Effect of growth factors on stromal CFU-f colony formation in mouse bone marrow cell cultures

N. F. Gamaleya Research Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 12, pp. 615–617, December, 1991.     

Effects of osteogenic growth factors on bone marrow stromal cell differentiation in a mineral-based delivery system

Delivering growth factors from bone-like mineral combines osteoinductivity with osteoconductivity. The effects of individual and sequential exposure of BMP-2 and FGF-2 on osteogenic differentiation, and their release from apatite were studied to design a dual delivery system. Bone marrow stromal cells were seeded on TCPS with the addition of FGF-2 (2.5, 10, 40 ng/ml) or BMP-2 (50, 150, 450 ng/ml) for 6 days. DNA content and osteogenic response were examined weekly for 3 weeks. FGF-2 increased DNA content; however, high concentrations of FGF-2 inhibited/delayed osteogenic differentiation, while a threshold concentration of BMP-2 was required for significant osteogenic enhancement. The sequence of delivery of BMP-2 (300 ng/ml) and FGF-2 (2.5 ng/ml) also had a significant impact on osteogenic differentiation. Delivery of FGF-2 followed by BMP-2 or delivery of BMP-2 followed by BMP-2 and FGF-2 enhanced osteogenic differentiation compared to the simultaneous delivery of both factors. Release of BMP-2 and FGF-2 from bone-like mineral was significantly affected by the concentration used during coprecipitation. BMP-2 also demonstrated a higher “burst” release compared to FGF-2. By integrating the results of the sequential delivery of BMP-2 and FGF-2 in solution, with the release of individual growth factors from mineral, an organic/inorganic delivery system based on coprecipitation can be designed for multiple biomolecules.     

The effects of bone marrow stromal cell transplants on tendon healing in vitro

The purpose of this study was to investigate the effect of bone marrow stromal cells (BMSCs) on tendon healing in a canine ex vivo model. Bone marrow was harvested and BMSCs were isolated and cultured according to established protocols. Cells were seeded into 0.5 mg/ml collagen gels and cultured for 24 h to allow gel contraction, and then implanted between the lacerated ends of repaired flexor digitorum profundus tendons. Tendons repaired with a gel patch alone and without a gel patch served as control groups. After 2 and 4 weeks in culture, the repaired tendons were evaluated for breaking strength and stiffness. Cell viability was assessed by labeling the cells with PKH26 red fluorescent cell linker. The maximal strength and stiffness of repaired tendons with the BMSC-seeded patch were significantly higher than the repaired tendons without a patch or with a patch without cells, at both 2 and 4 weeks (p < 0.05). Viable BMSC were present between the cut tendon ends at both 2 and 4 weeks. We conclude that BMSC-seeded gel patch transplantation has the potential to enhance flexor tendon healing, and we plan to investigate this effect in vivo.     

Engineering injectable bone using bone marrow stromal cell aggregates

With the increasing popularity of minimally invasive surgery, to develop an injectable bone would be highly preferable for the repair of bone nonunions and defects. However, the use of dissociated cells and exogenous carriers to construct injectable bone faces several drawbacks. To circumvent these limitations, we first harvested a cell sheet from rabbit bone marrow stromal cells using a continuous culture method and a scraping technique. The obtained sheet was then cut into fragments of multicellular aggregates, each of which was composed of a certain number of cells, extracellular matrix, and intercellular connections. The aggregates showed apparent mineralization properties, high alkaline phosphatase activity, increased osteocalcin content, and upregulated bone markers, implying their in vitro osteogenic potential. Then, serum-free medium (the control group), dissociated cell suspension (the cell group), and suspension of multicellular aggregates (the aggregate group) were injected subcutaneously on the back of the nude mice to evaluate ectopic bone formation. The results revealed that the aggregate group showed significantly larger and denser bone at the injection sites than the cell group, whereas bone formation did not occur in the control group. Additionally, when injecting them locally into the mandibular fracture gap of delayed healing in a rabbit model, we observed the most improved bone healing in the aggregate group. More cells survive and retain at the injection sites in the aggregate group than that in the cell group postoperatively. Our study indicates that the multicellular aggregates might be considered a promising strategy to generate injectable bone tissue and improve the efficacy of cell therapy.     

