骨髓间充质干细胞复合表面改性的3-羟基丁酸和3-羟基己酸共聚酯膜的体外相容性

目的: 研究大鼠骨髓间充质干细胞(BMSCs)在不同处理工艺的3-羟基丁酸和3-羟基己酸共聚酯(PHBHHx)膜材料上的生长、增殖情况,为组织工程学筛选出一种新型的适合细胞生长、增殖的生物材料.方法: 用全骨髓培养法获得BMSCs,并行免疫荧光鉴定,同时在成骨诱导液中诱导BMSCs,钙钴法检测碱性磷酸酶(ALP),茜素红矿化结节染色;成脂诱导后行油红O染色.将不同处理工艺的材料与第3代BMSCs复合培养1、 3、 7d,通过扫描电镜、MTT法和中性红染色法分别观察细胞的形态、增殖变化及分布情况.结果: BMSCs免疫荧光鉴定CD44、 CD106均呈阳性.经成骨诱导后,ALP呈阳性表达,矿化结节染色呈橘红色;经成脂诱导后,14d油红O染色呈阳性.细胞在经过碱处理或添加5%、 10%中药涂层的PHBHHx膜材料表面均生长状态良好,增殖活力强且分布均匀.结论: 经过碱处理的,且含5%、 10%中药涂层的PHBHHx膜,具有良好的BMSCs相容性,可以作为一种新型的生物材料应用于组织工程.     

经表面修饰的-TCP对BMSCs细胞增殖及分化作用的研究

目的研究三种材料,材料一：β-磷酸三钙支架（β—TCP）;材料二：明胶／1氐结晶度羟基磷灰石涂层-磷酸三钙支架（gel／lc—HA．β-TCP）;材料三：明胶／高结晶度羟基磷灰石涂层-β-磷酸三钙（gel／he-HA-β-TCP）。三种材料的细胞毒性及兔骨髓基质细胞（BMSCs）与材料共培养增殖的情况。方法取兔股骨骨髓腔细胞,进行贴壁培养BMSCs。在成骨诱导液中诱导BMSCs。将诱导后的BMSCs与三种支架材料在培养板内共培养1、3、5、7、9d。采用形态学观察、MTT法及ALP检测试剂盒等方法检测材料的BMSCs细胞毒性及BMSCs细胞在材料表面的增殖和分化能力。结果光镜及电镜下观察各组无显著差异。细胞毒性在0-1级。与细胞共培养,β-TCP组在第7．9天与阴性对照组差异有统计学意义。ALP检测,β-TCP组在第7、9天与阴性对照组差异有统计学意义（P〈0．05）,gel／lc—HA-β-TCP在第9天与阴性对照组差异有统计学意义（p〈0．05）。结论新型明胶／高结晶度羟基磷灰石涂层-β-磷酸三钙支架和明胶／低结晶度羟基磷灰石涂层-β-磷酸三钙支架与BMSCs生物相容性好,适合作为支架材料负载BMSCs构建组织工程骨。     

全骨髓贴壁法培养兔骨髓间充质干细胞体外定向成骨诱导分化及鉴定

背景：流式细胞仪分离法和免疫磁珠分离法对细胞活性影响较大,密度梯度离心法虽然能够获得纯度高的单核细胞,但由于多次离心可造成细胞的大量流失且对细胞活性有一定的影响使其应用值得商榷。 目的：采用全骨髓贴壁法分离兔骨髓间充质干细胞进行成骨诱导分化及鉴定。 方法：采用全骨髓贴壁法体外分离培养兔骨髓间充质干细胞,倒置显微镜下观察细胞形态学特征。在成骨诱导剂作用下,通过碱性磷酸酶染色试剂盒行碱性磷酸酶染色,Ⅰ型胶原免疫细胞化学染色,Von Kossa法及茜素红进行矿化结节染色以及电镜下检测兔骨髓间充质干细胞成骨诱导后的形态结构。 结果与结论：经诱导后细胞出现与成骨细胞相似的形态学特征,碱性磷酸酶染色阳性,Ⅰ型胶原免疫细胞化学染色,Von-Kossa法及茜素红矿化结节染色阳性。表明经成骨诱导剂诱导后全骨髓贴壁法体外分离纯化培养的兔骨髓间充质干细胞能向成骨细胞方向分化增殖。     

