运动诱导的低铁状态大鼠骨髓细胞铁摄入的变化

本文观察了运动性低铁状态大鼠骨髓细胞转铁蛋白 (Tf)结合铁和非Tf结合铁摄入的变化。大鼠随机分为 6个月的运动组 (EG)和对照组 (SG)。SG平均每个幼红细胞Tf受体数为 890 15 0± 16 4849个 ,而在EG为 2 17536 0± 46 2 737个 (P <0 0 5 ) ,但受体的解离常数不受运动影响。EG中Tf的内吞平台和胞内铁聚积速度显著高于SG ,胞浆和胞内膜性成分中Tf结合铁和Fe(Ⅱ )摄入增加。EG的胞浆内Fe(Ⅱ )摄入的米氏常数值降低 ;细胞膜性成分中Fe(Ⅱ )摄入的最大速度增加。上述结果表明 ,运动不仅通过增加Tf受体的表达促进Tf结合铁的内吞 ,而且增强非Tf结合铁的内吞途径。尽管这些变化的机制尚不清楚 ,但它们有利于运动时血红素的合成     

Polypeptide factors regulating osteogenesis and bone marrow repair

Osteogenic growth polypeptides regulate bone cell function in vitro and may act in vivo in an autocrine, paracrine, or endocrine manner. Several of these polypeptides are present in the blood in an inactive form. During postablation bone marrow regeneration these factors may be activated, released from the blood clot, and together with locally produced polypeptides mediate the initial intramedullary/systemic osteogenic phase of this process. Then, the same and/or other polypeptides expressed by stromal cells have the potential to promote the second phase of regeneration that consists of osteoclastogenesis, resorption of the transient intramedullary bone, and hemopoiesis. This may be an indirect influence since these polypeptides can regulate the stromal cell expression of some of the hemopoietic factors. Clinically, the osteogenic growth polypeptides that regulate osteogenesis and hemopoiesis have a potential role in osteoporosis therapy, implant bone surgery, and bone marrow transplantation. © 1994 Wiley-Liss, Inc.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.     

Osteocalcin promotes differentiation of osteoclast progenitors from murine long-term bone marrow cultures

Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either α-MEM with 2% heat-inactivated horse serum alone (α) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal α medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase–positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 μg/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 ± 14.2) than in GM-CSF alone (53.3 ± 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption. © 1994 Wiley-Liss, Inc.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

Understanding the pathology and mechanisms of type I diabetic bone loss

Type I (T1) diabetes, also called insulin dependent diabetes mellitus (IDDM), is characterized by little or no insulin production and hyperglycemia. One of the less well known complications of T1-diabetes is bone loss which occurs in humans and animal models. This complication is receiving increased attention because T1-diabetics are living longer due to better therapeutics, and are faced with their existing health concerns being compounded by complications associated with aging, such as osteoporosis. Both male and female, endochondrial and intra-membranous, and axial and appendicular bones are susceptible to T1-diabetic bone loss. Exact mechanisms accounting for T1-diabetic bone loss are not known. Existing data indicate that the bone defect in T1-diabetes is anabolic rather than catabolic, suggesting that anabolic therapeutics may be more effective in preventing bone loss. Potential contributors to T1-diabetic suppression of bone formation are discussed in this review and include: increased marrow adiposity, hyperlipidemia, reduced insulin signaling, hyperglycemia, inflammation, altered adipokine and endocrine factors, increased cell death, and altered metabolism. Differences between T1-diabetic- and age-associated bone loss underlie the importance of condition specific, individualized treatments for osteoporosis. Optimizing therapies that prevent bone loss or restore bone density will allow T1-diabetic patients to live longer with strong healthy bones.     

