Inhibited anabolic effect of insulin‐like growth factor‐I on stromal bone marrow cells in endothelial nitric oxide synthase‐knockout mice

Aims: Insulin‐like growth factor‐I (IGF‐I), parathyroid hormone (PTH) and PTH‐related protein (PTHrP) are hormones that have anabolic effects on bone formation. The aim of this study was to investigate whether production of nitric oxide (NO) is involved in the effect of IGF‐I and PTH/PTHrP on osteoblast‐like cells. Methods: Bone marrow stromal cells from adult endothelial nitric oxide synthase (eNOS)‐knockout (eNOSKO) mice and wild type (WT) counterparts were cultivated with osteogenic substances. The cells showed an osteoblastic phenotype measured as osteocalcin production and alkaline phosphatase activity. DNA synthesis was measured as [³H] thymidine incorporation in the bone marrow cells and in a human osteosarcoma cell‐line (SaOS‐2). Results: The stimulatory effect of IGF‐I on thymidine incorporation seen in WT animals was absent in eNOSKO mice. Addition of a NO donor to eNOSKO cells recovered the effect of IGF‐I on thymidine incorporation. PTH/PTHrP stimulated cell proliferation in both WT and eNOSKO mice. In SaOS‐2 cells, incubation with IGF‐I together with a NOS inhibitor resulted in an inhibition of the anabolic effect of IGF‐I on cell proliferation. Conclusions: The stimulatory effect of IGF‐I on WT cell proliferation was abolished in eNOSKO cells, recovered by an NO donor and inhibited in osteosarcoma cells by a NOS inhibitor. The results indicate that the effect of IGF‐I is dependent on NO production. The impaired IGF‐I response may contribute to the bone defect formation seen in eNOSKO animals.     

Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells

One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/10(6) cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.     

Beneficial effect of autologous transplantation of bone marrow stromal cells and endothelial progenitor cells on cerebral ischemia in rabbits

We tested the therapeutic effect of autologous transplanted bone marrow stromal cells (BMSCs) and endothelial progenitor cells (EPCs) on cerebral ischemia in rabbits. Rabbit permanent middle cerebral artery occlusion (MCAO) models were intravenously injected with ex vivo expanded autologous BMSCs (n = 8), EPCs (n = 8), or phosphate-buffered saline (n = 6). 14 days after the transplantation, both infusion groups witnessed a functional improvement, a decrease in the number of apoptotic cells and an increase in the microvessel density in the ischemic boundary area, as compared to vehicle-treated control group. The EPCs treated group also exhibited a diminished infarct area in comparison with the control group. Moreover, immunohistochemistry revealed that few transplanted BMSCs expressed markers for astrocytes (GFAP⁺) and neurons (NeuN⁺), and most of EPCs were capable of binding to UEA-1 lectin and were incorporated into capillaries. Our data suggest that both BMSCs and EPCs, despite differences in their action mechanism, can be functional cytoreagents for treatment of cerebral ischemia in rabbits.     

Modulation of the migration and differentiation potential of adult bone marrow stromal stem cells by nitric oxide

Nitric oxide (NO) is a diffusible free radical, which serves as a pluripotent intracellular messenger in numerous cell systems. NO has been demonstrated to regulate actin dependent cellular functions and functions as a putative inductive agent in directing stem cells differentiation. In this study, we investigated the effect of exogenous NO on the kinetics of movement and morphological changes in adult bone marrow stromal cells (BMSCs) in a wound healing model of cellular migration. Cellular migration and morphological changes were determined by measurement of changes in the area and fractal dimension of BMSCs monolayer as a function of time in the presence of an NO donor (S-Nitroso-N-Acetyl-D,l-Penicillamine, SNAP) compared to untreated BMSCs. Response of the BMSCs’ actin cytoskeleton and desmin to NO was assessed by determining changes in their integrated optical density (IOD) and fractal dimension at 24 h and 7 days. NO suppressed BMSCs’ migration accompanied by a reduction in cell size, with maintenance of their stellate to polygonal morphology. In response to NO, the actin cytoskeleton expressed an increase in randomness but maintained a constant amount of F-actin relative to the cell size. The presence of NO also induced an increase in randomly organized cytoplasmic desmin. These data suggest that NO has an apparent inductive effect on adult BMSCs and is capable of initiating phenotypic change at the gross cellular, cytoskeletal and molecular levels. It is apparent, however, that additional factors or conditions are required to further drive the differentiation of adult BMSCs into specific phenotypes, such as cardiomyocytes.     

