Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow‐derived mesenchymal stem cells

Human adult bone marrow‐derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi‐lineage differentiation potential. We compare the different basal culture media DMEM‐F12, DMEM‐high glucose (DMEM‐HG), DMEM‐low glucose (DMEM‐LG), knock‐out DMEM (DMEM‐KO) and Mesencult^® on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO‐SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up‐scaling to provide sufficient numbers for transplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering

The heterogeneous population of non-haematopoietic cells residing in the bone marrow (bone marrow stromal cells, BMSCs) and the different fractions and components obtained from platelet-rich plasma provide an invaluable source of autologous cells and growth factors for bone and other connective tissue reconstruction. In this study, we investigated the effect of an allogenic platelet lysate on human BMSCs proliferation and differentiation. Cell proliferation and number of performed cell doublings were enhanced in cultures supplemented with the platelet-derived growth factors (platelet lysate, PL), either with or without the concomitant addition of fetal bovine serum (FBS), compared to cultures performed in the presence of FBS and FGF2. Both in vitro and in vivo osteogenic differentiation were unaltered in cells maintained in medium supplemented with PL and not FBS (Only PL) and in cells maintained in medium containing FBS and FGF2. Interestingly, the in vitro cartilage formation was more effective in the pellet of BMSCs expanded in the Only PL medium. In particular, a chondrogenic differentiation was observed in pellets of some in vitro-expanded BMSCs in the Only PL medium, whereas pellets from parallel cell cultures in medium containing FBS did not respond to the chondrogenic induction. We conclude that the platelet lysate from human source is an effective and even more beneficial substitute for fetal bovine serum to support the in vitro expansion of human BMSCs for subsequent tissue-engineering applications.     

Vascular endothelial growth factor‐transfected adipose‐derived stromal cells enhance bone regeneration and neovascularization from bone marrow stromal cells

Vascular endothelial growth factor (VEGF)‐transfected adipose‐derived stromal cells (ADSCs^VEGF) were devised to promote bone regeneration and neovascularization of bone marrow stromal cells (BMSCs). ADSCs^VEGF were added to BMSCs and cocultured in variable proportions. ADSCs^VEGF alone or ADSCs^VEGF with BMSCs (BMSCs:ADSCs^VEGF ratio of 1:0.025–0.5) induced significantly greater tube formation in human umbilical vein endothelial cells than untransfected ADSCs. The cocultures of BMSCs and ADSCs^VEGF at ratios of 1: 0.025–0.1 showed significantly greater osteogenic differentiation and mineralization than BMSCs alone in vitro. Osteogenic markers COL1A1, OCN and BSP were most effectively induced at the BMSC: ADSC^VEGF ratio of 1:0.05. Of angiogenesis‐related genes, upregulation of cathepsin Z and downregulation of early growth response 1 were observed while two osteogenesis‐related genes, osteoactivin and tetranectin, were upregulated in BMSCs/ADSCs^VEGF compared to BMSCs/ADSCs. When critical size calvarial defects in rats were implanted with mixture of BMSCs and ADSCs^VEGF along with hydroxyapatite/β‐tricalcium phosphate granules, BMSCs and ADSCs^VEGF at the ratio of 1:0.05 showed better bone regeneration that BMSCs alone. The cotransplantation of ADSCs^VEGF with BMSCs significantly increased neovascularization on the regenerated bone of the repaired defect than BMSCs alone. In conclusion, ADSCs^VEGF added in small proportion to BMSCs effectively promote bone regeneration and neovascularization. Copyright © 2017 John Wiley & Sons, Ltd.     

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

Objective. Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. Material and methods. Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP‐accepted medium (EMEA medium) or in one of four other media. Results. FACS analysis showed that the plastic‐adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary‐like structures. Conclusions. We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.     

Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate–gelatine–hydroxyapatite scaffolds with anisotropic pore structure

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube‐like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube‐like pores were generated by diffusion‐controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 µm. To evaluate the potential use of alginate–gelatine–HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate–gelatine–HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10–14‐fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic‐induced hBMSCs. Furthermore, the expression of bone‐related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic‐induced hBMSCs on alginate–gelatin–HA scaffolds. Copyright © 2008 John Wiley & Sons, Ltd.     

