辛伐他汀促进大鼠BMSCs分化为成骨细胞

目的 探讨辛伐他汀对体外诱导培养的大鼠骨髓基质干细胞（BMSCs）成骨分化的作用,以及对大鼠BMSCs基因表达谱的影响。方法 取6周龄雄性SD大鼠股骨、胫骨BMSCs进行培养,第3天改用成骨诱导培养基,同时实验组（SIM）加入辛伐他汀（1×10-7mol/L）,对照组（V）加入等量溶剂。细胞培养的第14天进行碱性磷酸酶（ALP）比活性测定;第21天进行如下检测：Von Kossa染色观察细胞外基质矿化情况;提取、纯化mRNA,逆转录合成cDNA,荧光标记后与大鼠全基因组寡核苷酸芯片（G4130A）杂交、扫描后筛选出差异表达的基因,Real-time RT-PCR验证部分差异表达基因mRNA的表达。 结果 1. SIM组ALP比活性及细胞外基质矿化能力明显强于V组(p<0.05);2. 在22,575个基因中,共检测出678个差异表达基因,其中包括ALPl、TGFβ1、OCN、 DLX5、Axin2、BMP-2、IBSP、MMP13等与成骨分化相关的基因。结论 辛伐他汀能够促进体外诱导培养的BMSCs向成骨细胞分化,其作用机制可能与调控多种成骨相关基因的表达有关。     

In vivo osteogenic differentiation of rat bone marrow stromal cells in thermosensitive MPEG-PCL diblock copolymer gels

Methoxy poly(ethylene glycol)-poly(epsilon-caprolactone) (MPEG-PCL) diblock copolymers were prepared by ring-opening polymerization and their phase transition behavior characterized as a function of temperature. The MPEG-PCL solutions formed a sol at room temperature, and underwent sol-to-gel followed by gel-to-sol phase transitions as the temperature was increased. The temperature range over which the solutions were in a gel state could be extended simply by increasing the PCL chain length in the diblock copolymer. Scanning electron microscopy (SEM) images of MPEG-PCL solutions in the sol and gel states revealed near-regular and irregular porous structures, respectively. in vitro culture of rat bone marrow stromal cells (rBMSCs) on gel surfaces exhibited mostly round cells after 1 day of incubation. SEM images of the attached cells clearly showed the cell body and anchoring filopodia. Injection of room-temperature diblock copolymer solutions into Sprague-Dawley rats produced a gel at body temperature. In situ gel-forming scaffolds in vivo were successfully fabricated by simple subcutaneous injection of MPEG-PCL diblock copolymer solutions. The gel implants retained their original shape for 4 weeks without in- flammation at the injection site. Gel implants removed after 4 weeks were found to be surrounded by a thin fibrous capsule consisting of fibroblasts and blood vessels cells. Hematoxylin and eosin (H&E) and von Kossa staining revealed bone formation in gel implants containing both rBMSCs and dexamethasone, with the degree of bone formation increasing markedly with increasing dexamethasone concentration. Thus, our results show that in situ gel scaffolds fabricated from MPEG-PCL diblock copolymer solutions containing dexamethasone enable multipotent rBMSCs to produce viable bone when injected into rats.     

仙灵骨葆对骨质疏松大鼠骨量及骨髓基质干细胞成骨分化的影响

背景：双侧卵巢切除可造成大鼠骨量丢失,仙灵骨葆作为传统中药具有一定的促进骨形成作用。 目的：观察仙灵骨葆体内给药对骨质疏松大鼠骨量及骨髓基质干细胞成骨分化能力以及相关因子表达的影响。 方法：3月龄雌性SD大鼠24只随机数字表法均分为3组,卵巢切除组和仙灵骨葆组行卵巢切除,造模6周后仙灵骨葆组给予仙灵骨葆250 mg/(kg•d),干预8周;正常对照组不予干预。 结果与结论：卵巢切除组L2-L4椎体骨密度显著低于其他两组,仙灵骨葆组仍显著低于正常对照组(P < 0.05);卵巢切除组血清骨钙素、骨髓基质干细胞的骨形态发生蛋白2、骨钙素mRNA水平均低于其他两组(P < 0.05)。细胞外基质矿化能力亦明显低于正常对照组和仙灵骨葆组。提示大鼠去势14周后骨量丢失显著,仙灵骨葆可部分阻止其骨量丢失,其作用机制可能与促进大鼠骨髓基质干细胞的成骨分化有关。     

beta-TCP microporosity decreases the viability and osteoblast differentiation of human bone marrow stromal cells