Klotho对大鼠骨髓间充质干细胞增殖和分化的影响

目的 探讨延缓衰老基因Klotho对骨髓间充质干细胞（BMSCs）增殖和分化的影响。 方法 体外条件下培养大鼠骨髓间充质干细胞,构建分泌型Klotho(sKL)过表达的BMSCs。Western blotting检测细胞及培养基中sKL的表达;MTT法检测细胞增殖能力;Western blotting检测衰老标志物P53、P21蛋白的表达。利用成骨诱导液定向诱导BMSCs向成骨细胞分化,应用碱性磷酸酶(ALP)染色鉴定成骨效果;利用成脂诱导液定向诱导BMSCs向脂肪细胞分化,应用油红O染色鉴定成脂效果。 结果 Western blotting结果显示,sKL组细胞和培养基中sKL蛋白表达显著上调（P<0.05）,P53、P21表达显著下调（P<0.05）;MTT结果显示,各组细胞吸光度值（A值）差别无显著性（P>0.05）,sKL组成骨及成脂能力明显强于对照组（P<0.05）。 结论 sKL增强了大鼠BMSCs的延缓衰老能力,对BMSCs的成骨分化和成脂分化产生一定的促进作用,而对BMSCs的增殖无显著影响。     

超顺磁性氧化铁标记对骨髓间充质干细胞多向分化诱导的影响

研究兔骨髓间充质干细胞(BMSCs)经超顺磁性氧化铁(SPIO)标记后,体外成骨、成脂及成软骨诱导能力的变化。体外贴壁培养和扩增兔BMSCs,采用SPIO(25μg/ml)联合硫酸鱼精蛋白转染剂标记兔BMSCs,分别采用适宜的成骨、成脂及成软骨诱导培养液对磁标记BMSCs进行体外定向诱导培养3周,诱导过程中观察细胞形态学变化。3周后对成骨定向诱导组进行钙结节茜素红染色和碱性磷酸酶(ALP)组化染色;对成脂肪定向诱导组行油红-O染色;对成软骨定向诱导组行番红O染色、II型胶原免疫组化染色检测观察胞外基质糖胺多糖、II型胶原的分泌和表达。利用Image-Pro Plus图像分析系统对免疫细胞化学染色进行光密度半定量分析。结果表明:超顺磁性氧化铁粒子标记BMSCs后普鲁士染色和电镜检查显示细胞胞浆内含致密铁颗粒;磁标记BMSCs在成骨、成脂及成软骨潜在多向分化诱导能力上与对照组未标记细胞相比无统计学差异。提示,SPIO联合硫酸鱼精蛋白转染剂能成功标记BMSCs,超顺磁性氧化铁标记对兔BMSCs体外多向分化诱导能力无明显影响,合理应用这种新型细胞标记技术将促进对组织工程种子细胞的研究。     

细胞凝集态在钙结节形成过程中的显微结构观察

目的 观察兔骨髓基质细胞来源的细胞凝集态在体外钙结节形成过程的形态变化.方法 通过全骨髓培养方法分离培养兔骨髓基质细胞,并在培养瓶中进行扩增,使用成骨诱导液作为培养基;再通过四环素标记、碱性磷酸酶染色、茜素红染色、扫描电镜及透射电镜方法,研究细胞凝集态在钙结节形成过程中的形态变化.结果荧光显微镜下观察可见,四环素标记的...     

