辛伐他汀对人骨髓基质干细胞成骨分化功能的促进作用

目的观察辛伐他汀对体外培养的人骨髓基质干细胞(humanBoneMarrowStromalcells,hMSCs)成骨分化功能的影响,探讨其刺激成骨的作用机制。方法体外培养来自于股骨颈骨折患者的骨髓基质干细胞,传代后实验组加入1×10-7molL的辛伐他汀,于不同时间点采用Westernblot检测核转录因子1(CoreBindingFactor1,Cbfa1)的表达,碱性磷酸酶(AlkalinePhosphatase,ALP)试剂盒检测ALP的比活性,及放射免疫法检测骨钙素(Osteocalcin,OCN)含量。结果辛伐他汀作用后,实验组与对照组比较,实验组Cbfa1蛋白表达水平增高,ALP比活性增高且骨钙素含量增加。结论1×10-7molL辛伐他汀能够促进人骨髓基质细胞成骨分化,此种促进作用可能与辛伐他汀增强其分化过程中相关蛋白的表达有关。     

脉冲电磁场促进人骨髓间充质干细胞成骨的研究

目的 :观察脉冲电磁场 (PEMFs)对人骨髓间充质干细胞 (hMSCs)成骨能力的影响。方法 :hMSCs受频率 12Hz、场强 1.1mT的PEMFs刺激 8h/d ,检测细胞增殖、细胞周期 ,观察细胞超微结构、骨钙素、Ⅰ型胶原免疫组化染色 ,碱性磷酸酶 (ALP)活性、核固红钙染色。结果 :脉冲电磁场作用后的细胞增殖能力提高 ,细胞形态发生变化 ,透射电镜观察提示细胞成熟 ,核固红钙染色、骨钙素、Ⅰ型胶原免疫组化染色均阳性 ,ALP活性增高。结论 :hMSCs经合理的PEMFs体外刺激后 ,细胞增殖加速 ,符合成骨细胞的形态特征和生物学特性。     

生长板软骨细胞对骨髓基质干细胞体外分化的影响

目的 观察大鼠骨髓基质干细胞(BMSCs)在与大鼠生长板软骨细胞体外共培养条件下，BMSCs碱性磷酸酶(ALP)活性变化及成骨分化的标志骨钙素与Ⅰ型胶原mRNA水平表达的影响，从而认识生长板软骨细胞旁分泌作用对BMSCs分化的影响，以期帮助认识生长板损伤后骨桥形成的发生机制。方法 大鼠BMSCs在与生长板软骨细胞进行间接共培养，并设阴性对照。测定ALP活性．用RT-PCR方法，检测Ⅰ型胶原与骨钙素mRNA的表达。结果 BMSCs随共培养时间的延长，ALP活性明显升高，Ⅰ胶原mRNA表达丰度明显高于对照组，骨钙素mRNA在对照组中几乎未见PCR扩增产物，共培养组在终末期则有一定的表达。结论 BMSCs在与大鼠生长板软骨细胞体外共培养后，出现成骨分化倾向：随时间延长效应愈加显著，提示生长板软骨细胞的旁分泌作用可以促进BMSCs向成骨分化。     