Association of osteoprogenitor cells to calcium phosphate ceramics is currently under intense investigation, for its considered ability to induce bone formation and therefore to allow the successful repair of large bone defects. However, if the first experimental and clinical studies provided promising results, lack of new bone formation has been reported in a large number of animal experiments. In this context and since it has been reported that in some conditions, calcium phosphate ceramic microstructure induces ectopic bone formation, we investigated the effects of ceramic microporosity on the behavior of osteoprogenitor cells for the development of hybrid materials. Human bone marrow stromal cells (BMSCs) were seeded on beta-tricalcium phosphate (TCP) ceramics with 0, 25, or 45% microporosity and cell adhesion, viability, and osteoblastic differentiation have been studied for three weeks. Cell counts, measurement of MTS conversion, and LDH activity indicated that microporosity decreased the viability of BMSCs in a time and rate-dependent manner. In addition, microporosity inhibited osteoblastic differentiation as compared with nonmicroporous ceramics, as revealed by decreased alkaline phosphatase activity and osteocalcin secretion. Results of this in vitro study therefore highlight a negative role for beta-TCP microporosity in the behavior of human osteoprogenitor cells.     

Effect of different magnitudes of continuous tensile stress on osteogenic differentiation of rat bone marrow stromal cells

背景：研究证实力学刺激是影响骨改建的重要因素,可促进骨髓基质干细胞骨向分化;但不同幅度力学刺激对骨髓基质干细胞分化的影响尚不明确。 目的：观察持续张应力对大鼠骨髓基质干细胞成骨分化的影响。 方法：全血贴壁培养法获取大鼠骨髓基质干细胞。采用Flexercell-4000细胞体外应力加载系统对骨髓基质干细胞施加5%,10%,15%幅度的持续张应力,对照组则不加力培养,频率1 Hz,持续时间48 h。分别在加力后1,6,12,24,48 h检测成骨标记物碱性磷酸酶、Ⅰ型胶原、骨钙素mRNA及成骨特异性转录因子Runx2的mRNA及蛋白表达。 结果与结论：5%和10%持续张应力作用下,骨髓基质干细胞的成骨标记基因碱性磷酸酶、Ⅰ型胶原、骨钙素mRNA的表达较对照组升高(P < 0.05),10%张力组升高的时间均较5%张力组早、幅度较高。15%持续在加力6 h时可促进骨髓基质干细胞碱性磷酸酶、Ⅰ型胶原mRNA的表达(P < 0.05)、随后表达下降,加力48 h后上述指标均低于对照组(P < 0.05),骨钙素mRNA的表达加力6 h后均低于对照组(P < 0.05)。5%张力组仅加力24 h后骨髓基质干细胞Runx2蛋白表达高于对照组(P < 0.05),10%,15%张力组加力6 h后Runx2蛋白表达均高于对照组(P < 0.05)。结果证实,5%,10%,15%持续张应力均可更有效地促进骨髓基质干细胞的骨向分化,10%持续张力的促进效应更显著。     

