Bone formation using human adipose tissue-derived stromal cells and a biodegradable scaffold

Human adipose tissue, obtained by liposuction, was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). The ATSCs, as well as bone marrow-derived mesenchymal stem cells (BMSCs), have the capacity for renewal and the potential to differentiate into multiple lineages of mesenchymal tissues. These cells are capable of forming bone when implanted ectopically in an appropriate scaffold. The aim of this study was to evaluate a beta-tricalcium phosphate (beta-TCP) as a scaffold and to compare the potential of osteogenic differentiation of ATSCs with BMSCs. Both cell types were loaded into beta-TCP disk and cultured in an osteogenic induction medium. Optimal osteogenic differentiation in ATSCs in vitro, as determined by secretion of osteocalcin, scanning electron microscope, and histology, were obtained in the culturing with the beta-TCP disk. Furthermore, bone formation in vivo was examined by using the ATSC- or BMSC-loaded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts and to synthesize bone in beta-TCP disk as have BMSCs.     

Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source

Adult bone marrow contains mesenchymal stem cells (bone marrow-derived mesenchymal stem cells; BMSCs) which contribute to the generation of mesenchymal tissue such as bone, cartilage, muscle and adipose. However, using bone marrow as a source of stem cells has the limitation of a low cell number. An alternate source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Human adipose tissue obtained by liposuction was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). In this study, we compared the osteogenic differentiation of ATSCs with that of BMSCs. Both cell types were cultured in atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS scaffold) for three-dimensional culturing in a specific osteogenic induction medium. Optimal osteogenic differentiation in both cell types, as determined by alkaline phosphatase cytochemistry, secretion of osteocalcin, mineral (calcium phosphate) deposition and scanning electron microscopy, was obtained with the same three-dimensional culture. Furthermore, osteoblastic lining in vivo was examined using ATSC-seeded or BMSC-seeded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts to that of BMSCs.     

Expansion and characterization of adipose tissue-derived stromal cells cultured with low serum medium

Adipose tissue contains a population of cells that have extensive self-renewal capacity and the ability to differentiate along multiple lineages. In addition, adipose tissue-derived stromal cells (ATSCs) are able to differentiate into various cell types that may be useful for autologous cell transplantation for defects of bone, cartilage, adipose, and tendon, etc. Most protocols for in vitro cultures of ATSCs include fetal bovine serum (FBS) as a nutritional supplement. However, in some cell cultures, it involves multiple doses of FBS, which raises a concern over possible infections as well as immunological reactions that are caused by medium-derived FBS proteins, sialic acid, etc. In this study, we were able to expand mouse ATSCs using low mouse serum media containing collagen type I, heparin-carrying polystyrene, and fibroblast growth factor (FGF)-2. These expanded mouse ATSCs maintained their multilineage potential for differentiation into adipocytes, osteoblasts, and chondrocytes. Therefore, this method, which uses autologous cells and low serum media, may be able to be utilized for clinical cell therapies.     

Effects of serial passaging on the adipogenic and osteogenic differentiation potential of adipose-derived human mesenchymal stem cells

Adipose-derived human mesenchymal stem cells (hMSCs) will be more valuable for tissue engineering applications if they can be extensively subcultured without loss of phenotype and multilineage differentiation ability. This study examined the effects of serial passaging on growth rate, gene expression, and differentiation potential of adipose-derived hMSCs. Differentiation was assessed by analyzing changes in messenger RNA (mRNA) expression of osteogenic and adipogenic marker genes and by determining production of calcium deposits and lipid vacuoles. Cells cultured in osteogenic medium for 2 weeks upregulated expression of alkaline phosphatase mRNA relative to cells in growth medium, and deposited calcium. Calcium deposition decreased in cells from passages 4 to 6 but returned to levels near or above those of primary cells by passage 10. Cells cultured in adipogenic medium upregulated expression of lipoprotein lipase and peroxisome proliferator activated receptor-gamma mRNA relative to cells in growth medium, and formed lipid vacuoles at all passages. By passage 8, however, cells in adipogenic medium also deposited calcium. Growth rate was stable through passage 5, then decreased. The results of this study indicate that adipose-derived hMSCs are capable of both adipogenic and osteogenic differentiation through 10 passages (34 population doublings) but that osteogenic differentiation may start to dominate at later passages.     

