and beta- and gamma- catenins but not alpha-catenin was associated with the activation of caspase-3 and prevented by the caspase inhibitor Z-VAD-fmk. The pattern of cadherin/catenin cleavage detected in apoptotic osteoblasts was reproduced in vitro by recombinant caspase-3. The presence of a 90-kDa extracellular domain fragment of N-cadherin in conditioned medium from apoptotic cells indicates that additional extracellular or membrane-associated proteases also are activated. Disruption of N-cadherin-mediated cell-cell adhesion with function-blocking antibodies induced osteoblast apoptosis, activation of caspases, and cleavage of beta-catenin. These findings provide compelling evidence that N-cadherin-mediated cell-cell adhesion promotes osteoblast survival and suggest that the underlying mechanism may involve activation of beta-catenin signaling.     

PTH1–34 alleviates radiotherapy-induced local bone loss by improving osteoblast and osteocyte survival

Cancer radiotherapy is often complicated by a spectrum of changes in the neighboring bone from mild osteopenia to osteoradionecrosis. We previously reported that parathyroid hormone (PTH, 1–34), an anabolic agent for osteoporosis, reversed bone structural deterioration caused by multiple microcomputed tomography (microCT) scans in adolescent rats. To simulate clinical radiotherapy for cancer patients and to search for remedies, we focally irradiated the tibial metaphyseal region of adult rats with a newly available small animal radiation research platform (SARRP) and treated these rats with intermittent injections of PTH1–34. Using a unique 3D image registration method that we recently developed, we traced the local changes of the same trabecular bone before and after treatments, and observed that, while radiation caused a loss of small trabecular elements leading to significant decreases in bone mass and strength, PTH1–34 preserved all trabecular elements in irradiated bone with remarkable increases in bone mass and strength. Histomorphometry demonstrated that SARRP radiation severely reduced osteoblast number and activity, which were impressively reversed by PTH treatment. In contrast, suppressing bone resorption by alendronate failed to rescue radiation-induced bone loss and to block the rescue effect of PTH1–34. Furthermore, histological analyses revealed that PTH1–34 protected osteoblasts and osteocytes from radiation-induced apoptosis and attenuated radiation-induced bone marrow adiposity. Taken together, our data strongly support a robust radioprotective effect of PTH on trabecular bone integrity through preserving bone formation and shed light on further investigations of an anabolic therapy for radiation-induced bone damage.     

iNOS expression and osteocyte apoptosis in idiopathic,non-traumatic osteonecrosis

ResultsThe morphology of osteocytes in the areas close to the necrotic region changed and the number of apoptotic osteocytes increased in comparison with the same region in control groups. The expression of iNOS and cytochrome C in osteocytes increased while Bax expression was not detectable in osteonecrosis samples. Using spontaneously hypertensive rats, we found a positive correlation between iNOS expression and osteocyte apoptosis in the osteonecrotic region. iNOS inhibitor (aminoguanidine) added to the drinking water for 5 weeks reduced the production of iNOS and osteonecrosis compared to a control group without aminoguanidine.InterpretationOur findings show that increased iNOS expression can lead to osteocyte apopotosis in idiopathic, non-traumatic osteonecrosis and that an iNOS inhibitor may prevent the progression of the disease.Patients with non-traumatic osteonecrosis (ON) fall into 2 groups: those with apparent etiological or risk factors and those without any identifiable etiology (Malizos et al. 2007). There are several hypotheses about the pathogenesis of so-called idiopathic ON, such as marrow edema and hemorrhage, thrombi or emboli in the microvasculature, cytotoxicity, lipocyte hyperplasia, osteoblast and osteoclast coupling dysfunction, and most recently, osteocyte apoptosis (Zalavras et al. 2000, Assouline-Dayan et al. 2002, Youm et al. 2010). It has also been reported that alterations in glucose and lactate levels in synovial fluid are associated with ON of the femoral head, indicating that synovial fluid metabolites may be an effective method of monitoring the disease progression (Huffman et al. 2007).Osteocytes have many functions, and they act as an orchestrator in bone remodeling (Bonewald 2011). Recently, it has been reported that the incidence of osteocyte apoptosis is increased in the femoral head during ON, regardless of etiological factors (Mutijima et al. 2014). However, the detailed apoptosis pathways and potential regulators in the pathogenesis of osteocytic apoptosis are not fully understood in non-traumatic osteonecrosis.Apoptosis pathways can be initiated through either the receptor pathway (the exogenous signal pathway) at the plasma membrane by death receptor ligation or the mitochondrial pathway (the endogenous signal pathway) (Elmore 2007). In the exogenous signal pathway, stimulation of death receptors of the tumor necrosis factor (TNF) receptor superfamily results in receptor aggregation and recruitment of the adaptor molecule Fas-associated death domain (FADD) and caspase-8. Upon recruitment, caspase-8 becomes activated and initiates apoptosis by direct cleavage of downstream effector caspases. In the endogenous signal pathway, a stress signal is initiated through the binding of activated Bax to the outer membrane of mitochondria—to induce the release of apoptogenic factors such as cytochrome C into the cytoplasm. The release of cytochrome C into the cytosol triggers activation of caspase-3, leading to apoptosis.Nitric oxide (NO) has been identified to be an agent that could induce non-traumatic osteonecrosis (Calder et al. 2004, Pan et al. 2013). NO is a small molecule produced by the enzymatic action of NO synthase. There are 3 protein forms of nitric oxide synthase (NOS): neuronal NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). Neuronal NOS and eNOS usually exist as constitutive forms to maintain physiological effects, while iNOS is produced in response to stimulation (Li and Poulos 2005). A number of drugs can regulate the activities of NOS, and aminoguanidine (AMG) has been reported to be an inhibitor of NOS—mainly acting on iNOS (Suzuki et al. 1996). To our knowledge, AMG has not been investigated in non-traumatic osteonecrosis treatment. We investigated possible correlations between iNOS expression and osteocyte apoptosis and also the therapeutic effect of AMG in non-traumatic osteonecrosis.     