The effect of bioactive glasses on bone marrow stromal cells differentiation

Bone marrow is a mixture of hematopoietic, vascular, stromal and mesenchymal cells capable of skeletal repair/regeneration thanks to the ability of bone marrow cells to differentiate into osteoblasts and osteoclasts. This ability is important in tissue regeneration during fracture healing, or for successful osteointegration of implanted prostheses, and in bone remodelling. Therefore, bone marrow cell culture systems seem to be useful and relatively close to in vivo conditions models to study interactions occurring at the cell-material interface of implants directed to hard tissue engineering. The purpose of this study was to investigate the ability of three bioactive glasses (45S, 58S and 77S) to induce osteogenic differentiation and cell mineralisation. A significant effect of the 45S and 77S bioactive materials was seen on early differentiation of the marrow stromal cells into osteoblast-like cells. 45S bioglass evidenced also the highest effect on cell mineralisation at the same level as cells treated with dexametasone, used as positive control. 77S treated cells evidenced also a significant inhibition in the number of multinucleated TRAP-positive cells (ostoclast-like cells) in comparison with the control untreated cells and in marrow cells treated with 45S and 58S bioactive glasses. These findings have potential implications and applications for tissue engineering where three-dimensional bioactive glass substrates could be used as scaffolds for in vitro production of bioengineered bone.     

Adhesion and growth of bone marrow stromal cells on modified alginate hydrogels

Alginate is a biodegradable, immunocompatible biopolymer that is capable of immobilizing viable cells and bioactive factors. Few investigations have analyzed the efficacy of alginate gels as substrata for cell attachment and proliferation. Here we have compared the adhesion and subsequent growth of human and rat bone marrow stromal fibroblastic cells on unmodified alginate hydrogel surfaces. It was found that, in contrast to rat cells, human cells did not readily attach or proliferate on unmodified alginates. In attempts to enhance these features, or collagen type I was incorporated into the gels, with no significant improvements in prolonged human cell adherence. However, alginate gels containing both collagen type I and beta-tricalcium phosphate were found to enhance human cell adherence and proliferation. Furthermore, interactions between the collagen and beta-tricalcium phosphate prevented loss of the protein from the hydrogels. These results indicate that alginate gels containing collagen have potential uses as vehicles for delivery of adherent cells to a tissue site. In addition, gels containing beta-tricalcium phosphate, with or without collagen type I incorporation, have potential to support cell growth and differentiation in vitro before implantation. This study emphasizes the limitations of the uses of cells derived from experimental animals in certain model studies relating to human tissue engineering.     

外源性胰岛素样生长因子1基因在大鼠骨髓基质细胞中的表达及对其生物学行为的影响

目的：观察人胰岛素样生长因子1（hIGF-1）基因转染大鼠骨髓基质细胞（BMSC）后产物的表达及对BMSC特征的影响.方法：构建hIGF-1逆转录病毒载体并转染BMSC,嘌呤霉素筛选,RT-PCR和ELISA法检测hIGF-1表达,MTT及美蓝、碱性磷酸酶钙钴、苏丹Ⅲ染色观察转染后BMSC增殖、集落形成及分化,并分析药物筛选对其增殖的影响.结果：转染BMSC经筛选后,IGF-1表达的增幅高于未经筛选者（10.7倍：6.7倍）.与空质粒转染组比较,未经筛选的BMSC增殖加快,而经筛选者增殖和集落形成能力降低,向脂肪及成骨细胞分化增强.结论：基因修饰可成功构建高表达IGF-1的BMSC,并促进其增殖和自然分化,但嘌呤霉素筛选对其自我更新能力有负面作用.     