Whatever their differentiation status,human progenitor derived – or mature – endothelial cells induce osteoblastic differentiation of bone marrow stromal cells

Association of the bone‐forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co‐culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular‐like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co‐cultured with human endothelial progenitor cells in order to assess whether progenitor‐derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co‐culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non‐orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co‐cultures were undertaken: HBMSCs–PDECs, HBMSCs–HUVECs, HOPs–PDECs, HOPs–HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co‐culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real‐time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono‐ and co‐cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Differential effect of platelet-rich plasma and fetal calf serum on bone marrow-derived human mesenchymal stromal cells expanded in vitro

Mesenchymal stromal cells (MSCs) derived from various sources have great potential for use in cell-based therapies. Since the proportion of primary MSCs contained in bone marrow or adipose tissue is low, plastic adherence and in vitro expansion are necessary to expand MSCs prior to clinical application. Human platelet-rich plasma has been introduced as an alternative serum source but functional differences have so far not been described. Here we cultured MSCs derived from human bone marrow in medium supplemented with either 10% fetal calf serum (FCS) or 5% and 10% platelet-rich plasma (PRP) until the first or second passage. Parameters under investigation were cell yield, clonogenicity, phenotype as well as migratory and differentiation potential. In addition, the secretion of SDF-1α and the induced migration of CD34(+) haematopoietic stem cells (HSCs) were investigated with regard to the different serum source. The use of PRP resulted in a significantly higher expansion rate and yield at passages 0 and 1. In addition, the level of secreted SDF-1α was significantly increased in the supernatant of MSCs cultured with FCS instead of human PRP. Consistent with this, the migration capacity of MSCs cultured with 10% FCS as well as their capability to induce the migration of CD34(+) haematopoietic progenitors in a transwell assay was higher. Our results demonstrate that human PRP can be seen as an alternative serum source to FCS for MSC cultivation. However, the requirements of the specific clinical application must be carefully considered before the respective serum source is selected.     

Micro‐aggregates do not influence bone marrow stromal cell chondrogenesis

Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC‐based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro‐aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFβ₃ added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFβ, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro‐aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro‐aggregates, under all TGFβ conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro‐aggregates. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparing the osteogenic potential of bone marrow and tendon‐derived stromal cells to repair a critical‐sized defect in the rat femur

Despite significant advancements in bone tissue‐engineering applications, the clinical impact of bone marrow stromal cells (BMSCs) for the treatment of large osseous defects remains limited. Therefore, other cell sources are under investigation for their osteogenic potential to repair bone. In this study, tendon‐derived stromal cells (TDSCs) were evaluated in comparison to BMSCs to support the functional repair of a 5 mm critical‐sized, segmental defect in the rat femur. Analysis of the trilineage differentiation capacity of TDSCs and BMSCs cultured on collagen sponges revealed impaired osteogenic differentiation and mineral deposition of TDSCs in vitro, whereas chondrogenic and adipogenic differentiation was evident for both cell types. Radiographic assessment demonstrated that neither cell type significantly improved the healing rate of a challenging 5 mm segmental femoral defect. Transplanted TDSCs and BMSCs both led to the formation of only small amounts of bone in the defect area, and histological evaluation revealed non‐mineralized, collagen‐rich scar tissue to be present within the defect area. Newly formed lamellar bone was restricted to the defect margins, resulting in closure of the medullary cavity. Interestingly, in comparison to BMSCs, significantly more TDSC‐derived cells were present at the osteotomy gap up to 8 weeks after transplantation and were also found to be located within newly formed lamellar bone, suggesting their capacity to directly contribute to de novo bone formation. To our knowledge, this is the first study investigating the in vivo capacity of TDSCs to regenerate a critical‐sized defect in the rat femur. Copyright © 2015 John Wiley & Sons, Ltd.     

骨髓基质细胞对血管内皮细胞生长及微血管形成的影响

目的研究骨髓基质细胞（BMSCs）在体外对血管内皮细胞生长及对微血管形成的影响。方法体外分离培养成人BMSCs和脑血管内皮细胞，分为共培养组、培养液组和对照组，观察BMSCs对脑血管内皮细胞增殖及微血管网状结构形成的影响。结果培养液组和共培养组对内皮细胞的增殖和微血管形成均有促进作用，共培养组的作用更为明显。结论BMSCs在体外促进内皮细胞的生长及微血管的形成。     

成人骨髓间充质干细胞体外定向诱导分化为神经元样细胞的研究

目的研究人骨髓间充质干细胞（mesenchymal stem cells，MSCs）向神经细胞分化的可能性。方法利用Per-coil梯度分离及贴壁筛选法分离培养和扩增MSCs；利用bFGF、化学诱导剂DMSO和BHA联合诱导MSCs向神经元转化，观察分化过程中细胞形态的变化，利用免疫细胞化学和RT-PCR方法检测神经元特异性标志物的表达情况。结果经Perecoll梯度分离及贴壁筛选法获得了纯度较高的人MSCs，诱导分化后的细胞呈现双极、多极和锥形的典型神经元细胞的形态，并且分别从mRNA和蛋白水平上证明诱导分化后的细胞表达神经元标记物NSE和NF，不表达神经胶质细胞标记物GFAP。结论人MSCs可以在体外诱导分化为神经元样细胞，这种潜能使其有可能成为神经系统疾病细胞移植治疗的种子细胞。     