冲击波诱导人骨髓基质细胞成骨分化及机制的研究

目的 观察出生后人骨髓基质细胞(hIMSCs)在体外培养条件下增殖与分化的特点；研究适宜能量冲击波对出生后hMSCs成骨分化的作用及机制。方法 抽取健康自愿者髂骨骨髓，采用密度梯度离心法进行hMSCs体外培养。设冲击波组(SW组)与对照组，应用不同能量级冲击波对SW组原代细胞进行处理，根据细胞活力测定与集落形成数量确定适宜的冲击波能量值。应用适宜的冲击波能量处理hMSCs原代细胞并传代培养，采用倒置显微镜观察、细胞增殖活力测定、ELISA法检测细胞分泌TGF-81、茜素红染色、钙钴法染色、四环素荧光标记、细胞分泌碱性磷酸酶测定和逆转录一聚合酶链反应(RT-PCR)检测骨钙素mRNA表达等方法，对SW组和对照组的各代细胞形态、增殖与分化及其机制进行探讨。结果冲击波处理体外原代培养hMSCs的适宜能量为10kV(500)。SW组细胞在冲击波处理后早期分泌TGF-B1显著高于对照组(P＜0．001)。SW组与对照组细胞在形态学方面第3代前无明显差别；SW组各代细胞分泌碱性磷酸酶显著高于对照组(P＜0．01)；茜素红染色、钙钴法染色、四环素荧光标记等显示SW组细胞的成骨作用明显优于对照组；SW组细胞经冲击波处理后第10天应用RT-PCR方法可以检测到骨钙素mRNA的表达，与对照组比较差异有统计学意义(P＜0．001)。结论 冲击波对体外培养的出生后人骨髓基质细胞具有促进成骨分化的作用，适宜能量为10kv(500)，其机制之一为TGF-B1介导的促hMSCs成骨分化作用。10kV(500)能量级的冲击波对体外培养的hMSCs增殖无影响，大于该能量的冲击波具有抑制细胞增殖的作用。     

应用脉冲电磁场刺激人骨髓间充质干细胞体外培养的生物学特性的研究

目的　应用脉冲电磁场 (PEMFs)刺激体外分离培养的人骨髓间充质干细胞 (hMSCs) ,对其生物学特性进行研究。方法　PEMFs (1 2Hz、 1 1mT、 8h/d)刺激hMSCs体外培养 ,计算细胞的倍增时间 ,观察细胞超微结构 ,放免法测定骨钙素的分泌情况和钙化结节VonKossa染色。结果　脉冲电磁场作用后的细胞增殖能力提高 ,细胞形态发生变化 ,透射电镜观察提示细胞成熟 ,VonKossa钙染色阳性 ,骨钙素的分泌活性增高。结论　hMSCs经合理的PEMFs体外刺激培养后 ,不仅能够大量扩增细胞 ,同时能够向成骨细胞转化 ,具有一定的成骨活性 ,是一种较为理想的骨种子细胞来源     

人脱细胞骨复合骨髓基质细胞成骨活性的实验研究

目的 研究人脱细胞骨(HAB)复合经诱导的骨髓基质细胞的实验效果,观察细胞的黏附和生长情况,并对其成骨活性进行检测。方法 用15％的过氧化氢和乙醚去除人髂骨块内的结缔组织和细胞成分,消毒后制备人脱细胞骨。取材活体或新鲜尸体的骨髓行骨髓基质细胞培养,细胞纯化后加入β-甘油酸钠,地塞米松和抗坏血酸等向成骨方向诱导,并进行对照培养。通过碱性磷酸酶(ALP)和骨钙素(OCN)检测来确定骨髓基质细胞的增殖和分化情况,将诱导的骨髓基质细胞浓缩后复合到制备好的脱细胞骨块内进行复合培养。8d后通过光镜和电镜等形态学观察以及生化指标检测来确定细胞的成骨活性。结果 人髂骨块内细胞清除干净,骨基质保存良好;诱导后的骨髓基质细胞ALP和OCN水平明显高于对照组(P<0.05);复合后的人脱细胞骨培养液中ALP和OCN检测呈阳性;骨髓基质细胞在HAB支架内附着紧密,生长良好。结论 人脱细胞骨复合经诱导的骨髓基质细胞在体外具有有效的成骨功能,是一种较为理想的骨组织工程材料。     

转基因人骨髓间质干细胞在支架中增殖及矿化的研究

目的:观察携带骨形态发生蛋白2基因(BMP-2)的人骨髓间质干细胞(hMSCs)在生物衍生骨支架环境中的增殖及矿化.方法:利用BMP-2腺病毒载体转染hMSCs后种植生物衍生骨支架中,扫描电镜观察细胞生长增殖,能量谱仪测定钙质分泌,RT-PCR检测培养液上清中BMP-2表达.结果:转染后,hBMSC呈ALP、Ⅰ和Ⅱ型胶原阳性表达;细胞在支架中伸展良好,生长旺盛,并产生钙盐沉积;RT-PCR显示培养液上清中存在BMP-2基因表达.结论:转染BMP-2基因的hMSCs在生物衍生骨支架中能够立体生长、增殖,并继续表达目的基因,为基因修饰的组织工程骨修复骨缺损奠定了基础.     