大鼠骨髓间质细胞向Schwann-like细胞的诱导分化

目的探索成年大鼠骨髓间质细胞(MSCs)向Schwann—like细胞转化的方法和机理。方法分别用变性神经提取液和含有大鼠白血病抑制因子(rLIF)、上皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)等的诱导液诱导成年大鼠骨髓间质细胞,观察诱导过程中细胞形态学变化,利用免疫细胞化学染色鉴定诱导细胞性质;同时培养大鼠Schwann细胞作为对照。结果经变性神经提取液诱导后细胞转化为梭形细胞,并排列成网状;经细胞因子诱导后细胞呈梭形排列,S-100,GFAP染色呈阳性反应。结论变性神经提取液中含有促使骨髓间质细胞向Schwann-like细胞分化的必要成份;LIF可能是这些必要成份的重要组成之一。     

Effects of bisphosphonates on proliferation and osteoblast differentiation of human bone marrow stromal cells

Bisphosphonates are well known potent inhibitors of osteoclast activity and are widely used to treat metabolic bone diseases. Recent evidence from in vitro and in vivo studies indicates that bisphosphonates may additionally promote osteoblastic bone formation. In this study, we evaluated the effects of three FDA-approved and clinically utilized bisphosphonates, on the proliferation and osteogenic differentiation of human bone marrow stromal cells (BMSC). BMSC were obtained from patients undergoing primary total hip arthroplasty for end-stage degenerative joint disease. Cells were treated with or without a bisphosphonate (alendronate, risedronate, or zoledronate) and analyzed over 21 days of culture. Cell proliferation was determined by direct cell counting. Osteogenic differentiation of BMSC was assessed with alkaline phosphatase bioassay and gene expression analyses using conventional RT-PCR as well as real-time quantitative RT-PCR. All bisphosphonates tested enhanced the proliferation of BMSC after 7 and 14 days of culture. Steady-state mRNA levels of key genes involved in osteogenic differentiation such as bone morphogenetic protein-2 (BMP-2), bone sialoprotein-II, core-binding factor alpha subunit 1 (cbfa1) and type 1 collagen, were generally increased by bisphosphonate treatment in a type- and time-dependent manner. Gene expression levels varied among the different donors. Enhancement of osteogenic differentiation was most pronounced after 14 days of culture, particularly following zoledronate treatment (p < 0.05 for BMP-2). In conclusion, using a clinically relevant in vitro model we have demonstrated that bisphosphonates enhance proliferation of BMSC and initiate osteoblastic differentiation. When administered around joint replacements, bisphosphonates may potentially compensate for the deleterious effects of particulate wear debris at the bone-implant interface, by encouraging increased numbers of cells committed to the osteoblastic phenotype, and thus improve the longevity of joint replacements.     

持续张应力对骨髓基质干细胞增殖及骨向分化的影响

目的通过体外加力装置研究持续牵张应力对大鼠骨髓基质干细胞(bone marrow stromal cells,BMSCs)增殖及骨向分化能力的影响。方法选用3月龄健康SD雌性大鼠,采用全血贴壁培养法分离及培养BMSCs。取生长良好的第3～5代细胞接种于Flexercell应力加载系统(10%、1 Hz),根据应力作用时间不同分为1、6、12、24 h组和48 h组。观察并分析持续牵张力对于大鼠BMSCs形态、增殖活性以及成骨能力变化的影响。结果 (1)随着加力时间的延长,与对照组相比,实验组细胞形态呈现一定规律性,细胞长轴多垂直于受力径向。(2)10%持续张应力作用可抑制BMSCs增殖活性。(3)持续张应力可增高碱性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型胶原(collagenI,COLⅠ)、核心结合因子Cbfa1(core binding factor a1,又名Runx2)mRNA的表达量,且呈现时间依赖性。其中实验组ALP表达量在24 h明显高于相应对照组,COLⅠ表达量在24 h及48 h均明显高于对照组,Runx2表达量在6 h与对照组相比显著增高(P<0.05)。骨钙素(osteocalcin,OC)含量在加力起始阶段显著高于对照组,随时间推移逐渐下降,48 h时明显低于对照组(P<0.05)。(4)持续张力可以促使Runx2蛋白水平增高,且在6 h实验组明显高于对照组(P<0.05)。之后缓慢下降,在24 h时显著低于对照组水平(P<0.05)。结论持续牵张力作用下BM-SCs细胞形态呈现一定规律性排列,其增殖活性受到抑制,但早期成骨向分化能力却显著提高。     