骨髓间充质干细胞及其向肝样细胞分化的潜能

背景：研究表明,在一定条件下骨髓间充质干细胞可诱导分化为肝样细胞,为治疗急性肝衰竭等终末期肝病提供了新的思路。目的：对骨髓间充质干细胞的发现、分离培养、诱导分化及应用前景等方面做一综述。方法：计算机检索中国期刊网全文数据库以及PubMed数据库1999至2014年期间有关骨髓间充质干细胞及其向肝样细胞分化的文章。检索词分别为“肝样细胞,骨髓间充质干细胞,细胞分化”和“hepatocyte-like cel s, bone marrow mesenchymal stem cel s,differentiation”。最后选择52篇文章纳入结果分析。结果与结论：目前,肝组织工程的首要问题是寻找性状稳定具有肝特异性功能的种子细胞,成熟肝细胞获取难度较大,并具有产生免疫排斥反应、体外培养困难等缺陷,严重限制了肝移植的开展。骨髓间充质干细胞具有多向分化、自我更新快、易于扩增及培养等优点,被认为是最有前途的细胞来源。已有研究表明骨髓间充质干细胞在体内外均可分化为肝细胞,并具有肝细胞的合成和分泌功能。但如何大量扩增此类细胞的同时又能保持其良好的分化潜能、体外培养的最佳条件、诱导分化机制和临床应用的安全性等尚需进一步研究和探讨。     

Bone marrow mesenchymal stem cells increase skin regeneration efficiency in skin and soft tissue expansion

Background: Skin and soft tissue expansion has limitations such as long hospitalization time and flap retraction after expansion. Our previous study suggested that bone marrow-derived stem cells contribute skin regeneration in skin and soft tissue expansion. In this study, the authors explored the feasibility of applying the bone marrow mesenchymal stem cells (BMMSCs) to the treatment of skin and soft tissue expansion and increasing the skin regeneration efficiency.

Methods: Sixty silicone expanders were implanted in the backs of 15 pigs, and allogeneic BMMSCs were transplanted to skin shallow fascia layer (local transplantation, Group A) or via ear vein (systemic transplantation, Group B). Group C was the Sham operation control; and then the expanders were injected with normal saline (N.S.). Skin was obtained at different time points (days 0, 14, 21, 28, 35, and 42). The organizational structure changes of the target skin were observed in the expansion process. The distribution, differentiation, and paracrine function of labeled BMMSCs were detected.

Results: Comparing with Group B (25.00 ± 1.98 cm²) or Group C (24.00 ± 1.10 cm², no transplantation), the expanded skin area of Group A (28.82 ± 1.43 cm²) increased, with the morphology of epidermis thickened, and dermis thinned. The BMMSCs differentiated into vascular endothelial cells and dermal fibroblasts. The quantity of newborn cells was proportional to the number of transplanted cells. The gene expression of VEGF, bFGF, EGF, and SDF in Group A was higher than those in Group B or C. The most obvious changes were on day 35.

Conclusions: The local transplanted BMMSCs could increase the skin regeneration efficiency in skin and soft tissue expansion and reduce skin shrinkage effectively after removing the expander. Growth factors, VEGF, bFGF, EGF, and SDF, are favorable to this process.     

Bone regeneration in minipigs via calcium phosphate cement scaffold delivering autologous bone marrow mesenchymal stem cells and platelet‐rich plasma

