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.     

Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue.     

Influence of calcium phosphate crystal assemblies on the proliferation and osteogenic gene expression of rat bone marrow stromal cells

Calcium phosphates (CaPs) have been investigated as substrates to promote bone formation both in vitro and in vivo. The aim of this study was to examine the proliferation and differentiation of rat bone marrow stromal cells (BMSCs) cultured on three-dimensional (3D) octacalcium phosphate (OCP) crystal assemblies. The cytotoxicity of OCP crystal assemblies was evaluated by measuring the lactate dehydrogenase (LDH) release from BMSCs during 10h of incubation with OCP crystal assemblies. The proliferation of BMSCs on OCP crystal assemblies in medium with or without osteogenic supplements was also investigated using the MTT assay with tissue culture treated plastic (TP) as the control. The tissues formed by BMSCs cultured on OCP crystal assemblies for 24 days were examined following staining with haematoxylin and eosin (H&E), alkaline phosphatase (ALP) and Van Gieson's techniques. The influence of OCP crystal assemblies on mRNA expression of alpha chain of collagen type I (Coll-Ia), ALP and osteocalcin (OC), osteonectin (ON), osteopontin (OP), lumican, Cbfa1, EST317 and EST350 by the BMSCs were also investigated using semi-quantitative RT-PCR. Although OCP crystals were relatively cytotoxic compared with TP, proliferation of BMSCs occurred when seeded onto OCP crystal assemblies. BMSCs cultured on OCP demonstrated similar proliferation rates as found on the control and no significant difference (P<0.05) in the number of cells cultured in medium supplemented with or without osteogenic additives on TP and OCP. The deposition of collagen and ALP were detected in tissue synthesised by BMSCs cultured on OCP crystals assemblies. OCP crystal assemblies down-regulated basal bone ECM proteins, including Coll-Ia, ON and lumican, in the first week of culture, whilst up-regulation of the same genes was observed after 24 days of culture. The observed down-regulation of Cbfa1 on OCP substrates was consistent with the negative effect of OCP crystal assemblies on the genes encoding bone ECM proteins. The up-regulation of OC mRNA expression by OCP crystal assemblies could be related to the requirement for synthesis of more OC proteins to control the concentration of calcium ions in culture medium.     

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。     

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。     

Improved osteogenic differentiation of human marrow stromal cells cultured on ion-induced chemically structured poly-epsilon-caprolactone

The ability to control cell proliferation/differentiation, using material surface, is a main goal in tissue engineering. The objective of this study was to evaluate the attachment, proliferation and differentiation to the osteoblastic phenotype of human marrow stromal cells (MSC) when seeded on poly-epsilon-caprolactone (PCL) thin films before and after irradiation with 10 keV He+. The polymeric surface was characterized as surface chemical structure and composition, roughness and morphology on the micro- and nano-scale, wettability and surface free energy parameters. MSC were obtained from patients undergoing routine hip replacement surgery, expanded in vitro and cultured on untreated PCL and He+ irradiated PCL films for up to 4-5 weeks in osteogenic medium. He+-irradiation led to slight smoothening of the surface and different nanoscale surface chemical structure, while surface free energy resulted unchanged in comparison to untreated PCL. The results from biological testing demonstrated that early attachment and further proliferation, as well as osteoblastic markers, were higher for MSC on He+-irradiated PCL. In conclusion, the change of PCL surface properties induced by ion beam irradiation is confirmed to enhance the adhesion of MSC and support their differentiation.     