Proliferation, osteogenic differentiation, and distribution of rat bone marrow stromal cells in nonwoven fabrics by different culture methods

The proliferation, osteogenic differentiation, and distribution patterns of stromal cells from rat bone marrow were investigated in a three-dimensional nonwoven fabric of polyethylene terephthalate fiber by the static, agitated, and stirred culture methods; stirring speeds were 10, 50, and 100 rpm in the stirred culture method. The culture method affected the time profile of proliferation and osteogenic differentiation of cells or their distribution in the fabric. The extent of cell proliferation and osteogenic differentiation became higher in order of the stirred at 100 rpm = the stirred at 50 rpm > the stirred at 10 rpm > the agitated > the static methods. In addition, the cells were more uniformly proliferated in the fabric by the stirred culture method with time than they were proliferated in the fabric by other methods. The alkaline phosphatase (ALP) activity and calcium content were higher for cells cultured by the stirred culture method than those cultured by other methods. The total ALP activity, calcium content, and bone mineral density were higher for every stirred method than those for other methods. However, the distribution uniformity of cells differentiated was low irrespective of the culture method. It is concluded that the extent of proliferation and differentiation of cells or their distribution uniformity in the nonwoven fabrics was influenced by the culture method.     

大鼠骨髓间充质干细胞成骨诱导分化的超微结构变化

背景：目前对骨髓间充质干细胞成骨诱导分化的超微结构观察的报道甚少。 目的：采用全骨髓贴壁法分离培养大鼠骨髓间充质干细胞,成骨诱导并染色鉴定,利用电镜观察诱导前后细胞超微结构变化特点。 方法：全骨髓贴壁法体外分离、培养、纯化大鼠骨髓间充质干细胞,流式细胞仪检测细胞表面标记物,成骨染色鉴定向成骨方向诱导分化,扫描电镜及透射电镜观察成骨诱导前后细胞超微结构变化。 结果与结论：培养的第3代骨髓间充质干细胞纯度高、活力强,成骨诱导后的碱性磷酸酶活性染色、钙化结节染色均呈阳性。扫描电镜及透射电镜观察显示,经向成骨细胞诱导分化后,细胞形态铺展,不规则,其线粒体、粗面内质网、空泡明显增多,表明细胞功能活跃。     

Matrix-mediated retention of osteogenic differentiation potential by human adult bone marrow stromal cells during ex vivo expansion

During prolonged cultivation ex vivo, adult bone marrow stromal stem cells (BMSCs) undergo two probably interdependent processes, replicative aging and a decline in differentiation potential. Recently, our results with primary human fibroblasts indicated that growth on denatured collagen (DC) matrix results in the reduction of the rate of cellular aging. The present study has been undertaken to test whether the growth of human BMSCs under the same conditions would translate into preservation of cellular aging-attenuated functions, such as the ability to express HSP70 in response to stress as well as of osteogenic differentiation potential. We report here that growth of BMSCs on a DC matrix versus tissue culture polystyrene significantly reduced one of the main manifestations of cellular aging, the attenuation of the ability to express a major protective stress response component, HSP70, increased the proliferation capacity of ex vivo expanded BMSCs, reduced the rate of morphological changes, and resulted in a dramatic increase in the retention of the potential to express osteogenic-specific functions and markers upon treatment with osteogenic stimulants. BMSCs are a promising and increasingly important cell source for tissue engineering as well as cell and gene therapeutic strategies. For use of BMSCs in these applications, ex vivo expansion is necessary to obtain a sufficient, therapeutically useful, number of cells; however, this results in the loss of differentiation potential. This problem is especially acute in older patients where more extensive in vitro expansion of smaller number of stem/progenitor cells is needed. The finding that growth on certain biomaterials preserves aging-attenuated functions, enhances proliferation capacity, and maintains differentiation potential of BMSCs indicates a promising approach to address this problem.     