氯化锂对去卵巢骨质疏松大鼠骨微结构和骨髓基质细胞分化的影响

背景：骨质疏松是一种好发于绝经后妇女的慢性骨代谢疾病,随着世界人口老龄化的增加,如何预防和治疗绝经后骨质疏松是目前困扰医疗界的一大难题。 目的：探讨氯化锂对去卵巢骨质疏松大鼠骨微结构和骨髓基质细胞分化的影响。 方法：将30只3月龄雌性健康未孕SD大鼠去卵巢,因2只大鼠由于感染死亡,将剩下28只大鼠随机分为去卵巢体内组(9只)、去卵巢体外组(10只)和氯化锂组(9只)。手术后第11周,氯化锂组按照体质量每周腹腔注射3次氯化锂,剂量为15 mg/kg,干预8周后,用micro-CT检测9只去卵巢体内组和9只氯化锂组大鼠左侧股骨的骨微结构。10只去卵巢体外组大鼠的双侧股骨和胫骨用于骨髓基质细胞的培养,接种24 h后加入氯化锂,分为0 mmol/L(对照组)、1 mmol/L组和5 mmol/L组,培养至第6天和第8天更换培养基并加入相应浓度氯化锂,培养第10天处理细胞,用Western blot检测其SP7、Runx2和PPARγ2蛋白的表达水平。 结果与结论：①体内结果表明,氯化锂组体积骨密度、骨体积分数和骨小梁数目显著高于去卵巢体内组,骨小梁间隙显著低于去卵巢体内组,而骨小梁厚度和结构模型指数无显著变化。②体外结果表明,1 mmol/L和5 mmol/L氯化锂组骨髓基质细胞Sp7和Runx2蛋白表达水平显著高于对照组,而PPARγ2蛋白表达水平显著低于对照组。③以上实验结果表明,氯化锂可能是通过促进去卵巢骨质疏松大鼠骨髓基质细胞向成骨分化而改善去卵巢骨质疏松大鼠的骨微结构。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     

Effects on proliferation and differentiation of multipotent bone marrow stromal cells engineered to express growth factors for combined cell and gene therapy

A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF-BB), transforming growth factor β(1) (TGF-β(1) ), and vascular endothelial growth factor (VEGF), in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF-B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF-β(1) blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation.     

Three-dimensional model of bone marrow stromal cell culture

In a hematopoietic microenvironment in vivo, spatial organisation of hematopoiesis is possible due to the existence of a three-dimensional framework, the main part of which is formed by a branching population of stromal cells. Most of the previous in vitro studies, concerning long-term bone marrow cultures, were based on a previously prepared, flat adherent layer of stromal cells. There are only few reports concerning the three-dimensional growth pattern of the bone marrow stroma. In the present study we used a new three-dimensional model of the stromal cell culture. The framework for the cultured stromal cells was a structure of a nonliving trabecular bone (Unilab Surgibone). After a period of about four weeks the stromal cells created a spatial network which filled the intertrabecular spaces of the spongy bone.     

Osteogenic effects of bioactive glass on bone marrow stromal cells

Bioactive glass (BG) is an effective synthetic bone graft material. BG granules of narrow size range (300-355 mum) have the ability to form new bone tissue inside excavations produced by in vivo resorption. Previously, we demonstrated that BG stimulates the differentiation of cultured osteoblast precursors if the glass surface was biomimetically modified by the formation of bone-like apatite and adsorption of serum proteins. We now report that modified BG can also increase the rate at which multipotential rat bone marrow stromal cells (rMSC) will undergo osteogenesis. BG promoted rMSC osteogenesis both when cells were plated in contact with BG and when cells were not directly in contact with the BG. Alkaline phosphatase activity, a marker of bone cell differentiation, was used as an indicator for osteogenesis. Alkaline phosphatase activity of rMSCs exposed to osteoinducers such as ascorbate, dexamethasone, and BMP-2 was enhanced in the presence of BG. The stimulatory effect of BG was more pronounced in rMSC cultures with low basal alkaline phosphatase activity than in those with higher activity. The enhanced differentiation of rMSCs was associated with both a change in rMSC morphology and altered chemical composition of the cell culture media. rMSCs cultured on BG in the presence of BMP or dexamethasone exhibited a more rounded osteoblast-like appearance as compared with cells grown on tissue culture plastic. In the presence of BG, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period, suggesting a continuous dissolution of surface-modified BG and resulting release of BG dissolution products. The data suggest that both surface- and solution-mediated events play a role in the osteogenic effect of BG.     