Comparison of potentials of stem cells isolated from tendon and bone marrow for musculoskeletal tissue engineering

The use of tendon-derived stem cells (TDSCs) as a cell source for musculoskeletal tissue engineering has not been compared with that of bone marrow stromal cells (BMSC). This study compared the mesenchymal stem cell (MSC) and embryonic stem cells (ESC) markers, clonogenicity, proliferative capacity, and multilineage differentiation potential of rat TDSC and BMSC in vitro. The MSC and ESC marker profiles of paired TDSC and BMSC were compared using flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Their clonogenicity and proliferative capacity were compared using colony-forming and 5-bromo-2'-deoxyuridine assays, respectively. The expression of tenogenic, osteogenic, and chondrogenic markers at basal state were examined using qRT-PCR. Their osteogenic, chondrogenic, and adipogenic differentiation potentials were compared using standard assays. TDSC and BMSC showed similar expression of CD90 and CD73. TDSC expressed higher levels of Oct4 than BMSC. TDSC exhibited higher clonogenicity, proliferated faster, and expressed higher tenomodulin, scleraxis, collagen 1 α 1 (Col1A1), decorin, alkaline phosphatase, Col2A1, and biglycan messenger RNA levels than BMSC. There was higher calcium nodule formation and osteogenic marker expression in TDSC than BMSC upon osteogenic induction. More chondrocyte-like cells and higher glycosaminoglycan deposition and chondrogenic marker expression were observed in TDSC than BMSC upon chondrogenic induction. There were more oil droplets and expression of an adipogenic marker in TDSC than BMSC upon adipogenic induction. TDSC expressed higher Oct4 levels, which was reported to positively regulate mesendodermal lineage differentiation, showed higher clonogenicity and proliferative capacity, and had greater tenogenic, osteogenic, chondrogenic, and adipogenic markers and differentiation potential than BMSC. TDSC might be a better cell source than BMSC for musculoskeletal tissue regeneration.     

豚鼠骨髓间充质干细胞的分离培养及成骨诱导

背景：利用骨髓间充质干细胞的取材灵活性及快捷性,对已掌握的培养技术及成骨诱导进一步探索性研究。 目的：通过建立豚鼠骨髓间充质干细胞的体外分离培养法,探讨豚鼠骨髓间充质干细胞表型特征以及多项分化潜能。 方法：利用贴壁培养法分离纯化豚鼠骨髓间充质干细胞,传代扩增,流式细胞分析检测细胞表面分子CD29、CD44、CD45的表达。分别采用成骨诱导培养液和成脂诱导培养液定向诱导骨髓间充质干细胞向脂肪细胞、成骨细胞分化。 结果与结论：原代分离的骨髓间充质干细胞在接种后96 h贴壁,细胞形态为椭圆形,多角形及短梭形,8 d时细胞呈长梭形并达到90%单层融合。经传代扩增,细胞进一步纯化,细胞形态为均一的长梭形并呈漩涡状排列,而且生长速率加快。流式细胞检测 CD29、CD44阳性率分别为95.7%和65.7%。不同诱导剂定向诱导后,经油红O、茜素红S、碱性磷酸酶染色、免疫组织化学Ι型胶原酶鉴定,P3代骨髓间充质干细胞分别向脂肪细胞及成骨细胞分化。结果表明,通过贴壁筛选方法,体外分离培养的豚鼠骨髓间充质干细胞具有很强的增殖能力,并保持稳定的表型特征及多向分化潜能。     

冻存骨髓基质细胞复合材料体内成骨基质的合成能力

背景:课题组以往研究显示：体外培养条件下,冻存骨髓基质细胞复苏后仍保持较高的细胞存活率、细胞增殖及成骨分化能力。上述结果仍然需要进一步在体内环境下证实。 目的：观察经超低温冻存后的骨髓基质细胞和支架材料胶原膜BME-10X复合体植入裸鼠体内后Ⅰ型胶原的合成情况。 方法：体外分离培养Beagle犬骨髓基质细胞,冻存12个月后复苏,体外构建骨髓基质细胞和胶原膜材料复合体。分别经矿化诱导培养液、基础培养液培养5 d后,植入裸鼠体内,于术后第4周取出标本,进行大体观察、组织病理学和免疫组化分析,并应用图像分析系统对各组标本中的Ⅰ型胶原进行定量分析。以矿化诱导培养液培养的单纯胶原膜材料为对照组。 结果与结论：对照组在植入胶原膜后,胶原膜边界清晰,膜边缘及内部基本没有细胞生长,Ⅰ型胶原分布很少;在未诱导矿化组,术后第4周可见,胶原膜内有细胞长入,并有细小的条索状新生胶原形成,Ⅰ型胶原分布明显增多;在诱导矿化组,植入后也可见支架材料的分解降解和更多的细胞生长,大量新生的胶原形成类骨质样组织,与前两组对比,Ⅰ型胶原分布增多有显著性意义。结果表明冻存骨髓基质细胞复苏后进行体外培养扩增与诱导分化,并在体内环境下复合胶原支架材料,仍然具有较强成骨能力。     

Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro

Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (bone marrow stromal cells [BMSCs]) are negative for immunologically relevant surface markers and inhibit proliferation of allogenic T cells in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immunological characteristics of BMSCs have been the subject of various investigations, those of stem cells isolated from adipose tissue (ASCs) have not been adequately described. In addition, the influence of osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject of this article. Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of resting and activated allogenic peripheral blood mononuclear cells (PBMCs) was studied as a measure of the immune response (mixed lymphocyte culture). At the same points, the expression of immunologically relevant surface markers (e.g., major histocompatibility complex (MHC)-I, MHC-II, CD40, CD40L) was measured, and correlations between the different sets of results were sought. The pattern of surface antigen expression of BMSCs is the same as that of ASCs. Analogous to BMSCs, undifferentiated cells isolated from adipose tissue lack expression of MHC-II; this is not lost in the course of the osteogenic differentiation process. In co-culture with allogenic PBMCs, both cell types fail to lead to any significant stimulation, and they both retain these characteristics during the differentiation process. BMSCs and ASCs suppress proliferation on activated PBMCs before and after osteogenic differentiation. Our results confirm that MSCs are immune modulating cells. These properties are retained even after osteogenic induction in vitro and seem to be similar in BMSCs and ASCs. Our results suggest that allogenic transplantation of BMSCs and ASCs would be possible, for example, in the context of tissue engineering.     

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。     

体外诱导人肾间质成纤维细胞成骨分化

 目的: 观察在体外培养条件下,使用成骨诱导培养液或不同浓度钙离子诱导人肾间质成纤维细胞发生成骨分化的效果,初步探讨肾脏Randall斑形成可能的细胞机制。方法: 体外培养人肾间质成纤维细胞,实验分为5组:成骨诱导组(加成骨诱导液)、CaⅠ组(加0.5 mmol/L Ca²⁺液)、CaⅡ组(加1.5 mmol/L Ca²⁺液)、Ca Ⅲ 组(加2.5 mmol/L Ca²⁺液)和对照组(加PBS)。各组细胞分别培养至第1、3、6、9天时,采用MTT法检测细胞活力;诱导至第9天时,用细胞钙茜素红染色液和钙钴法磷酸酶染色液对各组细胞进行染色,观察钙结节形成和碱性磷酸酶的表达情况。另外,分别采用real-time PCR和Western blot检测各组细胞不同时间点Runt相关转录因子2(Runx2)的 mRNA和蛋白表达水平。 结果: 在1.5 mmol/L 和2.5 mmol/L Ca²⁺浓度条件下细胞的活力明显受抑制。细胞染色结果证实成骨诱导液组和钙离子组均可以见到典型的红色钙结节和黑色块状的硫化钴沉淀物。成骨诱导组细胞Runx2的mRNA和蛋白相对表达量(0~9 d)逐渐升高(P<0.05);诱导至第9天时,3个钙离子组细胞Runx2的mRNA和蛋白表达呈浓度依赖性升高(P<0.05)。结论: 在体外培养环境下,人肾成纤维细胞可以在成骨诱导培养液的刺激作用下发生成骨分化;另外,在高钙离子环境,人肾成纤维细胞也发生了类似的成骨分化。肾乳头组织中的成纤维细胞在高钙离子等因素的作用下发生成骨分化,这可能是肾脏Randall斑形成的细胞学基础。     

骨关节炎患者膝关节滑膜组织来源间充质干细胞的增殖与分化

背景：相对于其他来源的间充质干细胞,滑膜间充质干细胞来源丰富,并且滑膜组织可以在部分切除后迅速再生,并发症少,已逐渐成为近年来干细胞研究的热点。 目的：观察骨关节炎患者膝关节滑膜组织来源间充质干细胞的增殖和定向分化能力。 方法：对滑膜组织来源间充质干细胞进行分离及培养,采用MTT法检测细胞增殖能力,于成骨诱导第7天定量检测碱性磷酸酶活性,诱导第7,14,21天检测成骨相关基因表达,诱导第21天行茜素红染色。 结果与结论：①第3代滑膜间充质干细胞增殖速度较快,接种第1,2天处于潜伏期,3-6 d为对数增长期,第7天细胞增殖速度变慢,进入平台期。②成骨诱导后第3,7,10天诱导组碱性磷酸酶活性均显著高于对照组(P < 0.05)。成骨诱导21 d,茜素红染色阳性,细胞外基质中出现钙沉积以及钙结节。③成骨诱导第7天可见骨结合蛋白以及Runx-2的表达,至第21 天时,骨结合蛋白以及Runx-2的表达水平达到最大值。④以上结果表明来源于晚期骨关节炎患者膝关节滑膜组织的间充质干细胞具有很强的增殖能力,在体外可成功诱导分化为成骨细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

