Cell differentiation of a human bone marrow cell culture under the influence of UHMW-PE debris

AIM: The purpose of the present study was to evaluate the influence of ultra-high-molecular-weight polyethylene (UHMW-PE), which is the major constituent of the material debris formed as a result of orthopaedic implant wear, on the cellular differentiation in a modified in vitro model. METHODS: UHMW-PE particles (? < or = 7.5 microm) were suspended in soluble collagen type I and subsequently solidified in different concentrations (10(5), 10(6) and 10(7) particles per well) on the bottom of the wells. Human bone marrow cells in a concentration of 3 x 10(6) cells per well were seeded on the collagen-particle substrate and maintained for up to 72 h. The response of the cells to the particles was examined by light microscopy, scanning electron microscopy and FACS analysis compared to cells on control collagen surfaces without any particles. RESULTS: Light and scanning microscopic evaluation revealed that the UHMW-PE particles, which had built large conglomerates (? 7.5 microm), were mainly surrounded by the cells and less phagocytosed. The results of the FACS analysis revealed significant differences in CD3/CD4 positive, CD14 positive and CD19 positive cells (p < 0.05). A significant elevation of CD3/CD4 positive and CD14 positive cells (p < 0.05) was observed after the period of culture (72 h) whereas a significant decrease could be detected in the case of CD19 positive cells. CONCLUSION: The results demonstrate that the particle-induced response by UHMW-PE limits itself not only to the particle macrophage contact but influences also the differentiation of the bone marrow. Moreover, the results confirm that the present method is useful to evaluate the in vitro effects of UHMW-PE wear particles with direct particle cell contact. Although the particles built large conglomerates, it could be shown that a change of the immune-competent cells also occurred.     

Long-term bone marrow culture as a stem cell source for transplantation

Stem cells in bone marrow capable of inducing spleen colonies in lethally irradiated mice can be maintained in liquid culture for up to 100 days if an appropriate marrow-derived feeder layer is provided. Marrow from some mouse strains cannot be maintained in such cultures. Neither duration of culture nor in vitro cultivation of marrow on allogeneic feeder layer in any way modulates the occurrence of graft-versus-host disease. Cocultivation of marrow with allogeneic feeder layers does not induce tolerance to feeder-layer-type skin grafts in lethally irradiated syngenic recipients. Though an excellent source of stem cells, long-term marrow cultures require further modification before successful allogeneic marrow transplantation without graft-versus-host reaction can be carried out.     

Effect of chitosan particles and dexamethasone on human bone marrow stromal cell osteogenesis and angiogenic factor secretion

Chitosan is a polysaccharide scaffold used to enhance cartilage repair during treatments involving bone marrow stimulation, and it is reported to increase angiogenesis and osteogenesis in vivo. Here, we tested the hypotheses that addition of chitosan particles to the media of human bone marrow stromal cell (BMSC) cultures stimulates osteogenesis by promoting osteoblastic differentiation and by favoring the release of angiogenic factors in vitro. Confluent BMSCs were cultured for 3 weeks with 16% fetal bovine serum, ascorbate-2-phosphate and disodium β-glycerol phosphate, in the absence or presence of dexamethasone, an anti-inflammatory glucocorticoid commonly used as an inducer of BMSC osteoblast differentiation in vitro. As expected, dexamethasone slowed cell division, stimulated alkaline phosphatase activity and enhanced matrix mineralization. Added chitosan particles accumulated intra- and extracellularly and, while not affecting most osteogenic features, they inhibited osteocalcin release to the media at day 14 and interfered with mineralized matrix deposition. Interestingly, dexamethasone promoted cell attachment and suppressed the release and activation of matrix metalloprotease-2 (MMP-2). While chitosan particles had no effect on the release of angiogenic factors, dexamethasone significantly inhibited (p < 0.05 to p < 0.0001) the release of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), interleukins 1β, 4, 6, and 10 (IL-1β, IL-4, IL-6, IL-10), and a host of other inflammatory factors that were constitutively secreted by BMSCs. These results demonstrate that chitosan particles alone are not sufficient to promote osteoblast differentiation of BMSCs in vitro, and suggest that chitosan promotes osteogenesis in vivo through indirect mechanisms. Our data further show that continuous addition of dexamethasone promotes osteoblastic differentiation in vitro partly by inhibiting gelatinase activity and by suppressing inflammatory cytokines which result in increased cell attachment and cell cycle exit.     