新型骨形态发生蛋白7多肽对骨髓基质干细胞生物学行为的影响

背景:国内外对骨形态发生蛋白7的研究倾向于其在组织工程方面的应用,但是骨形态发生蛋白7除具有很强的骨诱导活性之外,还影响动物生殖系统的生长发育及生理功能,如何在充分发挥其骨诱导活性的同时,避免其他生物学作用,是骨组织工程领域面临的一个难题。目的:观察人工合成的新型骨形态发生蛋白7多肽对骨髓基质干细胞增殖、成骨分化及矿化等生物学行为影响。方法:分离培养大鼠骨髓基质干细胞并用流式细胞仪进行鉴定。将传至第3代的骨髓基质干细胞分为实验组和对照组,实验组在培养基加入终质量浓度为100mg/L的骨形态发生蛋白7多肽,对照组不加多肽。分别在培养的2,4,6,8,10d对各组细胞进行MTT检测,评价其增殖情况;培养7,14d后,通过检测细胞内钙含量和碱性磷酸酶活性评价其成骨分化情况,并进行钙黄绿素染色观察其矿化情况。结果与结论:原代培养大鼠骨髓基质干细胞生长良好,其纯度达到91.5%。细胞增殖实验显示骨髓基质干细胞在实验组和对照组中均增殖良好,差异无显著性意义(P0.05);诱导培养7d和14d后,实验组碱性磷酸酶活性及钙含量显著高于对照组(P0.05),实验组细胞矿化明显强于对照组。实验结果表明新型骨形态发生蛋白7多肽对骨髓基质干细胞增殖没有显著影响,但可以促进其向成骨方向分化及矿化。     

不同浓度的骨碎补总黄酮对大鼠骨髓间充质干细胞向成骨细胞分化的影响

目的:通过在体外条件下对大鼠骨髓间充质干细胞(BMSCs)实施干预诱导,探寻骨碎补总黄酮对BMSCs的成骨诱导分化能力及不同浓度下可能存在的剂量效应关系。方法:应用全骨髓贴壁培养法对BMSCs进行分离、纯化,并通过流式细胞术对BMSCs进行鉴定,再通过设立含不同浓度骨碎补总黄酮条件培养基,分组对BMSCs进行干预,观察各组7d和14d碱性磷酸酶(ALP)的表达,同时通过ALP染色和矿化结节染色进行定性分析。结果:用全骨髓贴壁法成功分离获得了均一稳定的BMSCs,各浓度骨碎补总黄酮作用BMSCs7d和14d后,诱导不同程度的ALP表达,经10-5g/L骨碎补总黄酮诱导,ALP染色及矿化结节染色均阳性。结论:低浓度骨碎补总黄酮能促进BMSCs向成骨细胞分化,其ALP生成量随着骨碎补总黄酮浓度的降低大致呈增加趋势。     

人骨髓间充质干细胞向成骨细胞分化中26RFa的作用

背景:研究表明26RFa在骨形成、痛觉、内分泌、心血管以及能量代谢等方面都有重要的调节作用。目的:观察26RFa在成骨培养体系中对人骨髓间充质干细胞增殖分化的影响。方法:通过MTT实验,以此确定26RFa对人骨髓间充质干细胞是否有促增殖作用及其起作用的最大活性浓度;将人骨髓间充质干细胞接种于6孔培养板上,实验分为2组:实验组含有10-11mol/L的26RFa,对照组不含26RFa。取成骨诱导8,12,16 d细胞用碱性磷酸酶试剂盒测定细胞内碱性磷酸酶活性;成骨诱导21,28 d后行茜素红染色和Von Kossa染色,计算每张玻片钙化结节数,Western blot检测cbfa1的表达。结果与结论:成骨诱导8,12,16 d后细胞内碱性磷酸酶活性实验组高于对照组(P0.05,P0.01,P0.05);21,28 d后茜素红染色和Von Kossa矿化结节数实验组均高于对照组;实验组细胞cbfa1表达明显高于对照组(P0.05)。说明在适宜的培养条件下,26RFa可促进人骨髓间充质干细胞向成骨细胞分化。     