Inhibition of osteoblast apoptosis by thrombin

The multifunctional serine protease thrombin has been shown to be a specific agonist for a variety of functional responses of cells including osteoblasts. The current study was conducted to determine if thrombin was capable of inhibiting apoptosis in osteoblasts, and if so, to examine the mechanism by which this occurred. Thrombin (20-100 nM) significantly inhibited apoptosis in serum-starved cultures of the human osteoblast-like Saos-2 cell line and cultures of primary osteoblasts isolated from mouse calvariae, as well as dexamethasone-treated primary mouse osteoblasts. Inhibition of serum deprivation-induced apoptosis was shown to require thrombin's specific proteolytic activity. Primary mouse osteoblasts were found to express two functional thrombin receptors, PAR-1 and PAR-4. Thrombin inhibited serum deprivation-induced apoptosis in osteoblasts isolated from PAR-1 null mice to the same degree as in osteoblasts isolated from wild-type mice. Treatment of serum-deprived osteoblasts, isolated from either PAR-1 null or wild-type mice, with a PAR-4-activating peptide failed to significantly inhibit apoptosis compared to the relevant control. Medium conditioned by thrombin-treated osteoblasts, in which thrombin had been inactivated, was able to inhibit serum deprivation-induced osteoblast apoptosis almost as well as thrombin itself. Blocking protein synthesis, by cycloheximide pretreatment of the conditioning cells, prevented this action. The ability of known osteoblast survival factors, such as transforming growth factor beta1, fibroblast growth factor-2, insulin-like growth factor-II, and interleukin-6, to inhibit serum deprivation-induced osteoblast apoptosis was also tested. None of these factors was able to inhibit serum deprivation-induced osteoblast apoptosis to the same extent as thrombin. The results presented here demonstrate that thrombin treatment of osteoblasts inhibits apoptosis induced either by dexamethasone or by serum deprivation. Furthermore, it does so independently of the known thrombin receptors by bringing about the synthesis and/or secretion of an unknown survival factor or factors, which then act in an autocrine fashion to inhibit apoptosis.     

The effects of storage media and perfusion on osteoblast and osteocyte survival in free composite bone grafts

Twenty-two adult mongrel dogs were used to investigate the effects of storage alone and storage and perfusion in 3 different storage media on the survival of osteocytes and osteoblasts in free bone grafts revascularized by microvascular anastomoses. Evaluation of the grafts at 2 weeks demonstrated that storage in chilled (+ 5°C) physiologic saline or Collins-Terasaki solution resulted in greater survival of osteocytes and osteoblasts than did storage in chilled BGJb solution or in room temperature air. No beneficial effect could be detected from perfusing the bone grafts with their storage media. On the contrary, deleterious effects were noted at the sites of the anastomoses, with thromboses of vessels secondary to endothelial damage.     