Osteogenic differentiation of rat bone marrow stromal cells cultured on Arg-Gly-Asp modified hydrogels without dexamethasone and beta-glycerol phosphate

In this study, we investigated the effect of signaling peptides incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels on in vitro differentiation and mineralization of marrow stromal cells (MSCs) cultured in media without soluble osteogenic supplements (dexamethasone and beta-glycerol phosphate). When MSCs were cultured for 16 days on OPF hydrogels modified with Arg-Gly-Asp (RGD) containing peptides, the normalized cell number was dependent on the peptide concentration between days 0 and 5 and reached comparable values at day 10 regardless of the concentration. The alkaline phosphatase (ALP) activity of MSCs on the peptide-modified OPF hydrogels was also concentration-dependent: ALP activity showed peaks on day 10 or day 13 on OPF hydrogels modified with 2.0 and 1.0 micromol peptide/g, which were significantly greater than those on the OPF hydrogels modified with 0.1 micromol peptides/g or no peptide. A characteristic marker of osteoblastic differentiation, osteopontin (OPN), was detected for all the test groups. However, OPN secretion between days 0 and 10 was significantly higher on the peptide modified hydrogels compared to that on tissue culture-treated polystyrene. Taken together, the results indicate that the presence of signaling peptide allows for a favorable microenvironment for MSCs to differentiate into osteoblasts and produce mineralized matrix, although the soluble factors may further enhance calcium deposition. These findings further support the usefulness of OPF hydrogels as scaffolds for guided bone regeneration, and represent an initial step in exploring the complex relationship between soluble and insoluble factors in osteogenic differentiation on biodegradable materials.     

Osteogenic differentiation of cultured rat and human bone marrow cells on the surface of zinc-releasing calcium phosphate ceramics

Rat and human bone marrow cells (BMCs) were cultured on a composite ceramic of zinc-containing beta-tricalcium phosphate and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 and varying zinc contents. After a 2-week culture of the BMCs in the presence of beta-glycerophosphate and dexamethasone, many macroscopic mineralized areas with high alkaline phosphatase (ALP) activity were seen on the ZnTCP/HAP ceramic disks. The ALP activity increased with increasing zinc content in the ceramics. The highest ALP activity was observed when the BMCs were cultured on the ceramics with 1.26 wt % zinc, and the ceramics released zinc ions at concentrations from 2.2 to 7.2 microg/mL into the culture medium. Zinc ions were incorporated into mineralized areas produced by BMCs. BMCs also were directly cultured onto the culture dish surface, and the addition of 100 microM of free ZnCl(2) (6.5 microg/mL) to the culture medium significantly increased the ALP activity of the BMCs relative to the culture medium without the ZnCl(2)addition. The maximum zinc concentration required to enhance mineralization was higher in human BMCs than in rat BMCs. The present study demonstrates the superiority of ZnTCP/HAP ceramics over TCP/HAP in supporting the osteogenic differentiation of BMCs, and thus these ceramics are safe and useful in clinical settings, such as for bone reconstructive surgery.     

Selective differentiation of mammalian bone marrow stromal cells cultured on three-dimensional polymer foams

Bone marrow stromal cells (BMSC) are pluripotent progenitor cells that can regenerate different skeletal tissues in response to environmental signals. In this study, we used highly porous, structurally stable three-dimensional polymer foams in conjunction with specific regulatory molecules to selectively differentiate mammalian BMSC into either cartilaginous or bone-like tissues. Bovine BMSC were expanded in monolayers and cultured on 5-mm-diameter, 2-mm-thick foams made of poly(lactic-co-glycolic acid) and poly(ethylene glycol). Constructs maintained their original size and shape for up to 4 weeks of culture and supported BMSC growth and production of extracellular matrix (ECM). By proper use of chondrogenic (dexamethasone, insulin, transforming growth factor-beta1) or osteogenic (dexamethasone, beta-glycerophosphate) medium supplements, we could control whether the generated ECM was cartilaginous (containing collagen type II and sulfated glycosaminoglycans) or bone-like (containing osteocalcin, osteonectin, and mineralized foci). After 4 weeks of cultivation, cartilaginous and bone-like ECM were uniformly distributed throughout the construct volume and respectively represented 34.2 +/- 9.3% and 12.6 +/- 3.2% of the total available area. BMSC culture on poly(lactic-co-glycolic acid)/poly(ethylene glycol) foams provides a three-dimensional model system to study the development of mesenchymal tissues in vitro and has potential applications in engineering autologous grafts for skeletal tissue repair.     