应用脉冲电磁场刺激人骨髓间充质干细胞体外培养的生物学特性的研究

目的应用脉冲电磁场(PEMFs)刺激体外分离培养的人骨髓间充质干细胞(hMSCs)，对其生物学特性进行研究。方法PEMFs(12Hz、1．1mT、8h／d)刺激hMSCs体外培养，计算细胞的倍增时间，观察细胞超微结构，放免法测定骨钙素的分泌情况和钙化结节Von Kossa染色。结果脉冲电磁场作用后的细胞增殖能力提高，细胞形态发生变化，透射电镜观察提示细胞成熟，Von Kossa钙染色阳性，骨钙素的分泌活性增高。结论hMSCs经合理的PEMFs体外刺激培养后，不仅能够大量扩增细胞，同时能够向成骨细胞转化，具有一定的成骨活性，是一种较为理想的骨种子细胞来源。     

成骨诱导的兔骨髓基质干细胞成骨活性的表达及维持

目的观察成骨诱导的兔骨髓基质干细胞(BMSCs)体内、外环境下成骨活性的表达及维持。方法观察BMSCs在体外成骨诱导培养条件下的成骨分化特性;构建兔BMSCs与活骨组织共培养模型模拟体内“成骨环境”,将成骨诱导的MSCs置于共培养及普通传代培养条件下进行传代培养,观察经成骨诱导的BMSCs在体外及模拟体内的培养条件下细胞的表型维持情况。结果药物成骨诱导培养的BMSCs,其ALP活性及骨钙素均显著高于普通培养组(P<0.05);经过诱导培养的BMSCs,其Ⅰ型胶原、骨钙素免疫组化阳性。RT-PCR法半定量测定Ⅰ型胶原mRNA,成骨诱导培养的Ⅰ型胶原mRNA表达量明显高于普通传代培养对照组。药物成骨诱导后的细胞在体外普通传代培养传5代后,细胞碱性磷酸酶(ALP)活性、骨钙素水平及Ⅰ型胶原表达稳定维持在较高水平,保持其成骨细胞的表型;在共培养条件下,ALP活性、骨钙素水平Ⅰ型胶原表达保持在高水平,且ALP活性、骨钙素水平在大部分时间点均高于普通传代培养。结论药物成骨诱导培养呈现促BMSCs向成骨方向转化的特点,能使ALP、骨钙素及Ⅰ型胶原表达短期内达到高水平;经成骨诱导的BMSCs在体外或模拟的体内传代培养条件下,均能维持成骨表型,保持成骨活力。     

辛伐他汀在人骨髓基质干细胞向成骨细胞分化过程中的作用

目的观察辛伐他汀对体外培养的人骨髓基质干细胞（Human Bone Marrow Stromal cells,hBMCs）成骨分化功能的影响,探讨其刺激成骨的作用机制。方法体外培养来自于外伤所致股骨颈骨折患者的骨髓基质干细胞,传代后实验组加入1×10^-7mol/L的辛伐他汀,在不同时间点采用ELISA检测核转录因子1（Core Binding Factor1,Cbfa1）与DNA的结合活性,碱性磷酸酶（Alkaline Phosphatase,ALP）比活性,及放射免疫法检测骨钙素（Osteocalcin,OCN）含量。结果在辛伐他汀作用后,实验组与对照组比较,实验组Cbfa1因子与DNA的结合活性增高,ALP比活性增高且骨钙素含量增加。结论1×10^-7mol/L辛伐他汀能够促进人骨髓基质干细胞成骨分化,此种促进作用可能与辛伐他汀增加其分化过程中相关转录因子的活性有关。     

Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering.