Changes in osteocyte density correspond with changes in osteoblast and osteoclast activity in an osteoporotic sheep model

Summary  

Histomorphometric assessment of trabecular bone in osteoporotic sheep showed that bone volume, osteoid surface area, bone formation rate, and osteocyte density were reduced. In contrast, eroded surface area and empty lacunae density were increased. Changes in osteocyte density correlated with changes in osteoblast and osteoclast activity.     

Molecular mechanism of nitric oxide-induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. Our previous study showed that NO induced osteoblast apoptosis [Chen RM, Liu HC, Lin YL, Jean WC, Chen JS, Wang JH. Nitric oxide induces osteoblast apoptosis through the de novo synthesis of Bax protein. J Orthop Res 2002;20:295-302]. This study was further aimed to evaluate the mechanism of NO-induced osteoblast apoptosis from the viewpoints of mitochondrial functions, intracellular oxidative stress, and the anti-apoptotic Bcl-2 protein using neonatal rat calvarial osteoblasts as the experimental model. Exposure of osteoblasts to sodium nitroprusside (SNP), an NO donor, significantly increased amounts of lactate dehydrogenase in the culture medium, and decreased cell viability in concentration- and time-dependent manners. Administration of SNP in osteoblasts time-dependently led to DNA fragmentation. The mitochondrial membrane potential was significantly reduced following SNP administration. SNP decreased complex I NADH dehydrogenase activity in a time-dependent manner. Levels of cellular adenosine triphosphate (ATP) were suppressed by SNP. In parallel with the mitochondrial dysfunction, SNP time-dependently increased levels of intracellular reactive oxygen species. Immunoblotting analysis revealed that SNP reduced Bcl-2 protein levels. Exposure to lipopolysaccharide (LPS) and IFN-γ significant increased endogenous nitrite production. In parallel with the increase in endogenous NO, administration of LPS and IFN-γ suppressed cell viability, mitochondrial membrane potential, and ATP synthesis. Results of this study show that NO released from SNP can induce osteoblast insults and apoptosis, and the mechanism may involve the modulation of mitochondrial functions, intracellular reactive oxygen species, and Bcl-2 protein.     

TNF-α对成骨细胞凋亡作用的探讨

目的 探讨TNF-α诱导成骨细胞凋亡的作用,为绝经后妇女骨质疏松的发生机理和治疗方法的研究提供切实可行的依据。方法 在体外培养成骨细胞的过程中,分别加人不同浓度的TNF-α,用流式细胞仪检测其对成骨细胞的凋亡率。结果 TNF-α15ng/mL组、30ng/mL组对成骨细胞的生长均有显著的抑制作用,其中又以30ng/mL组最明显。结论 TNF-α在一定浓度中对成骨细胞的生长有显著的抑制作用, 尤以30ng/mL组最明显。     

Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast: implication of different focal adhesion formation.

The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real-time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow-induced calcium may be related to differences in focal adhesion formation. INTRODUCTION: To explore the immediate response to mechanical stress in a bone cell population, we examined flow-induced calcium transients. In addition, the involvement of focal adhesion-related calcium transients in response to fluid flow in the cells was studied. MATERIALS AND METHODS: Bone cells were isolated from 16-day-old embryonic chicken calvaria by serial treatment with EDTA and collagenase. Single cells on glass without intercellular connections were subjected to fluid flow, and intracellular calcium concentration was measured using imaging with fluo-3. The identification of cell populations in the same field was performed with a chick osteocyte-specific antibody, OB7.3, and an alkaline phosphatase substrate, ELF-97, for osteoblast identification afterward. Immunofluorescence staining of vinculin was performed to visualize focal adhesions. RESULTS: The percentage of cells responding to fluid shear stress at 1.2 Pa was 5.5% in osteocytes, 32.4% in osteoblasts, and 45.6% in OB7.3/ELF-97-negative cells. Furthermore, osteoblasts and OB7.3/ELF-97-negative cells were more responsive to 2.4 Pa than 1.2 Pa, whereas osteocytes were less responsive. The elevation of calcium transients over baseline did not show any significant differences in the populations. To elucidate the mechanism accounting for the fact that single osteocytes are less sensitive to fluid shear stress of up to 2.4 Pa than osteoblasts, we studied focal adhesion-related calcium transients. First, we compared focal adhesion formation between osteocytes and osteoblasts and found a larger number of focal adhesions in osteoblasts than in osteocytes. Next, when the cells were pretreated with GRGDS (0.5 mM) before flow treatment, a significant reduction of calcium transients in osteoblasts (18%) was observed, whereas calcium transients in osteocytes were not changed by GRGDS. Control peptide GRGES did not reduce the calcium transients in either cell type. Furthermore, we confirmed that osteoblasts in calvaria showed a marked formation of vinculin plaques in the periphery of the cells. However, osteocytes in calvaria showed faint vinculin plaques only at the base of the processes. CONCLUSIONS: On glass, single osteocytes are less sensitive to fluid shear stress up to 2.4 Pa than osteoblasts. The difference in calcium transients might be related to differences in focal adhesion formation. Shear stress of a higher magnitude or direct deformation may be responsible for the mechanical response of osteocytes in bone.     