Development of specific collagen scaffolds to support the osteogenic and chondrogenic differentiation of human bone marrow stromal cells

Type I Collagen matrices of defined porosity, incorporating carbonate substituted hydroxyapatite (HA) crystals, were assessed for their ability to support osteo- and chondrogenic differentiation of human bone marrow stromal cells (HBMSCs). Collagen-HA composite scaffolds supported the osteogenic differentiation of HBMSCs both in vitro and in vivo as demonstrated by histological and micro-CT analyses indicating the extensive penetration of alkaline phosphatase expressing cells and new matrix synthesis with localised areas immunologically positive for osteocalcin. In vivo, extensive new osteoid formation of implant origin was observed in the areas of vasculature. Chondrogenic matrix synthesis was evidenced in the peripheral regions of pure collagen systems by an abundance of Sox9 expressing chondrocytes embedded within a proteoglycan and collagen II rich ECM. The introduction of microchannels to the scaffold architecture was seen to enhance chondrogenesis. Tissue specific gene expression and corresponding matrix synthesis indicate that collagen matrices support the growth and differentiation of HBMSCs and suggest the potential of this platform for understanding the ECM cues necessary for osteogenesis and chondrogenesis.     

Human bone marrow stromal cell responses on electrospun silk fibroin mats

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.     

Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells

One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/10(6) cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.     

外源性胰岛素样生长因子1基因在大鼠骨髓基质细胞中的表达及对其生物学行为的影响

目的：观察人胰岛素样生长因子1（hIGF-1）基因转染大鼠骨髓基质细胞（BMSC）后产物的表达及对BMSC特征的影响.方法：构建hIGF-1逆转录病毒载体并转染BMSC,嘌呤霉素筛选,RT-PCR和ELISA法检测hIGF-1表达,MTT及美蓝、碱性磷酸酶钙钴、苏丹Ⅲ染色观察转染后BMSC增殖、集落形成及分化,并分析药物筛选对其增殖的影响.结果：转染BMSC经筛选后,IGF-1表达的增幅高于未经筛选者（10.7倍：6.7倍）.与空质粒转染组比较,未经筛选的BMSC增殖加快,而经筛选者增殖和集落形成能力降低,向脂肪及成骨细胞分化增强.结论：基因修饰可成功构建高表达IGF-1的BMSC,并促进其增殖和自然分化,但嘌呤霉素筛选对其自我更新能力有负面作用.     

Adhesion and growth of bone marrow stromal cells on modified alginate hydrogels

Alginate is a biodegradable, immunocompatible biopolymer that is capable of immobilizing viable cells and bioactive factors. Few investigations have analyzed the efficacy of alginate gels as substrata for cell attachment and proliferation. Here we have compared the adhesion and subsequent growth of human and rat bone marrow stromal fibroblastic cells on unmodified alginate hydrogel surfaces. It was found that, in contrast to rat cells, human cells did not readily attach or proliferate on unmodified alginates. In attempts to enhance these features, or collagen type I was incorporated into the gels, with no significant improvements in prolonged human cell adherence. However, alginate gels containing both collagen type I and beta-tricalcium phosphate were found to enhance human cell adherence and proliferation. Furthermore, interactions between the collagen and beta-tricalcium phosphate prevented loss of the protein from the hydrogels. These results indicate that alginate gels containing collagen have potential uses as vehicles for delivery of adherent cells to a tissue site. In addition, gels containing beta-tricalcium phosphate, with or without collagen type I incorporation, have potential to support cell growth and differentiation in vitro before implantation. This study emphasizes the limitations of the uses of cells derived from experimental animals in certain model studies relating to human tissue engineering.     