The use of multipotent human mesenchymal stem cells (hMSCs) for tissue engineering has been a subject of extensive research. The donor variation in growth, differentiation and in vivo bone forming ability of hMSCs is a bottleneck for standardization of therapeutic protocols. In this study, we isolated and characterized hMSCs from 19 independent donors, aged between 27 and 85 years, and investigated the extent of heterogeneity of the cells and the extent to which hMSCs can be expanded without loosing multipotency. Dexamethasone-induced ALP expression varied between 1.2- and 3.7-fold, but no correlation was found with age, gender, or source of isolation. The cells from donors with a higher percentage of ALP-positive cells in control and dexamethasone-induced groups showed more calcium deposition than cells with lower percentage of ALP positive cells. Despite the variability in osteogenic gene expression among the donors tested, ALP, Collagen type 1, osteocalcin, and S100A4 showed similar trends during the course of osteogenic differentiation. In vitro expansion studies showed that hMSCs can be effectively expanded up to four passages (approximately 10-12 population doublings from a P0 culture) while retaining their multipotency. Our in vivo studies suggest a correlation between in vitro ALP expression and in vivo bone formation. In conclusion, irrespective of age, gender, and source of isolation, cells from all donors showed osteogenic potential. The variability in ALP expression appears to be a result of sampling method and cellular heterogeneity among the donor population.     

Proliferative and osteogenic differentiation capacity of mesenchymal stromal cells: Influence of harvesting site and donor age

Human mesenchymal stromal cells (hMSCs) are the cellular source of new bone formation and an essential component of autologous bone grafts. Autologous bone graft harvesting is routinely conducted at the iliac crest, although alternative donor sites with lower complication rates are available. Thus, the aim of this study was to compare hMSCs harvested from the iliac crest and the proximal tibia regarding their proliferative and osteogenic differentiation capacity. Furthermore, we investigated the influence of donor age on these biological properties.HMSCs were isolated from iliac crest or proximal tibia bone grafts of 46 patients. Proliferative capacity was assessed by cumulative population doublings, population doubling time, colony forming units and cell proliferation assays. Osteogenic capacity was assessed by quantification of extracellular calcium deposition and marker gene expression levels. The number of hMSCs per gram harvested tissue was determined. Furthermore, the adipogenic and chondrogenic differentiation capacity were quantified using BODIPY and Safranin Orange staining, respectively. Additional analyses were carried out after grouping young (18–49 years) and aged (≥50 years) donors.HMSCs derived from the proximal tibia featured a comparable proliferative and osteogenic differentiation capacity. No significant differences were found for any analysis conducted, when compared to hMSCs obtained from the iliac crest. Furthermore, no significant differences could be revealed when comparing young and aged donors. This was equally true for hMSCs from both donor sites after comparison within the same age group.Our study demonstrates comparable biological properties of hMSCs derived from both donor sites, the iliac crest and the proximal tibia. Furthermore, aging does not alter proliferative and osteogenic differentiation capacity. Consequently, the proximal tibia should be considered more closely as an alternative donor site in patients of all age groups.     

Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.

We used the patch-clamp technique and RT-PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L-type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L-type Ca2+ channel function. INTRODUCTION: During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. MATERIALS AND METHODS: Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique. RESULTS: mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. CONCLUSION: These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.     

Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation

Human mesenchymal stem cells (hMSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications by virtue of their capacity for self‐renewal and multipotent differentiation. Our intent was to characterize the effect of pulsed electromagnetic fields (PEMFs) on the proliferation and osteogenic differentiation of hMSCs in vitro. hMSCs isolated from the bone marrow of adult patients were cultured with osteogenic medium for up to 28 days and exposed to daily PEMF stimulation with single, narrow 300 µs quasi‐rectangular pulses with a repetition rate of 7.5 Hz. Relatively greater cell numbers were observed at late stages of osteogenic culture with PEMF exposure. The production of alkaline phosphatase (ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMF treatment in both basal and osteogenic cultures as compared to untreated controls. Furthermore, the expressions of other early osteogenic genes, including Runx2/Cbfa1 and ALP, were also partially modulated by PEMF exposure, indicating that osteogenesis in hMSCs was associated with the specific PEMF stimulation. Based on ALP and alizarin red S staining, the accumulation of ALP protein produced by the hMSCs as well as calcium deposits reached their highest levels at day 28. Our results indicate that extremely low‐frequency PEMF stimulation may play a modulating role in hMSC osteogenesis. Taken together, these findings provide insights on the development of PEMF as an effective technology for regenerative medicine. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells.