The effect of sodium salicylate on the osteoclast-like cell formation and bone resorption in a mouse bone marrow culture

Salicylates are reported to have an inhibitory effect on bone resorption in vivo and in vitro. The present study examined the effect of sodium salicylate on the formation of osteoclast-like cells in vitro. When mouse bone marrow cells were cultured for 8 days with 10^-8 M 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), numerous clusters of mononuclear and multinucleated cells (MNCs) formed, which stained positive for tartrate-resistant acid phosphatase (TRAP-positive). In similar cultures using sodium salicylate, the number of both TRAP-positive mononuclear and TRAP-positive MNCs were found to diminish in proportion to the concentration of sodium salicylate. A time-course experimental model showed that the number of TRAP-positive MNCs decreased slightly when sodium salicylate was given early in the culture period, and decreased markedly when the drugs were given later in the culture period. Pit formation and bone-resorption area on the bone slices were also inhibited by adding sodium salicylate continuously with 1,25(OH)₂D₃. The sodium salicylate showed no cytotoxic effect because the total number of adherent cells, including both TRAP-positive and TRAP-negative cells, was independent of the presence of sodium salicylate. These results suggest that sodium salicylate has an inhibitory effect on the recruitment of osteoclast-like MNCs and that this inhibition is greater during the later stage of mouse bone marrow culture.     

Cytokine profiles in conditioned media from cultured human intervertebral disc tissue. Implications of their effect on bone marrow stem cell metabolism

BACKGROUND: Cytokines released from intervertebral discs cultured in vitro have not been profiled, and the effect of these cytokines on human bone marrow stem cells is yet to be studied. MATERIALS AND METHODS: Intervertebral discs from 14 patients who had undergone spinal fusion surgery were cultured separately in vitro. Conditioned media were collected after 48 and 96 h of culture in serum-free Minimum Essential Medium (MEM). Profiling of the cytokines was conducted using pooled media. Conditioned medium from each patient was also tested in human bone marrow stem cell culture, and incorporation of alkaline phosphatase and 3H-thymidine incorporation was evaluated. RESULTS: Of the 18 cytokines screened, 12 were found to be positive, but only eotaxin, IP-10, Rantes IL-6 and IL-8 seemed to be present at high levels. There was a close correlation between IL-6 and IL-8 levels in the medium (R = 0.90, p < 0.001). When the conditioned media were added to human bone marrow stem cell cultures, cellular proliferation was stimulated (p = 0.02), but alkaline phosphatase activity remained unchanged. Cellular proliferation correlated negatively with IL-6 levels (R = -0.44, p = 0.04). INTERPRETATION: Intervertebral discs secrete certain cytokines into the medium when cultured in vitro, and conditioned media from cultured intervertebral discs stimulate proliferation of bone marrow stem cells.     

Mesenchymal stem cell content of human vertebral bone marrow

Mesenchymal stem cells (MSCs) are capable of down-regulating alloimmune responses and promoting the engraftment of hematopoietic stem cells. MSCs may therefore be suitable for improving donor-specific tolerance induction in solid-organ transplantation. Cells from cadaveric vertebral bone marrow (V-BM), aspirated iliac crest-BM, and peripheral blood progenitor cells were compared. Cells were characterized by flow cytometry and colony assays. MSCs generated from V-BM were assayed for differentiation capacity and immunomodulatory function. A median 5.7 x 10(8) nucleated cells (NCs) were recovered per vertebral body. The mesenchymal progenitor, colony-forming unit-fibroblast, frequency in V-BM (11.6/10(5) NC, range: 6.0-20.0) was considerably higher than in iliac crest-BM (1.4/10(5) NC, range: 0.4-2.6) and peripheral blood progenitor cells (not detectable). MSC generated from V-BM had the typical MSC phenotype (CD105(pos)CD73(pos)CD45(neg)CD34(neg)), displayed multilineage differentiation potential, and suppressed alloreactivity in mixed lymphocyte reactions. V-BM may be an excellent source for MSC cotransplantation approaches.     