In vitro study of osteogenic differentiation of bone marrow stromal cells on heat-treated porcine trabecular bone blocks

This in vitro study investigated the potential of the heat-treated porcine trabecular bone block as a bone substitute for the treatment of bone defects or related diseases. Chemical, mechanical, and morphological studies of bone blocks were performed. The resultant properties were compared with the properties of currently available commercial products from bovine trabecular bones. The major component of the bone block was hydroxyapatite, and the ratio of Ca/P was 1.65-1.66. The average values of the compressive Young's modulus and the ultimate strength were 346.33 +/- 83.15 and 6.66 +/- 1.62 MPa, respectively. The pore size of the heat-treated bone blocks was approximately 300-500 microm. For the biological investigations, expanded bone marrow stromal cells (BMSCs) were isolated from the femurs of New Zealand White rabbits and were dynamically seeded into the heat-treated porcine bone block (10x10x5 mm3). Before the cells were seeded, the heat-treated porcine bone blocks were divided into two groups: collagen coated blocks (n=16) and uncoated blocks (n=16). Within each group, the blocks were again divided into two groups, depending on the culture method, i.e., static or rotating culture. Cells were cultured in the blocks for up to 6 weeks. Scanning electron microscopic examination after 4 weeks showed that the cell layers attached to the porcine bone block. Proliferation and osteogenic differentiation were analyzed by cell counting, an MTT assay, alkaline phosphatase activity, and total protein content. The deposition of extracellular substances and osteoid formation surrounded by osteoblast-like cuboidal cells were confirmed through histochemical staining and transmission electron microscopy. Based on the results of this study, we conclude that heat-treated porcine trabecular bone has great potential as a bone substitute and may even be superior to currently available commercial products.     

姜黄素调控大鼠骨髓间充质干细胞的成骨分化

背景：姜黄素能明显降低破骨细胞的骨重吸收,诱导破骨细胞凋亡,抑制破骨细胞形成。 目的：观察不同浓度姜黄素对大鼠骨髓间充质干细胞成骨分化的影响。 方法：采用贴壁法分离培养大鼠骨髓间充质干细胞,传至第3代后采用成骨培养基进行成骨诱导,在诱导前3 d于诱导培养基中分别加入0(对照),10,15 μmol/L姜黄素。 结果与结论：接种培养7 d,倒置显微镜下可见分散的骨髓间充质干细胞小集落,集落细胞呈放射状生长。随培养时间延长,细胞集落增大,细胞形态为长梭形。加入成骨培养基7 d后细胞形态逐渐由长梭形变为多角形,随成骨诱导时间延长形态变化更加明显。加入不同浓度姜黄素后骨髓间充质干细胞增殖无变化。姜黄素呈剂量依赖性促进骨髓间充质干细胞碱性磷酸酶活性及Runx-2、血红素单加氧酶1的表达。提示姜黄素能有效促进大鼠骨髓间充质干细胞早期成骨分化,其机制可能与提高干细胞血红素单加氧酶1的表达有关。     

Transwell小室环境下兔成骨样细胞与骨髓基质干细胞的共培养及成骨分化

背景：以往在体外采用地塞米松、生长因子或用成骨样细胞与骨髓间充质干细胞按1∶1混合培养诱导成骨均存在种种局限。 目的：观察在Transwell小室环境下成骨样细胞与骨髓基质干细胞体外共培养及成骨样细胞定向诱导骨髓基质干细胞向成骨细胞分化的可行性。 方法：取第3代乳兔成骨样细胞与第3代兔骨髓基质干细胞接种共培养于Transwell小室内,成骨样细胞接种于培养板底层,骨髓基质干细胞接种于Transwell膜内膜上作为实验组。以骨髓基质干细胞单独接种于Transwell小室内,下层为基础培养液作为对照组。 结果与结论：实验组共培养骨髓基质干细胞明显向成骨细胞分化,细胞碱性磷酸酶活性显著高于对照组(P < 0.05)。实验组骨髓基质干细胞茜素红染色强阳性,可见呈红色结节,经PT-PCR扩增后,可见成骨启动基因核心结合因子α1的表达;对照组未见矿化结节。说明应用Transwell小室可实现成骨样细胞与骨髓基质干细胞体外共培养,并能定向诱导骨髓基质干细胞向成骨细胞分化。关键词：成骨样细胞;Transwell小室;骨髓基质干细胞;共培养;成骨分化;兔 doi:10.3969/j.issn.1673-8225.2012.19.004     