Icariin promotes osteogenic differentiation of bone marrow stromal cells and prevents bone loss in OVX mice via activating autophagy

Osteoporosis (OP) results from the impaired function of endogenous bone marrow mesenchymal stem cells (BMSCs). Icariin (ICA) has shown potential osteoprotective effects. However, the molecular mechanism for the anabolic action of ICA remains largely unknown. The objective of the present study is to investigate whether ICA prevents bone loss by acting on BMSCs via affecting the level of autophagy after ovariectomy (OVX). The BMSCs were extracted from BALB/c mice treated with ICA, chloroquine (CQ, an autophagy inhibitor) or ICA + CQ. The OVX mice were injected with ICA, CQ, or ICA + CQ for 1 month. We performed Alizarin Red staining and alkaline phosphatase staining to detect osteogenic differentiation of BMSCs. Micro-CT, hematoxylin and eosin staining, Oil Red O staining, and tartrate-resistant acid phosphatase staining were used to assess the bone mass, lipid droplets and osteoclasts in femurs. Autophagy activity in BMSCs from different groups was evaluated by Western blot analysis. The osteogenic differentiation of BMSCs from OVX-induced OP mice was decreased. Treatment with ICA reduced bone loss and formation of osteoclasts and increased osteogenic differentiation of BMSCs in vitro and vivo. In addition, autophagy was enhanced in BMSCs of OVX mice treated with ICA. Our results indicate that ICA prevents OVX-induced bone loss possibly by strengthening the osteogenic differentiation of BMSCs via increasing autophagic activity.     

Menopause transition promotes distinct modulation of mRNAs and miRNAs expression in calvaria and bone marrow osteoblastic cells

Investigation on functional genome research may contribute to the knowledge of functional roles of different mRNAs and miRNAs in bone cells of osteoporotic animals. Currently, few studies indicate the changes in gene modulation that osteoporosis causes in osteoblastic cells from different sites. Thus, the purpose of this investigation was to evaluate cell viability, alkaline phosphatase activity and modulation of mRNAs/miRNAs in osteoblastic cells from calvaria and bone marrow by means of microarray technology. Wistar female rats were divided in sham operated and ovariectomized groups. After 150 days of ovariectomy, cells were isolated from both sites to perform cell culture. Results showed that calvaria cells from ovariectomized rats had a decrease in viability when compared to control groups and to bone marrow cells from osteoporotic rats after 3 days. Alkaline phosphatase activity decreased in calvaria cells from ovariectomized rats whereas it was increased in bone marrow osteoblastic cells in the same group. Microarray data analysis showed 5447 differentially expressed mRNAs and 82 differentially expressed miRNAs in calvaria cells. The same way, 4399 mRNAs and 54 miRNAs were expressed in bone marrow cells. mRNAs associated with bone metabolism such as Anxa5, Sp7, Spp1, Notch1 were distinctively modulated in both sites, as well as miRNAs such as miR‐350, miR‐542‐3p, miR‐204‐5p, and miR‐30e‐3p. The RNA species identified in this study could be further used as targets for treatment or prevention of osteoporosis.     

Cathepsin K deficiency promotes alveolar bone regeneration by promoting jaw bone marrow mesenchymal stem cells proliferation and differentiation via glycolysis pathway

ObjectivesTo clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC).Materials and MethodsTooth extraction models of Ctsk knockout mice (Ctsk ^‐/‐) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK‐8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk^‐/‐ JBMMSC was sent to RNA‐seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis.ResultsWe found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up‐regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA‐seq results showed that coding genes of key enzymes in glycolysis were significantly up‐regulated in Ctsk^‐/‐ JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC’s regeneration was blocked subsequently.ConclusionsOur findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.     

Matrix remodeling associated 7 promotes differentiation of bone marrow mesenchymal stem cells toward osteoblasts