Long-term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS-treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi-treated hMSC cultures revealed patchy groups of cells displaying disorganized cytoskeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.     

A proteomic analysis during serial subculture and osteogenic differentiation of human mesenchymal stem cell.

Although previous studies have reported the effects of extensive subculturing on proliferation rates and osteogenic potential of human mesenchymal stem cells (hMSCs), the results remain controversial. The aim of our study was to characterize the proliferation and osteogenic potential of hMSCs during serial subculture, and also to identify proteins that are differentially regulated in hMSCs during serial subculture and osteogenic differentiation using proteome analysis. Here we show that the proliferation and osteogenic capacity of hMSCs decrease during serial subculturing. Several proteins were shown to be differentially regulated during serial subculture; among these the expression of T-complex protein 1 alpha subunit (TCP-1alpha), a protein known to be associated with cell proliferation, cell cycle, morphological changes, and apoptosis, gradually decreased during serial subculture. Among proteins that were differentially regulated during osteogenic differentiation, chloride intracellular channel 1 (CLIC1) was downregulated only during the early passages eukaryotic translation elongation factor, and acidic ribosomal phosphoprotein P0 was downregulated during the middle passages, while annexin V, LIM, and SH3 domain protein 1 (LASP-1), and 14-3-3 protein gamma (YWHAG) were upregulated during the later passage. These studies suggest that differentially regulated passage-specific proteins may play a role in the decrease of osteogenic differentiation potential under serial subculturing.     

Effects of matrix elasticity and cell density on human mesenchymal stem cells differentiation

Human mesenchymal stem cells (hMSCs) can differentiate into various cell types, including osteogenic and chondrogenic cells. The matrix elasticity and cell seeding density are important factors in hMSCs differentiation. We cultured hMSCs at different seeding densities on polyacrylamide hydrogels with different stiffness corresponding to Young's moduli of 1.6 ± 0.3 and 40 ± 3.6 kPa. The promotion of osteogenic marker expression by hard gel is overridden by a high seeding density. Cell seeding density, however, did not influence the chondrogenic marker expressions induced by soft gel. These findings suggest that interplays between cell–matrix and cell–cell interactions contribute to hMSCs differentiation. The promotion of osteogenic differentiation on hard matrix was shown to be mediated through the Ras pathway. Inhibition of Ras (RasN17) significantly decreased ERK, Smad1/5/8 and AKT activation, and osteogenic markers expression. However, constitutively active Ras (RasV12) had little effect on osteogenic marker expression, suggesting that the Ras pathways are necessary but not sufficient for osteogenesis. Taken together, our results indicate that matrix elasticity and cell density are important microenvironmental cues driving hMSCs proliferation and differentiation. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1360–1365, 2013     

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent

Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50–65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.     

Inhibition of osteogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (hMSCs) have been shown to differentiate into osteoblasts that, in turn, are capable of forming tissues analogous to bone. The present study was designed to investigate the inhibition of osteogenesis by hMSCs. Bone marrow-derived hMSCs were treated with transforming growth factor beta-3 (TGFbeta3) at various doses during or after their differentiation into osteogenic cells. TGFbeta3 was encapsulated in poly(DL-lactic-co-glycolic acid) (PLGA) microspheres and released via controlled delivery in the osteogenic culture of hMSCs and hMSC-derived osteoblasts for up to 28 days. Controlled release of TGFbeta3 inhibited the osteogenic differentiation of hMSCs, as evidenced by significantly reduced alkaline phosphatase activity and staining, as well as decreased mineral deposition. After hMSCs had been differentiated into osteoblasts, controlled release of TGFbeta3 further inhibited not only alkaline phosphatase and mineral deposition but also osteocalcin expression. These findings demonstrate the potential for sustained modulation of the behavior of stem cells and/or stem cell-derived lineage-specific cells via controlled release of growth factor(s). The attenuation of osteogenic differentiation of MSCs may facilitate understanding not only the regulation and patterning of osteogenesis in development but also several pathological models such as osteopetrosis, craniosynostosis, and heart valve calcification.