脱基质小牛骨粉复合骨髓基质干细胞的成骨作用

背景：利用骨组织工程技术在上颌窦提升中的成骨研究是目前口腔种植学到研究热点。 目的：探讨骨组织工程在上颌窦底提升同期牙种植中的成骨效果。 方法：体外分离培养犬骨髓基质干细胞,将细胞与脱基质小牛骨粉复合培养并向成骨细胞定向诱导分化。健康成年犬12只行双侧上颌窦底提升同期牙种植,一侧植入复合物,另一侧植入脱基质小牛骨粉做对照。 结果与结论：种植体稳固无松动,上颌窦黏膜完整。实验侧可见新生骨形成较早、骨量较多;对照侧新生骨形成较慢。X射线显示实验侧新生骨质致密,与种植体结合紧密。随时间的增加,牵出力增大,12,24周时两侧差异有显著性意义(P < 0.05)。组织形态学检测显示新生骨面积逐渐增加,12,24周时差异有显著性意义(P < 0.05)。提示组织工程化骨在上颌窦提升同期牙种植中可获得良好的成骨效果。     

Hypoxia increases VEGF-A production by prostate cancer and bone marrow stromal cells and initiates paracrine activation of bone marrow endothelial cells

Hypoxia develops at sites of rapid cancer growth near sites of poorly organized vasculature. Heparin binding growth factors (HBGFs) support neoangiogenesis of tumors. We examined the effect of culturing bone-targeted, metastatic C4-2B prostate cancer cells and bone stromal derived HS27a cells under hypoxic conditions on expression of vascular endothelial growth factor (VEGF) family members. A sealed chamber infused with 1% (hypoxic) or 20% (normoxic) O(2) was used. Both cell lines produced VEGF-A in normoxia, but little or no HB-EGF, another HBGF. HS27a cells produced low levels of FGF-2 and HGF, but little or none was secreted by C4-2B cells. Levels of VEGF-A in conditioned medium (CM) from both cell lines doubled when cultured in hypoxia. Similar changes in VEGF-A mRNA levels were seen. Receptor expression was unchanged by hypoxia. Changes in VEGF-A expression during hypoxia were preceded by nuclear accumulation of hypoxia inducible factor-1alpha (HIF-1alpha). Bone marrow endothelial (BME) cells express high levels of VEGFR2/flk-1, and are targets of VEGF-A induced neovascularization. BME cells proliferated in response to treatment with HS27a CM, but not C4-2B CM. BME cells formed tube-like angiogenic structures on growth factor reduced Matrigel in response to CM from HS27a or C4-2B cells. This response was greater when CM was produced under hypoxia, and was reduced by VEGF-A or FGF-2 neutralizing antibodies. We conclude that hypoxia triggers a physiologically relevant increase in VEGF-A by prostate cancer and bone marrow stromal cells which involves a paracrine loop that recruits and activates BME to support tumor neovascularization-related processes.     

Age-related increases in LPS-stimulated nitric oxide production from cultured rat bone marrow cells

Objective: To understand bone metabolism during senescence, we examined age-related change in nitric oxide (NO) production from bone marrow cells stimulated by lipopolysaccharide (LPS). Methods: We evaluated the age-related change in the NO production and expression of iNOS protein and mRNA of LPS-stimulated bone marrow cells collected from the tibiae of young and retired female and young and retired male rats. In addition, we used flow cytometry to assess changes in the distribution of CD14, a cell surface receptor of LPS. Results: The results revealed that NO production from bone marrow cells stimulated with LPS changed with aging. The NO levels in old rats were significantly higher (P<0.05) than those in young rats. Polymerase chain reaction (PCR) analysis indicated that the LPS-induced expression of iNOS mRNA was augmented in retired rats. Although the distribution pattern of the bone marrow cells was similar between young and retired rats, the percentage of CD14-positive cells in specific populations differed between the age groups. Specifically, in the granule-containing bone marrow cells, the percentage of CD14-positive cells was increased in retired rats. Conclusion: Our results indicate that LPS-stimulated NO production from rat bone marrow cells increased with age and that the difference in responsiveness might be due to changes in the percentage of CD14-positive cells in the bone marrow.     