去卵巢骨质疏松大鼠骨髓间充质干细胞的体外成骨诱导

背景：细胞学研究表明,骨髓间充质干细胞在绝经后骨质疏松症的发病过程中起有重要作用。 目的：观察去卵巢骨质疏松大鼠骨髓间充质干细胞的体外成骨分化。 方法：将6月龄雌性SD大鼠双侧卵巢切除建立骨质疏松模型。实验分为4组：正常干细胞组、骨质疏松干细胞组、正常干细胞成骨诱导组、骨质疏松干细胞成骨诱导组。全骨髓贴壁法培养正常和骨质疏松大鼠骨髓间充质干细胞至第3代细胞用于实验。倒置相差显微镜观察细胞形态,流式细胞仪测定细胞周期、增殖指数。加成骨诱导液进行成骨诱导,检测各组骨髓间充质干细胞碱性磷酸酶活性,茜素红染色法比较各组钙结节的形成。 结果与结论：正常干细胞成骨诱导组、骨质疏松干细胞成骨诱导组均具有成骨细胞的形态特征,但骨质疏松干细胞成骨诱导组形态变化相对缓慢。正常干细胞组细胞增殖指数高于骨质疏松干细胞组(P < 0.05);成骨诱导组碱性磷酸酶活性均明显高于相应的正常或骨质疏松干细胞组(P < 0.05);正常干细胞成骨诱导组明显高于骨质疏松干细胞成骨诱导组(P < 0.05)。成骨诱导组茜素红染色均呈阳性,相应的正常或骨质疏松干细胞组呈阴性;且正常干细胞成骨诱导组染色强于骨质疏松干细胞成骨诱导组。提示去卵巢骨质疏松大鼠骨髓间充质干细胞的增殖能力和成骨分化能力明显降低,可能与去卵巢大鼠骨质疏松的发生相关。     

羌活鱼研粉物、水提及酸提物含药血清对骨髓间充质干细胞增殖及向成骨分化的影响

背景：羌活鱼经大量的临床观察和报道有促进骨折愈合的作用,其具体的作用机制及其有效成分的研究报道尚少。 目的：分析羌活鱼研粉物、水提及酸提物含药血清对体外培养骨髓间充质干细胞增殖与成骨性分化的影响。 方法：制备羌活鱼研粉、水提物、酸提物,分别采用高、中、低剂量灌服大鼠制得羌活鱼研粉物、水提及酸提物含药血清,以灌服等体积生理盐水制得对照血清;采用贴壁筛选法培养大鼠骨髓间充质干细胞,通过MTT法检测细胞增殖;于干预后的不同时间点分别测定细胞碱性磷酸酶活性、钙盐沉积量、成骨性分化基因表达以及钙化结节数、并比较各组间差异。 结果与结论：羌活鱼研粉物及水提物含药血清具有刺激骨髓间充质干细胞增殖活性及促进其成骨性分化的能力,其中尤其以研粉物中剂量组最为明显,而酸提物含药血清并无此功效。主要表现在细胞增殖、碱性磷酸酶活性、钙盐沉积量、成骨性分化基因和钙化结节数显著高于空白对照组(P < 0.05)。     

Unitary bioresorbable cage/core bone graft substitutes for spinal arthrodesis coextruded from polycaprolactone biocomposites

A unitary bioresorbable cage/core bone graft substitute consisting of a stiff cage and a softer core with interconnected porosity is offered for spinal arthrodesis. Polycaprolactone, PCL, was used as the matrix and hydroxyapatite, HA, and β-tricalcium phosphate, TCP, were used in the formulation of the cage layer to impart modulus increase and osteoconductivity while the core consisted solely of PCL. The crystallinity, biodegradation rate (under accelerated conditions) and mechanical properties, i.e., the uniaxial compression, relaxation modulus upon step compression and cyclic compressive fatigue properties, of the co-extruded cage/core bone graft substitutes could be manipulated by changes in the concentration of HA/TCP in the cage layer. The cyclic fatigue behavior of the cage/core bone graft substitutes were also compared to the behavior of bovine vertebral cancellous bone characterized under similar testing conditions. The biocompatibility of the cage/core bone graft substitutes were assessed via in vitro culturing of human bone marrow derived stromal cells, BMSCs. The cell proliferation rates, time dependencies of the alkaline phosphates (ALP) activity and the expressions of bone markers, i.e., Runx2, ALP, collagen type I, osteopontin and osteocalcin, and the collected μ-CT images demonstrated the differentiation of BMSCs via osteogenic lineage and formation of mineralized bone tissue to indicate the biocompatibility of the cage/core bone graft substitutes.     

Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold

To investigate the potential application of bone marrow stromal cells (BMSCs) and an injectable sodium alginate/gelatin scaffold for bone tissue engineering (BTE). The phenotype of osteogenic BMSCs was examined by mineralized nodules formation and type I collagen expression. Cell proliferation was evaluated by MTT assay. The biocompatibility of scaffold and osteogenic cells were examined by hematoxylin and eosin (H&E) staining. Ectopic bone formation as well as closure of rabbit calvarial critical-sized defects following scaffold-cell implantation were analyzed by histological examination and computed tomography (CT) scanning. Spindle-shaped osteogenic cells of high purity were derived from BMSCs. The osteogenic cells and sodium alginate/gelatin (2:3) scaffold presented fine biocompatibility following cross-linking with 0.6% of CaCl(2). After implantation, the scaffold-cell construct promoted both ectopic bone formation and bone healing in the rabbit calvarial critical-sized defect model. Our data demonstrated that the sodium alginate/gelatin scaffold could be a suitable biomaterial for bone engineering, and the scaffold-osteogenic cells construct is a promising alternative approach for the bone healing process.     

富血小板血浆促进脂肪间充质干细胞的体内外成骨

背景：添加富血小板血浆可促进细胞体外成骨表型的快速转化,从而有效成骨。 目的：观察富血小板血浆对脂肪间充质干细胞体内和体外成骨能力的影响。 方法：第3代兔脂肪间充质干细胞进行成骨诱导培养,分为对照组和富血小板血浆组。细胞接种到钙磷陶瓷支架上后,体内外观察细胞/载体复合物的成骨情况。 结果与结论：两组细胞随着诱导时间的延长碱性磷酸酶活性增高,达到高峰值后随后逐渐下降,诱导后14 d时,富血小板血浆组即达到高峰值,对照组18 d达到高峰值。细胞/载体复合体切片Von Kossa染色显示两组载体的孔隙内衬面呈多层黑染状,有大量钙盐沉积。甲苯胺蓝染色显示载体的孔隙中可见成熟的骨质存在,周边区域较中心多。体外钙盐沉积对照组多,体内成骨面积富血小板血浆组多(P < 0.05)。说明,富血小板血浆可有效诱导脂肪间充质干细胞体内和体外成骨。     

Differential osteogenic activity of osteoprogenitor cells on HA and TCP/HA scaffold of tissue engineered bone

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering.     

In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering

Mineralized and partially or fully demineralized biomaterials derived from bovine bone matrix were evaluated for their ability to support human bone marrow stromal cell (BMSC) osteogenic differentiation in vitro and bone-forming capacity in vivo in order to assess their potential use in clinical tissue-engineering strategies. BMSCs were either seeded on bone-derived scaffolds and cocultured in direct cell-to-scaffold contact, allowing for the exposure of soluble and insoluble matrix-incorporated factors, or cocultured with the scaffold preparations in a transwell system, exposing them to soluble matrix-incorporated factors alone. Osteoblast-related markers, alkaline phosphatase (ALP) activity and bone sialoprotein (BSP) and osteopontin (OP) mRNA expression were evaluated in BMSCs following 14 days of cocultivation in both systems. The data demonstrate that BMSCs from some donors express significantly higher levels of all osteoblast-related markers following cocultivation in direct cell-to-scaffold contact with mineralized scaffolds in comparison to fully demineralized preparations, while BMSCs from other donors display no significant differences in response to various scaffold preparations. In contrast, BMSCs cocultured independently with soluble matrix-incorporated factors derived from each scaffold preparation displayed significantly lower levels of ALP activity and BSP mRNA expression in comparison to untreated controls, while no significant differences were observed in marker levels between cells cocultured similarly with different biomaterial preparations. In addition, BMSCs were seeded directly on mineralized and partially or fully demineralized biomaterials and implanted in subcutaneous sites of athymic mice for 8 weeks to evaluate their in vivo bone-forming capacity. The ex vivo incorporation of BMSCs into all bone-derived scaffold preparations substantially increased the mean extent and frequency of samples containing de novo bone formation over similar nonseeded controls, as determined by histological and histomorphometrical analysis. No statistically significant differences were observed in the extent or frequency of bone formation between various scaffold preparations seeded with BMSCs from different donors. These results demonstrate that the in vivo osteoinductivity of bone-derived scaffolds can be modulated by ex vivo incorporated BMSCs and the extent of scaffold demineralization plays a significant role in influencing in vitro osteogenic differentiation of BMSCs depending on the coculture system and BMSC donor.