永生化人骨髓基质干细胞系的生物学特征

[目的]明确永生化人骨髓基质干细胞系MSCxj的生物学特征.[方法]MSCxj细胞在连续体外培养过程中,相差显微镜及透射显微镜观察细胞的形态特征;计算群体倍增时间,测定细胞增殖周期,计算克隆形成率等生长增殖特征并与原代人骨髓基质干细胞BMSCs-2比较.[结果]细胞形态为成纤维细胞样,细胞器发达;群体倍增时间40.8 h,较BMSCs-2的43.82 h短,MSCxj生长增殖活跃;MSCxj克隆形成率10.9%,BMSCs-2为11.5%,具有克隆形成能力.[结论]MSCxj细胞保持了人骨髓基质干细胞的生物学特征.     

Importance of bone marrow cell dose in bone marrow transplantation

The importance of the size of the infused marrow cell dose (MCD) was investigated in 274 patients undergoing allogeneic BMT between 1975 and 1990. Among those, 65 had acute myelogenous leukemia (AML), 79 acute lymphoblastic leukemia (ALL), 58 chronic myelogenous leukemia (CML) and 25 severe aplastic anemia (SAA). MCD was analyzed in bivariate and multivariate analysis together with 6 other clinical factors. In multivariate analysis a low MCD was significantly associated with increased incidence of acute graft-versus-host disease (GvHD) in all patients (p = 0.005) and in ALL patients (p = 0.02) whereas in CML a high dose was instead correlated to acute GvHD. A low MCD was also correlated to an increased incidence of symptomatic cytomegalovirus (CMV) infection (p = 0.001). A low MCD was also correlated to death in acute GvHD in all patients (p = 0.01) and to a poor survival in all patients (p = 0.04) (AML, p = 0.07).     

人骨髓基质干细胞与表面置换珊瑚羟基磷灰石培养的定向诱导分化

目的观察人骨髓基质干细胞（hMSC）与表面置换的珊瑚羟基磷灰石（SCHA）体外培养的细胞粘附、增殖和分化,寻找理想的骨修复材料。方法海南天然滨珊瑚在特定温度和压力下部分水热反应,制成表面置换的珊瑚羟基磷灰石,将SCHA薄片与人骨髓基质干细胞体外培养,经诱导后于4、8、12、16d分别用荧光显微镜和扫描电镜（SEM）观察细胞活性、粘附和分化过程。结果SCHA保留原有的珊瑚贯穿多孔的三维结构。荧光显微镜可见hMSC在SCHA的表面和孔道内生长良好,第16d达最高水平;电镜显示细胞粘附良好,分化为成骨细胞,分泌大量胶原纤维,可见合成的钙结节。结论hMSC在表面置换的珊瑚羟基磷灰石内粘附、增殖、分化良好,两者具有较好的生物相容性,SCHA是一种良好的骨组织工程支架材料。     

Chemotransport contributes to the effect of oscillatory fluid flow on human bone marrow stromal cell proliferation

Mechanical loads produce a diverse set of biophysical signals that may regulate bone cell activity, but accumulating evidence suggests that interstitial fluid flow is the primary signal that bone cells perceive. Because we previously demonstrated that oscillatory fluid flow increases human bone marrow stromal cell proliferation, we investigated the contribution of fluid shear stress and chemotransport, two stimuli induced by interstitial fluid flow. Alterations in flow rate at a constant peak shear stress were associated with decreases in oscillatory fluid flow‐induced marrow stromal cell proliferation, while variations in peak fluid shear stress had no significant effect. Modulation of marrow stromal cell proliferation by flow rate may be attributed to changes in the release of ATP and intracellular calcium signaling. We found that if the flow rate is decreased while maintaining a constant peak fluid shear stress, marrow stromal cells release less ATP into the extracellular environment. Moreover, as the flow rate decreased fewer cells respond to fluid flow with an increase in intracellular calcium concentration. These data suggest that chemotransport is a prerequisite for marrow stromal cells to respond to interstitial fluid flow. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:918–924, 2008     