The matrix remodeling associated 7 (MXRA7) gene had been ill-studied and its biology remained to be discovered. Inspired by our previous findings and public datasets concerning MXRA7, we hypothesized that the MXRA7 gene might be involved in bone marrow mesenchymal stem cells (BMSCs) functions related to bone formation, which was checked by utilizing in vivo or in vitro methodologies. Micro-computed tomography of MXRA7-deficient mice demonstrated retarded osteogenesis, which was reflected by shorter femurs, lower bone mass in both trabecular and cortical bones compared with wild-type (WT) mice. Histology confirmed the osteopenia-like feature including thinner growth plates in MXRA7-deficient femurs. Immunofluorescence revealed less osteoblasts in MXRA7-deficient femurs. Polymerase chain reaction or western blot analysis showed that when WT BMSCs were induced to differentiate toward osteoblasts or adipocytes in culture, MXRA7 messenger RNA or protein levels were significantly increased alongside osteoblasts induction, but decreased upon adipocytes induction. Cultured MXRA7-deficient BMSCs showed decreased osteogenesis upon osteogenic differentiation induction as reflected by decreased calcium deposition or lower expression of genes responsible for osteogenesis. When recombinant MXRA7 proteins were supplemented in a culture of MXRA7-deficient BMSCs, osteogenesis or gene expression was fully restored. Upon osteoblast induction, the level of active β-catenin or phospho-extracellular signal-regulated kinase in MXRA7-deficient BMSCs was decreased compared with that in WT BMSCs, and these impairments could be rescued by recombinant MXRA7 proteins. In adipogenesis induction settings, the potency of MXRA7-deficient BMSCs to differentiate into adipocytes was increased over the WT ones. In conclusion, this study demonstrated that MXRA7 influences bone formation via regulating the balance between osteogenesis and adipogenesis in BMSCs.     

Transplantation with bone marrow stromal cells promotes wound healing under chemotherapy through altering phenotypes

Stem cell transplantation is a promising strategy for delayed wound healing caused by chemotherapy. However, the fate of stem cells under chemotherapy has not been fully elucidated. Herein we characterized human fetal bone marrow stromal cells (hBMSCs) during wound healing in mice treated with cyclophosphamide (CTX). The isolated hBMSCs expressed the phenotype of CD11b(low)/CD14(low)/CD34(low)/CD45(low)/CD29(high)/CD44(high)/CD90(high)/CD105(high)/CD146(high)/STRO-1(low). Following in vitro exposure to CTX, hBMSCs showed decreased cell growth in a dose- and time-dependent manner, accompanied by increased expressions of collagen-I/III, and CD31. After transplantation, wounds closed as early as 8 days and were positive for α-smooth muscle actin (α-SMA), implicating the enhanced re-epithelialization and wound contraction. Moreover, proliferating cell nuclear antigen (PCNA) and CD31 showed co-localization with α-SMA, suggesting the differentiation of hBMSCs into epithelial cells and myofibroblasts/fibroblasts. Taken together, our results indicate hBMSCs can accelerate wound healing under chemotherapy through altering their phenotypes.     

A dual role of cholesterol in osteogenic differentiation of bone marrow stromal cells

Osteoblasts, the chief bone-forming cells, are differentiated from mesenchymal stromal/stem cells. Disruption of this differentiation process can cause osteoporosis, a bone disease characterized by low bone mass and deteriorated bone structure. Cholesterol has been implicated in pathogenesis of osteoporosis, and was recently identified as an endogenous activator of Hedgehog (Hh) signaling. However, its pathological and physiological roles in osteoblast differentiation are still poorly understood. Moreover, it is unclear whether these potential roles played by cholesterol are related to its capability to modulate Hh pathway. In this study, we investigated the role of exogenous versus endogenous cholesterol in osteogenesis and Hh pathway activation using ST2 cells, a bone marrow stromal cell line. We found that exogenous cholesterol significantly inhibited alkaline phosphatase (ALP) activity and messenger RNA expression of osteoblast markers genes (Alpl, Sp7, and Ibsp) while modestly activating expression of Gli1 (a readout of Hh signaling) under both basal osteogenic culture condition and Wnt3a treatment. Similarly, exogenous cholesterol suppressed osteogenic response of ST2 cells to sonic Hh (Shh) or purmorphamine (Purmo) treatment, which, however, was accompanied by diminished induction of Gli1, indicating the involvement of a Hh-dependent mechanism. Interestingly, depletion of endogenous cholesterol also reduced Shh-induced ALP activity and Gli1 expression. Likewise, cholesterol depletion inhibited osteogenic response to Purmo, although it did not affect Gli1 induction. Taken together, our findings have demonstrated that cholesterol plays a dual role in osteoblast differentiation likely through both Hh-dependent and -independent mechanisms.