Osteoblast differentiation of bone marrow stromal cells cultured on silica gel and sol-gel-derived titania

Primary cultures of osteogenic precursor cells derived from rat bone marrow stroma were performed on commercially available pure titanium discs (Ti c.p.) and surface modified Ti c.p.using a sol-gel technique (Ti sol). In separate repeated experimental runs, cell behavior and in vitro mineralization were compared with cultures on silica gel bioactive glass discs (S53P4). All substrates were incubated in simulated body fluid prior to the experiment. Overall, variable effects between experimental runs were seen. Apparently, this was due to the heterogeneous nature of the used cell population. Therefore, only careful conclusions can be made. Initial cell adhesion and growth rates between 3 and 5 days of culture--analyzed by cell numbers--were in general comparable for the two titanium substrates, while initial growth up to day 3 is suggested to be higher in Ti c.p. compared to Ti sol. Although initial cell adhesion on the S53P4 glass discs was lower than the titanium substrates, cell growth rates appeared to be higher on the silica gel compared to the two titanium substrates. Further, there were some indications that the early and late osteoblast differentiation markers, alkaline phosphatase and osteocalcin, monitored up to day 24, were elevated in Ti c.p cultures compared to Ti sol cultures. There were no differences observed in in vitro mineralization between the titanium groups. S53P4 seemed to display a substantially higher differentiating capacity for both osteogenic cell markers as well as in vitro mineralization compared to the two titanium substrates.     

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.     

Stimulation of the differentiation of osteogenic rat bone marrow stromal cells by osteoblast cultures

The effect of conditioned medium (CM) from rat calvaria (RC) cel cultures on the growth and differentiation of osteogenic cells in rat bone marrow stromal cell (BMSC) cultures was investigated. Control cultures received either CM from periodontal ligament fibroblast cultures or fresh medium. RCCM stimulated the formation of nodules of bonelike tissue in bone marrow stromal cell cultures in a dose-dependent manner,and the maximal stimulation was associated with the osteoblast-enriched cell populations of the RC cultures. Ultrafiltration demonstrated that activity was confined to a CM fraction of 10- to 30-kilodalton molecular size. The activity was sensitive to boiling and trypsin treatments, but was not affected by neutralizing antibodies to transforming growth factor beta or insulin-like growth factor I or II. RCCM was found to initially increase the number and proportion of cells that expressed alkaline phosphatase activity, although the proportion of alkaline phosphatase-positive cells subsequently declined. These data were consistent with an initial stimulation of proliferation of a subpopulation of osteoprogenitor cells within the cultures, followed by their differentiation. The results suggest that mature osteoblasts may produce a paracrine growth factor that can stimulate the differentiation of osteoblasts from precursor cells.     

TAK-778 enhances osteoblast differentiation of human bone marrow cells cultured on titanium

TAK-778 induces bone growth in in vitro and in vivo models. The aim of this study was to evaluate the osteogenic potential of TAK-778 on human bone marrow cells cultured on commercially pure titanium (cpTi). Cells were cultured either in absence or in presence of TAK-778 (10(-5)M) on cpTi in supplemented alpha-MEM. For attachment evaluation, cells were cultured for 4 and 24h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. TAK-778 did not affect cell attachment and viability. Cell number was reduced by TAK-778. ALP activity, total protein content, and bone-like formation were increased by TAK-778. These results suggest that initial cell events such as cell attachment are not affected by TAK-778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone-like formation are enhanced by TAK-778 in presence of cpTi. It means that TAK-778 could be a useful drug to improve the osseointegration of implants by both enhancing and accelerating bone formation on Ti surface.     