骨髓间充质干细胞成骨分化中血管内皮生长因子和骨形态发生蛋白2的表达

背景：有研究表明血管内皮生长因子可促进内皮细胞增殖、分化和迁移,而骨髓间充质干细胞经诱导分化后细胞自身的血管内皮细胞生长因子及骨形态发生蛋白2如何表达,却鲜见报道。 目的：观察兔骨髓间充质干细胞成骨诱导分化过程中血管内皮细胞生长因子和骨形态发生蛋白2的表达。 方法：取第3代新西兰大白兔骨髓间充质干细胞,1 d后将细胞分为两组：对照组不更换培养基;诱导组加入成骨诱导培养基进行成骨诱导,流式细胞仪检测其表面抗原。RT-PCR和Western-Blot检测细胞成骨分化过程中血管内皮细胞生长因子和骨形态发生蛋白2的表达。 结果与结论：兔骨髓间充质干细胞成骨诱导后,细胞形态由长梭形逐渐变为短梭形,多角形,最后成集落层叠生长;在诱导过程中,碱性磷酸酶活性逐渐增高,与对照组比较有显著性差异(P < 0.05);诱导7 d时Ⅰ型胶原免疫细胞化学染色、14 时骨钙素免疫细胞化学染色、21 d时钙化结节染色阳性;RT-PCR和Western-Blot结果显示两组均有骨形态发生蛋白2和血管内皮细胞生长因子表达,诱导后第7,14,21天表达比较,差异有显著性意义(P < 0.05),第7天,两者表达最强。说明兔骨髓间充质干细胞分离、提取操作简便易行,且有较强的生长增殖能力,可诱导分化为成骨细胞。其在成骨诱导过程中,骨形态发生蛋白2和血管内皮生长因子表达均先增强后减弱,血管内皮细胞生长因子表达可能对血管生成有促进作用。     

骨髓间充质干细胞体外诱导向内皮和上皮细胞的分化

背景：骨髓来源的间充质干细胞在体外具有多系分化潜能,但其在体外向肺组织细胞的分化能力尚存在争议。 目的：体外诱导验证小鼠骨髓间充质干细胞向内皮细胞和上皮细胞分化的能力。 方法：分离小鼠骨髓来源的间充质干细胞,以内皮诱导液向内皮细胞分化。另外将小鼠骨髓间充质干细胞分别在以下诱导培养基中进行上皮诱导3周：单纯上皮诱导培养液,上皮诱导培养液加10 μg/L 转化生长因子β1,并以未经诱导的小鼠骨髓间充质干细胞作为阴性对照,肺泡上皮作为阳性对照。 结果与结论：小鼠骨髓间充质干细胞在上皮诱导培养液中诱导培养3周后,部分细胞由梭形变为典型的卵石样上皮细胞形态,诱导后约60%细胞表达广谱上皮细胞标志pan-CK,RT-PCR结果显示分化后的细胞表达上皮细胞特异标志CK18,未经诱导的间充质干细胞未表达。小鼠骨髓间充质干细胞在内皮诱导24 h后即出现了典型的血管网状结构,vWF免疫荧光染色显示约70%的细胞呈阳性,RT-PCR显示分化后的细胞表达内皮细胞特异性标志CD31、vWF和CD34。提示骨髓来源的间充质干细胞体外诱导培养具有跨胚层多系分化能力。     

Osteogenic Effects of Rest Inserted and Continuous Cyclic Tensile Strain on hASC Lines with Disparate Osteodifferentiation Capabilities

We investigated the effects of two types of cyclic tensile strain, continuous and rest inserted, on osteogenic differentiation of human adipose-derived adult stem cells (hASCs). The influence of these mechanical strains was tested on two hASC lines having different mineral deposition potential, with one cell line depositing approximately nine times as much calcium as the other hASC line after 14 days of culture in osteogenic medium on tissue culture plastic. Results showed that both continuous (10% strain, 1 Hz) and rest inserted cyclic tensile strain (10% strain, 1 Hz, 10 s rest after each cycle) regimens increased the amount and rate of calcium deposition for both high and low calcium depositing hASC lines as compared to unstrained controls. The response was similar for both types of tensile strain for a given cell line, however, cyclic tensile strain had a much stronger osteogenic effect on the high calcium depositing hASC line, suggesting that mechanical loading has a greater effect on cell lines that already have an innate ability to produce bone as compared to cell lines that do not. This is the first study to investigate the osteodifferentiation effects of cyclic tensile strain on hASCs and the first to show that both continuous (10%, 1 Hz) and rest inserted (10%, 1 Hz, 10 s rest) cyclic tensile strain accelerate hASC osteodifferentiation and increase calcium accretion.