大鼠骨髓基质细胞在脑内的迁移和分化

目的研究骨髓基质细胞(bMSCs)在大鼠脑内的迁移和分化情况。方法体外分离培养大鼠四肢骨的bMSCs,原代培养8~10 d后,同时用含增强型绿色荧光蛋白(EGFP)的反转录病毒上清转导bMSCs并进行抗性筛选,7~10 d可得到稳定表达EGFP的bMSCs,然后将EGFP标记的bMSCs通过侧脑室和尾静脉2种注射方式分别注入纹状体损伤的大鼠体内,进而观察bMSCs向脑内迁移和分化的情况。结果在移植后的大鼠脑内均观察到2种方式植入大鼠体内的bMSCs,这些EGFP阳性的细胞呈现出一些神经元和神经胶质细胞的形态并能表达一些神经元和神经胶质细胞的特异性标志物。结论骨髓基质细胞中确实含有能够向神经元方向分化的干细胞。     

The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml^?1 Dex-loaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.     

A bioactive triphasic ceramic-coated hydroxyapatite promotes proliferation and osteogenic differentiation of human bone marrow stromal cells

Hydroxyapatite (HA) ceramics are widely used as bone graft substitutes because of their biocompatibility and osteoconductivity. However, to enhance the success of therapeutic application, many efforts are undertaken to improve the bioactivity of HA. We have developed a triphasic, silica-containing ceramic-coated hydroxyapatite (HASi) and evaluated its performance as a scaffold for cell-based tissue engineering applications. Human bone marrow stromal cells (hBMSCs) were seeded on both HASi and HA scaffolds and cultured with and without osteogenic supplements for a period of 4 weeks. Cellular responses were determined in vitro in terms of cell adhesion, viability, proliferation, and osteogenic differentiation, where both materials exhibited excellent cytocompatibility. Nevertheless, an enhanced rate of cell proliferation and higher levels of both alkaline phosphatase expression and activity were observed for cells cultured on HASi with osteogenic supplements. These findings indicate that the bioactivity of HA endowed with a silica-containing coating has definitely influenced the cellular activity, projecting HASi as a suitable candidate material for bone regenerative therapy.     

补骨脂素对大鼠骨髓间充质干细胞成骨分化的影响

目的研究不同浓度的补骨脂素对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)成骨分化的影响,为临床应用补骨脂素治疗骨质疏松症提供理论依据。方法选取8周龄健康雄性SD大鼠,取双侧股骨、胫骨,对大鼠的BMMSCs进行分离、原代培养和细胞表型鉴定。取第3代大鼠BMMSCs,体外利用成骨诱导培养基进行不同浓度补骨脂素的药物诱导,MTT法检测不同浓度的补骨脂素对大鼠BMMSCs生长的影响;通过碱性磷酸酶(ALP)活性测定、Western blot法和茜素红染色方法评价不同浓度补骨脂素对大鼠BMMSCs成骨分化能力。结果第3代大鼠BMMSCs表面抗原符合干细胞鉴定标准,成骨诱导后给予15μmol/L补骨脂素对大鼠BMMSCs的增殖作用最佳,ALP活性、Runx-2表达和钙化结节数目均明显高于经典成骨诱导组、5μmol/L和10μmol/L和20μmol/L补骨脂素组。结论15μmol/L补骨脂素成骨诱导促进BMMSCs向成骨分化,从而起到防治骨质疏松的作用。