bFGF对大鼠骨髓基质细胞的诱导分化作用

目的 观察bFGF对大鼠骨髓基质细胞的诱导分化作用。方法 从大鼠骨髓中分离培养基质细胞并传至第3代，用含10ng/ml碱性成纤维细胞生长因子(bFGF)的诱导培养基诱导，然后观察细胞形态的变化，并采用免疫组织化学法检测诱导后24h、48h、72h、96h细胞神经丝蛋白(NF-200)和胶质纤维酸性蛋白(GFAP)的表达。结果 诱导后24h的部分细胞不仅在形态上表现为神经元样，而且呈NF-200阳性表达，GFAP阴性。48h、72h、96h NF-200阳性细胞数与24h间无显著性差异(P＞0．05)。结论bFGF可以在体外诱导骨髓基质细胞向神经元样细胞分化。     

Trophic effects of insulin-like growth factor-I on fetal rat hypothalamic cells in culture

The hypothesis that insulin-like growth factor-I is a trophic factor for primary fetal rat hypothalamic cells was tested, since we previously reported a potent mitogenic effect of this peptide on virally-transformed hypothalamic cells. It was found that insulin-like growth factor-I produced significant and dose-dependent increases in the survival of fetal hypothalamic neurons in primary mixed glial/neuronal cultures. By 48 h in vitro, cultures treated with insulin-like growth factor-I (6 nM) had twice as many neurite-bearing cells as controls, while by day 15 a five-fold difference was present. The peptide was similarly active in promoting neuronal survival in neuron-enriched (98% neurons) hypothalamic cultures. Mixed hypothalamic cultures had specific binding sites for insulin-like growth factor-I. In addition, the neurons grown in the presence of insulin-like growth factor-I had a more differentiated morphology and had significantly higher levels of protein kinase C, an enzyme that increases during neurite formation and synaptogenesis. Finally, glial-enriched cultures (greater than 99% glial cells) obtained from the fetal hypothalamus showed increased [3H]thymidine incorporation in response to insulin-like growth factor-I. These results further support the contention that insulin-like growth factor-I is a neurotrophic factor and suggest that it may participate in the normal development of the hypothalamus by increasing neuronal survival/differentiation and stimulating glial growth.     

低氧对大鼠骨髓基质细胞分泌神经生长因子的影响

目的:研究大鼠骨髓基质细胞(BMSCs)在低氧培养条件下神经生长因子(NGF)的表达.方法:采用全骨髓贴壁培养法体外分离、纯化大鼠的BMSCs.置于常氧和低氧(3%)条件下培养,用cell counting kit (CCK-8)测定细胞增殖情况;台盼蓝染色检测细胞的活力;提取2种培养条件5个时间点的细胞蛋白,免疫印迹法检测细胞蛋白中NGF的含量;收集2种培养条件下5个时间点的细胞上清液,ELISA法检测细胞上清液中NGF的含量.结果:在3%低氧条件下培养BMSCs,其细胞活力较好,细胞增殖加快;免疫印迹法检测结果表明,与常氧条件相比,随培养时间的延长,低氧条件下NGF的表达明显增加,其24h分泌达高峰;ELISA检测结果表明,随培养时间的延长,常氧下细胞上清液中NGF的浓度逐渐增加,低氧12h时NGF的浓度显著增高,明显高于常氧培养组.结论:BMSCs在3%低氧条件下培养细胞生长迅速,细胞活力较好;3%的低氧条件可增强BMSCs分泌NGF的能力.     