MicroRNA调控成骨细胞增殖、凋亡的研究进展

微小RNA( microRNA)是一类非编码小分子RNA,在基因表达调控中起重要作用,它通过与靶mRNA的特异性结合,导致靶mRNA降解或者抑制其翻译,对基因进行转录后调控,从而控制细胞的增殖、分化、凋亡等,参与疾病的发生发展。成骨细胞是骨形成过程中的重要细胞,其数量或功能的改变明显影响骨代谢。近年来.microRNA与骨代谢的关系备受关注,诸多研究表明microRNA在成骨细胞的分化中发挥重要调控作用,但其调节成骨细胞增殖和凋亡的研究相对较少。本文就 microRNA调控成骨细胞增殖、凋亡的研究进展进行综述。     

Role of macrophages in LPS-induced osteoblast and PDL cell apoptosis

In periradicular lesions and periodontal disease, bacterial invasion leads to chronic inflammation resulting in disruption of the structural integrity of the periodontal ligament and progressive alveolar bone destruction. The pathogenesis of these conditions has been attributed not only to bacterial-induced tissue destruction but also to a defect in periodontal tissue repair. Accumulated data have also shown that lipopolysaccharide (LPS) can directly induce cell death or apoptosis in many cell types, including macrophages, osteoblasts, vascular endothelial cells, hepatocytes and myocytes. The present study hypothesized that bacterial LPS-induced apoptosis in osteoblasts and periodontal ligament fibroblasts (PDL cells) is an important contributing factor to the defect in periodontal tissue repair in periodontal and periapical disease. Macrophages have been shown to respond to bacterial LPS by increasing the production of proinflammatory cytokines. In addition, large numbers of macrophages are present in inflamed periodontal tissue. We speculated that macrophages were a potential candidate cell for mediating apoptosis in osteoblasts and PDL cells in response to bacteria-derived LPS. The macrophage-like cell line, RAW 264.7, was stimulated with LPS, and the conditioned medium was used to treat osteoblasts and PDL cells. Bacterial LPS had no direct apoptotic effect on mouse osteoblasts or PDL cells, whereas the conditioned medium from LPS-activated macrophages was able to induce apoptosis in these cells. To evaluate the contribution of tumor necrosis factor-alpha (TNF-) released from macrophages on osteoblast and PDL cell apoptosis, cells were incubated with conditioned medium from LPS-treated macrophages in the presence and absence of anti-TNF- neutralizing antibodies. TNF- neutralizing antibody pretreatment inhibited the effect of conditioned medium from LPS-treated macrophages on osteoblast and PDL cell apoptosis in a dose-dependent manner. These results suggest that LPS could indirectly induce apoptosis in osteoblasts and PDL cells through the induction of TNF- release from macrophages. These studies provide insight into a potential mechanism by which bacterial-derived LPS could contribute to defective periodontal and bone tissue repair in periodontal and periapical disease.     

Glucocorticoids and osteocyte autophagy

Glucocorticoids are used for the treatment of inflammatory and autoimmune diseases. While they are effective therapy, bone loss and incident fracture risk are high. While previous studies have found GC effects on both osteoclasts and osteoblasts, our work has focused on the effects of GCs on osteocytes. Osteocytes exposed to low dose GCs undergo autophagy while osteocytes exposed to high doses of GCs or for a prolonged period of time undergo apoptosis. This paper will review the data to support the role of GCs in osteocyte autophagy.This article is part of a Special Issue entitled "The Osteocyte".