Cytokines transduced bone marrow stromal cell lines promote immunohematopoietic reconstitution in mice after allogeneic bone marrow transplantation

Impaired immune reconstitution following allogeneic T-cell depleted bone marrow transplantation (allo-TCD-BMT) is a major obstacle to its clinical application. Stromal cell line QXMSC1, established from bone marrow cells of BALB/c(H-2d), was transfected with murine IL-3 and/ or IL-2 gene, and injected into lethally irradiated C57BL/6(H2b) mice. We evaluated its effects on immunologic and hematopoietic reconstitution after allo-TCD-BMT. The results showed that QXMSC1-IL-3 + IL-2 could significantly increase the numbers of hematopoietic primitive progenitors (CFU-S), committed progenitors (CFU-GM, and BFU-E), and lymphocytes (CD8+ cells, CD4+ cells, and B cells). Similarly, immune functions of recipient mice were significantly enhanced in the QXMSC1-IL-3 + IL-2 group. In addition, QXMSC1-IL-3 or QXMSC1-IL-2 also exerted apparent effects on accelerating immune reconstitution, but these effects were far less than that of QXMSC1-IL-3 + IL-2. Our results demonstrated that stromal cell-mediated IL-3 and IL-2 gene therapy may be a potent approach in promoting immunologic and hematopoietic reconstitution after allo-TCD-BMT.     

兔骨髓基质细胞诱导成骨细胞复合同种异体生物衍生骨实验研究

目的探讨体外培养的骨髓基质细胞与自体来源的生物衍生骨复合后的生长特性，为进一步研究骨髓基质细胞作为种子细胞，以及探索一种良好的支架材料提供实验依据。方法分离纯化兔骨髓基质细胞并诱导成成骨细胞后与同种异体来源的生物衍生骨复合后体外培养，并在相差显微镜和扫描电镜下观察其生长情况。结果骨髓基质细胞在生物衍生骨上贴附生长、增殖良好。结论骨髓基质细胞可作为骨组织工程的理想种子细胞，与生物衍生骨复合后可作为骨组织工程的载体。     

Effect of platelet growth factors on proliferation of guinea pig bone marrow and splenic stromal precursor cells and proliferation of cultural descendants from bone marrow precursor cells

Elimination of platelets from guinea pig splenocyte suspension (laking megakaryocytes) with EDTA considerable reduces the efficiency cloning of splenic stromal precursor cells. It means that platelet-derived growth factors are necessary for stromal precursor cells from different organs (bone marrow, thymus, and spleen). The dependence on the platelet growth factors can vary within a wide range in descendants from cultured bone marrow precursor cells (passaged bone marrow fibroblasts at different stages of differentiation.     

骨髓基质细胞对"缺血/再灌注"海马片的保护作用及其机制

目的:探讨骨髓基质细胞对"缺血/再灌注"海马片的保护作用及其机制。方法:建立新生大鼠海马片"缺血/再灌注"模型。取成年大鼠骨髓, 培养、分离、纯化骨髓基质细胞。观察海马片细胞死亡及骨髓基质细胞表达脑源性神经营养因子(BDNF)的动态变化。结果:"缺血"后"再灌注"1、3、7d, 碘化丙啶荧光排斥法显示海马片大量细胞死亡。与骨髓基质细胞联合培养后1、3、7d, 仅有少量细胞死亡。加入抗BDNF抗体后, 骨髓基质细胞对"缺血/再灌注"海马片的保护作用明显减弱。正常骨髓基质细胞在自然条件下微弱表达BDNF;与"缺血/再灌注"海马片联合培养后骨髓基质细胞表达BDNF逐渐增加, 在第3、7d更明显;与正常海马片联合培养的骨髓基质细胞仅出现持续低水平的BDNF表达。结论:骨髓基质细胞可以减少缺血海马片细胞死亡;"缺血/再灌注"海马片与骨髓基质细胞联合培养可以促进骨髓基质细胞表达BDNF, 可能由此发挥对缺血海马片的保护作用。