Clastogenic effect of fenfluramine in mice bone marrow cells in vivo

Fenfluramine, an amphetamine derivative used in the treatment of obesity, has been evaluated in vivo in the bone marrow cells of Swiss albino mice using two cytogenetic endpoints for assessing its genotoxic and clastogenic potentials. Concentrations of 0.75, 1.5, 3.0, and 5.0 mg/kg b.w. were administered orally for the study of sister chromatid exchange frequencies and chromosome aberrations (CA). SCE frequencies showed a positive dose response; 1.5 mg/kg being the minimum effective concentration. Fen caused a prolongation of cell cycle at all concentrations. Except for the minimum therapeutic dose (0.75 mg), all other doses (1.5, 3.0, and 5.0 mg) showed a significant increase in the percentage of damaged cells over that of the vehicle control. The degree of clastogenicity was directly proportional to the dosage used and inversely related with the duration of treatment. A gradual reduction of the clastogenic potential was observed after 12 and 24 hr of exposure, indicating that the maximum effect occurs at the middle or late synthetic phase of the cell cycle. This study, probably the first detailed screening of the drug for its genotoxicity, shows that Fen is moderately clastogenic and a DNA damaging agent in vivo.     

Ghrelin抑制小鼠骨髓基质细胞成脂分化

目的观察胃促生长素ghrelin对原代培养小鼠骨髓基质细胞成脂分化的影响。方法体外培养小鼠骨髓基质细胞,MDI诱导剂诱导细胞成脂分化,显微镜观察细胞形态变化;油红O染色法检测细胞甘油三酯含量,化学比色法测定碱性磷酸酶(ALP)活性;MTT法检测细胞克隆化扩增活动,RT-PCR检测过氧化物酶体增殖物激活受体γ2(PPARγ2)和丝氨酸蛋白酶脂肪因子adipsinmRNA表达,Westernblot检测PPARγ2蛋白表达。结果Ghrelin能够剂量依赖性(10-9、10-8、10-7mol/L)地抑制骨髓基质细胞成脂分化,升高ALP活性(P<0·05或P<0·01)。Gh-relin能够降低PPARγ2和adipsinmRNA以及PPARγ2蛋白表达(P<0·05)。Ghrelin对细胞成脂分化早期的克隆化扩增没有显著影响。结论Ghrelin能够显著抑制体外培养小鼠骨髓基质细胞成脂分化,该作用可能与抑制PPARγ2表达有关。     

Simultaneous injection of bone marrow cells and stromal cells into bone marrow accelerates hematopoiesis in vivo

We have previously demonstrated that stromal cells can support the proliferation and differentiation of hematopoietic cells in vitro and in vivo and that a major histocompatibility complex restriction exists between hematopoietic stem cells and stromal cells. We have also found that intra-bone marrow (IBM) injection of allogeneic bone marrow cells (BMCs) leads to more rapid reconstitution of hematopoietic cells than intravenous injection. In the present study, we examine the effect of simultaneous injection of stromal cells and BMCs into the same bone marrow on the recovery of donor hematopoietic cells and demonstrate that simultaneous IBM injection of BMCs plus stromal cells is more effective in reconstituting recipients with donor hematopoietic cells than intravenous injection of BMCs plus stromal cells or IBM injection of BMCs alone.     

Expression of insulin-like growth factor-I (IGF-I) in aneurysmal bone cyst.

The effects of insulin-like growth factor-I on bone tissue and its role in bone development have been extensively investigated, but there is little information on its role in the pathogenesis of aneurysmal bone cyst. Therefore, using the techniques of immunohistochemistry and in situ hybridization, the authors studied the expression of insulin-like growth factor-I in 19 specimens of aneurysmal bone cyst. Insulin-like growth factor-I or specific mRNA sequences encoding for insulin-like growth factor-I were detectable in all specimens tested and were mainly localized in multinucleate giant cells. In contrast, only insignificant levels of insulin-like growth factor-I expression were detectable in normal human bone tissue. Taken together with the previously reported role of insulin-like growth factor-I in the pathogenesis of giant cell tumor, the findings of this study suggest that insulin-like growth factor-I may play a role in the pathogenesis of aneurysmal bone cyst.