人骨髓基质干细胞体外诱导培养的新方法研究

目的为骨组织工程的临床应用提供一种新的人骨髓基质干细胞(BMSCs)培养方法,以满足细胞培养过程中对各类细胞因子的需求,同时尽量减少细胞培养过程中动物源性抗原物质的引入。方法采用10%富血小板血浆(PRP)替代动物血清配比高糖DMEM培养基,体外诱导培养(50μg/mL抗坏血酸、10-8mol/L地塞米松、10-3mol/Lβ-甘油磷酸钠)人BMSCs,快速扩增后,倒置相差显微镜、扫描电镜观察各组细胞形态及细胞增殖情况。ALP染色与钙结节染色等方法对细胞进行生物学特性检测。结果人BMSCs24h开始贴壁,7d左右细胞融合。诱导培养后细胞能较快地扩增;ALP染色与钙结节染色结果显示细胞具有良好的成骨细胞生物学特性。结论以自体PRP替代动物血清体外诱导培养人BMSCs是一种良好的培养方法,所培养的细胞数量及其生物学特性能快速达到临床应用的需求。     

Assessment of mixed lymphocyte reactivity in human bone marrow cell cultures

The use of monoclonal anti-T-cell antibodies has been proposed as a means of eliminating T cells from bone marrow inocula, and thereby avoiding graft-versus-host reactions following transplantation. Since the mixed lymphocyte reaction (MLR) provides a measurement of alloreactive mature T cells, we have attempted to apply this assay to bone marrow populations before and after treatment with anti-T-cell antibodies and complement. However, initial studies showed MLR to be very difficult to measure using bone marrow as a responder cell population, and a systematic analysis of the reasons for this difficulty was therefore carried out. The first major problem in performance of standard one-way MLRs using bone marrow responder cells was found to be due to the presence of numerous non-T marrow cells that maintained high levels of background proliferation. Proliferation of these populations was found to be variable during MLR culture, leading to aberrant results. This problem was overcome by removing the rapidly proliferating population of marrow cells either by density centrifugation or by susceptibility to cryopreservation and thawing. A second problem causing variability even after removal of these proliferating cells was found to be due to additional non-T cells in the marrow that responded to soluble mediators produced by peripheral blood lymphocyte stimulator cells during an MLR. Such non-T-cell stimulation was not eliminated by removal of bone marrow T cells, obscuring the results of T cell depletion of the marrow. This problem was overcome by the use of HLA-defined B cell lines as stimulators. A mixture of such lines provided a reliable stimulator source that did not produce soluble mediators capable of stimulating additional marrow cells. These refinements of MLR conditions permit a reproducible and reliable assay of bone marrow MLR, and provide a means for assessment of elimination of such alloreactive cells by monoclonal antibodies and complement.     

Stimulatory effect of ipriflavone on formation of bone-like tissue in rat bone marrow stromal cell culture

The effects of ipriflavone (IP) (10^–5 M) on bone formation were studied in stromal cells from the femoral bone marrow of young adult rats cultured for 21 days in the presence of -glycerophosphate and dexamethasone. Stereoscopic microscopy showed nodule formation after 14 days of culturing, and both the number and the size of the nodules increased with time. The alizarin-red-stained calcified area in the nodules in the IP group was nearly 4 times as large as that in the control after 21 days. Light and electron microscopy revealed the presence of many osteoblast-like cells with developed rough endoplasmic reticulum and Golgi apparatus in the nodules in the control group after 14 days, and a collagenous fibril network was seen among the cells. After 21 days, calcification of the dense collagenous fibril network and bone matrix-like tissue were observed in many nodules, resulting in the formation of bone-like tissue containing osteocyte-like cells. In the IP group, the collagenous fibril network area in the nodules was greater than that in the control after 14 days, and a further increase in both the dense collagenous fibril network area and calcified bone-like tissue area was observed after 21 days. These findings indicate that IP stimulates bone-like tissue formation in the rat bone marrow stromal cell culture, suggesting that the promotion of collagen production by osteoblasts is involved in the stimulation of bone-like tissue formation by IP.     