Macroporous calcium phosphate cement (CPC) with stem cell seeding is promising for bone regeneration. The objective of this study was to investigate the effects of co‐delivering autologous bone marrow mesenchymal stem cells (BMSCs) and autologous platelet‐rich plasma (PRP) in CPC scaffold for bone regeneration in minipigs for the first time. Twelve female adult Tibet minipigs (12–18 months old) were used. A cylindrical defect with 10 mm height and 8 mm diameter was prepared at the femoral condyle. Two bone defects were created in each minipig, one at each side of the femoral condyle. Three constructs were tested: (1) CPC scaffold (CPC control); (2) CPC seeded with BMSCs (CPC‐BMSC); (3) CPC seeded with BMSCs and PRP (CPC‐BMSC‐PRP). Two time points were tested: 6 and 12 weeks (n = 4). Good integration of implant with surrounding tissues was observed in all groups. At 12 weeks, the CPC‐BMSC‐PRP group had significantly less residual CPC remaining in the defect than the CPC‐BMSC group and the CPC control (p < 0.05). The residual CPC volume for the CPC‐BMSC‐PRP group was half that of the CPC control. New bone formation for CPC‐BMSC‐PRP was more than two‐fold that of the CPC control (p < 0.05). CPC‐BMSC‐PRP had new blood vessel density that was nearly two‐fold that of the CPC control (p < 0.05). In conclusion, CPC scaffold with autologous BMSC‐PRP doubled the new bone regeneration and blood vessel density in minipigs compared with the CPC control. In the present study, the new macroporous CPC system with co‐delivered BMSC‐PRP has been shown to promote scaffold resorption and bone regeneration in large defects. Copyright © 2017 John Wiley & Sons, Ltd.     

大鼠骨髓MSCs表面分子检测及诱导分化研究

目的通过建立大鼠骨髓间充质干细胞（MSCs）的体外分离培养方法，探讨大鼠MSCs表型特征以及多向分化潜能。方法利用密度梯度离心法结合贴壁培养法分离纯化SD大鼠骨髓MSCs，传代扩增，进行形态学观察，测定生长曲线。免疫细胞组织化学及流式细胞分析检测细胞表面分子的表达。定向诱导MSCs向脂肪细胞、成骨细胞及软骨细胞分化。结果原代分离的MSCs在接种后48h贴壁，细胞形态为椭圆形，多角形及短梭形，12天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，Pl代细胞群体倍增时间为20．3h。细胞免疫组化结果显示P2代细胞表面分子CD44、FN表达阳性，而CD14、CD34阴性，流式细胞检测CD44、FN阳性率分别为92．09％和90．55％。不同诱导剂定向诱导2周，经油红O、阿新兰及茜素红S染色鉴定，P3代MSCs分别向脂肪细胞，软骨细胞及成骨细胞分化。结论通过密度梯度离心结合贴壁筛选方法，体外分离培养的大鼠骨髓MSCs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。     

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM‐MSCs) combined with calcium phosphate microparticles and enveloped in a platelet‐rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose‐derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM‐MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM‐MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM‐MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un‐aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM‐MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose‐derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.     

Optimization of the use of a pharmaceutical grade xeno‐free medium for in vitro expansion of human mesenchymal stem/stromal cells

Human bone marrow‐derived mesenchymal stem/stromal cells (hMSCs) are considered promising therapeutic agents in the field of cell therapy and regenerative medicine, mainly due to their relative facility to be isolated, multi‐differentiation potential, and immunomodulatory role. However, their application in clinics requires a crucial step of in vitro expansion. Most of the protocols for hMSCs in vitro culture use foetal bovine serum as medium supplement that, being from animal origin, presents several safety concerns and may initiate xenogeneic immune responses after cells transplantation. This work reports the optimization of a pharmaceutical‐grade xeno‐free strategy for hMSCs in vitro expansion based on the supplementation of basal medium with a pharmaceutical‐grade human plasma‐derived supplement for cell culture (SCC) and 2 human growth factors (bFGF and TGFβ1), plus a coating of human plasma fibronectin (Fn). After 4 weeks in culture, this strategy improves hMSCs expansion yield about 4.3‐fold in comparison with foetal bovine serum supplementation and 4.5‐fold compared with a commercially available xeno‐free medium. hMSCs expanded in SCC‐based formulation maintained their phenotype and differentiation capacity into osteogenic, adipogenic, and chondrogenic lineages, without alterations in cell karyotype. Overall, the SCC‐based medium appears to be an excellent alternative for the xeno‐free expansion of hMSCs as therapeutic agents for clinical applications.     

改良法培养骨髓基质干细胞向成骨细胞分化的实验研究

目的:研究大鼠骨髓基质干细胞的生长特点和在诱导条件下的成骨能力。方法:通过改良的骨髓培养法分离成年大鼠骨髓基质干细胞,应用含地塞米松、β-甘油磷酸钠和维生素C的诱导分化培养液定向诱导传代细胞向成骨细胞分化,并检测骨钙素的分泌、碱性磷酸酶的活性和细胞矿化作用。结果:原代培养基质干细胞首先形成细胞集落,12d时集落间接近融合;传代细胞体积变大,约5～7d传代一次。诱导条件下,细胞碱性磷酸酶活性明显增高,并出现了矿化结节。结论:骨髓基质干细胞易于分离培养及体外扩增,成骨能力肯定,可作为骨组织工程的种子细胞