Influence of rhBMP-2 on rat bone marrow stromal cells cultured on titanium fiber mesh

Titanium (Ti) fiber mesh is a candidate scaffold material for the creation of bone graft substitutes (BGS). Two densities (3.54 x 10(4) cells/cm(2) [LD or low density] and 3.54 x 10(5) cells/cm(2) [HD or high density]) of rat bone marrow stromal cells were seeded on Ti-fiber mesh discs. Cells were cultured for up to 16 days, 7 days of which the cells were in the presence of various concentrations of rhBMP-2 (0, 10, 100, and 1,000 ng/mL) in order to evaluate osteogenic expression. Scanning electron microscopy (SEM), light microscopy (LM), energy dispersive spectroscopy (EDS), DNA and calcium (Ca) content measurements, and x-ray diffraction (XRD) analysis were performed. SEM and EDS evaluation showed that a confluent layer of cells was present on top of the meshes together with collagen bundles and calcified globular accretions. Light microscopical evaluation showed a densely stained layer in the upper part of the mesh. SEM and Ca content measurement showed that calcification starts at 8 days. In addition, it was demonstrated that DNA content peaked at 8 days. LM, SEM, and Ca content evaluation revealed positive effects of increasing the cell seeding density, the rhBMP-2 concentration and the culture time on mineralization. Increasing the cell seeding density also showed a positive effect on DNA content. No effects of rhBMP-2 concentration were seen on DNA content. Finally, XRD revealed that the deposited matrix contained a precipitate of a stable calcium phosphate phase. We conclude that (1) titanium fiber mesh sustains excellent osteogenic expression in vitro, (2) increasing the cell seeding density has a positive effect on osteogenic expression in titanium mesh in vitro, and (3) in high density specimens, rhBMP-2 concentrations of 100 ng/mL and 1,000 ng/mL stimulate extracellular matrix calcification in a dose-responsive manner.     

Relationship between osteogenic characteristics of bone marrow cells and calcium phosphate surface relief and solubility

The capacity of mouse bone marrow cells to adhere to calcium phosphate surfaces and form tissue plates depending on the surface relief and solubility was studied in ectopic bone formation test. Calcium phosphate coating of titanium disks, made by the anodic spark (microarch) oxidation in 10% orthophosphoric acid with hydroxyl apatite particles, differed by the structure (thickness of coating, size of pores, and roughness) and solubility (level of in vitro oxidation of 1-week extracts of implants). Chemical (phasic and element) composition of the studied calcium phosphate coatings was virtually the same. The findings indicate that histogenesis is regulated by physicochemical characteristics of the implant surface. It seems that the osteogenic potential of calcium phosphate surfaces is largely determined by their relief, but not by pH of degradation products. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 1, pp. 107–112, January, 2006     

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.     

17β-雌二醇对大鼠骨髓间充质干细胞向成骨细胞分化的诱导调控*****☆

背景：研究发现骨髓间充质干细胞上存在雌激素受体,雌激素通过调节骨髓间充质干细胞的增殖、分化特性发挥促成骨作用。 目的：观察17β-雌二醇对骨髓间充质干细胞向成骨细胞分化的诱导作用,并探讨其作用机制。 方法：在基础成骨诱导培养基培养的第3代大鼠骨髓间充质干细胞中分别加入0(对照组),0.001,0.01,0.1 nmol/L 17β-雌二醇干预。Elisa法检测培养的骨髓间充质干细胞分泌Ⅰ型胶原的水平,RT-PCR及Western blotting法分别检测Runx2因子mRNA及蛋白水平。 结果与结论：与对照组比较,在给予0.001,0.01,0.1 nmol/L 17β-雌二醇干预后第5天,骨髓间充质干细胞Ⅰ型胶原表达显著升高(P < 0.05),第7天仍旧呈高表达(P < 0.05)。同时,在17β-雌二醇干预的第5,7天,Runx2因子mRNA及蛋白表达水平随17β-雌二醇浓度的增加而升高(P < 0.05),并呈剂量依赖性。表明17β-雌二醇可诱导骨髓间充质干细胞向成骨细胞转化,其可能通过上调Runx2因子表达发挥促成骨作用。 关键词：诱导分化;骨髓间充质干细胞;17β-雌二醇;Ⅰ型胶原;Runx2 doi:10.3969/j.issn.1673-8225.2012.10.003     

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。     

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