Functional assessment of T cell depletion from bone marrow prior to therapeutic transplantation using limiting dilution culture methods

We evaluate the usefulness of limiting dilution culture methods in assessing the extent of T lymphocyte depletion from bone marrow inocula, prior to transplant, using either ex vivo antibody/complement-mediated depletion or immunotoxin treatment. Complement-mediated depletion using anti-Leu-1 antibody was shown to result in a consistent decline of 99%-99.9% in the frequencies of T cells able to proliferate in mitogen-stimulated, interleukin-2 (IL-2)-supplemented cultures. Equivalent declines were demonstrated in frequencies of "helper" T cells able to respond to mitogen by making IL-2, and in "killer" T cells able to give rise to clones of cytotoxic effectors. In most experiments, a second cycle of anti-Leu-1 + complement treatment did not further diminish the fraction of proliferating cells, although T cells able to secrete IL-2 were additionally depleted following a second cycle of antibody and complement. The limiting dilution methods were found to be at least as sensitive as flow cytometric (FACS) methods for detecting residual T cell contamination after protocols involving complement-mediated lysis, and superior to FACS analysis for protocols involving T cell depletion by a ricin A chain-anti-T101 immunotoxin, in which treated T cells suffer functional impairment and eventual death after exposure to immunotoxin, but remain phenotypically detectable during FACS analysis. Although limiting dilution methods do not provide data as rapidly as FACS analyses, they do not require a cytofluorimeter, provide equal or greater sensitivity, and can assess functional impairment, for both helper and killer T cell sets, even in situations in which the depletion procedure does not lead to immediate cytolysis.     

体外培养对人骨髓间充质干细胞生物学行为的影响

目的了解体外培养对人骨髓间充质干细胞(MSCs)生物学行为的影响。方法MSCs来自5个不同个体。体外培养扩增传代后。取2代细胞6份、3代细胞9份、9代细胞2份进行形态学观察和细胞周期检测;取1代细胞2份、2代细胞3份、3至6代细胞各1份进行刀豆蛋白A(ConA)凝集试验;另取第3、9代细胞各1份,接种于严重联合免疫缺陷小鼠,分别在接种后第1、2、3、4周进行大体观察并取接种部位组织进行显微镜下观察。结果随着体外培养时间增加,细胞增殖变缓,体积变大。S期细胞数增高,G0～G1期细胞数减少。但所有检测细胞均为二倍体。ConA试验均未发现细胞凝集。不同培养时间细胞皮下接种均未见有炎性细胞浸润,但局部有胶原组织形成,至第4周消失。结论长期体外培养,细胞增殖能力降低,但并不会引起培养细胞发生恶性变。     

Induction of bone marrow mesenchymal stem cell chondrogenesis following short‐term suspension culture

The objective of this study was to evaluate mesenchymal stem cell (MSC) chondrogenesis following incubation in chondrogenic suspension cultures from which single cells were obtained. MSCs were maintained in suspension over a nonadherent surface for 3 days, dissociated into a suspension, and then evaluated for chondrogenesis in agarose in the presence or absence of transforming growth factor beta (TGFβ). In a second experiment, MSCs from suspension culture were returned to monolayer expansion for 2 days prior to testing for chondrogenesis. In both cases, undifferentiated MSCs were evaluated as controls. Suspension culture alone did not stimulate chondrogenesis. Suspension followed by expansion stimulated a four‐ to ninefold increase in extracellular matrix (ECM) synthesis in TGFβ‐free cultures, a finding that was attributed to an increase in viable MSCs that secreted a proteoglycan‐rich ECM. Gene expression of aggrecan and type II collagen increased with suspension culture, but decreased with postsuspension expansion. Therefore, stimulation of ECM synthesis without additional TGFβ exposure could not be attributed to an enhancement of chondrogenesis with monolayer culture. ECM synthesis of suspension/expansion‐conditioned MSCs without additional TGFβ exposure was less than samples maintained in TGFβ throughout the differentiation culture. Based on these findings, a better understanding of factors associated with early‐stage chondrogenesis and MSC differentiation to a highly active phenotype may lead to improved methods for stimulating chondrogenesis during short‐term culture. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:26–32, 2011