细胞间直接接触诱导BMSC软骨分化的初步研究

目的探讨细胞间直接接触是否能够单独诱导骨髓基质干细胞(Bone Marrow Stromal Cells,BMSC)软骨分化。方法体外培养扩增猪BMSC与关节软骨细胞,将1.0×106的细胞总量以2000r/min离心8min,制成细胞团块,即Pellet培养。实验分为5组,实验组:经0.5%多聚甲醛固定的软骨细胞与BMSC以1∶1混合;对照组1:同等数量的未经多聚甲醛固定的软骨细胞与BMSC1:1共培养制成Pellet;对照组2:同等数量的单纯软骨细胞制成Pellet;对照组3:同等数量的单纯BMSC制成Pellet;对照组4:同等数量的单纯经多聚甲醛固定的软骨细胞制成Pellet。每3天换液一次,每组3例。培养4周后,以大体观察、组织学、免疫组织化学、RT-PCR等方法对Pellet进行全面评价。结果培养4周后,实验组形成的Pellet,明显缩小,形状略不规则,颜色灰暗,柔软且没有弹性,组织学检测提示主要为纤维性成分及死细胞样结构,Safranin O染色及Ⅱ型胶原免疫组化均为阴性,RT-PCR检测未表达Ⅱ型胶原。对照组1和对照组2均形成圆盘状组织块,表面光滑,触之有一定弹性,组织学检测软骨陷窝形态规则,Safranin O染色及Ⅱ型胶原免疫组化均有阳性表达,RT-PCR检测Ⅱ型胶原高表达。对照组3的组织块明显收缩,呈褐色,无弹性。对照组4,细胞松散,不能形成Pellet。对照组3和4的各种软骨特异性相关检测均为阴性。结论细胞间直接接触不能够单独诱导BMSC向软骨分化,不是软骨细胞与BMSC混合共培养中发挥诱导作用的主要因素。     

骨髓基质细胞在藻酸盐凝胶中的生物学行为

目的：探讨骨髓基质细胞（Bone Marrow Stromal Cells,MSC)在海藻酸盐（Alginate)中的生物学行为，为在组织工程研究中选用海藻酸盐做为骨髓基质细胞的载体提供理论依据。方法：将地塞米松诱导后的骨髓基质细胞种植在1%海藻酸盐中，以HE染色，BMP-2免疫组化染色，扫描电镜观察骨髓基质细胞在凝胶中的生物学行为。结果：凝胶中细胞呈“悬浮”生长状态，可见到细胞分裂和核分裂相；细胞在凝胶中增殖，4d后可以见到基质分泌；海藻酸盐中培养组与常规培养组MSC的BMP-2阳性表达率无显著差异。结论：在海藻酸盐中骨髓基质细胞的生物学行为表现良好。此特征适宜作为骨组织工程的载体。     

脱细胞真皮基质作为Beagle犬骨髓基质细胞移植载体的实验研究

目的探讨脱细胞真皮基质（Acellular dermal matrix,ADM）作为Beagle犬骨髓基质细胞（Bone marrow stromalcells,BMSCs）移植载体的可行性。方法将BMSCs复合到ADM载体上,体外观察ADM与BMSCs的生物相容性：ADM—BMSCs复合物植入裸鼠皮下,以单纯ADM为对照组,分别于术后4周和8周进行大体标本观察及组织学分析。结果体外观察ADM与BMSCs的生物相容性良好。裸鼠皮下植入实验显示,实验组4周后ADM部分降解吸收,BMSCs生长良好,出现新生组织,部分形成类骨样结构;8周后,ADM大部分吸收,形成大量的新生组织和类骨样结构;对照组新生组织形成较少,ADM支架材料降解速度与实验组类似。结论ADM可作为Beagle犬BMSCs的移植载体。     

大鼠骨髓基质细胞的体外培养

目的：观察大鼠骨髓基质细胞在体外培养的条件,为骨组织工程学研究奠定一定实验基础。方法：取幼年SD大鼠,处死后分离骨髓,原代培养骨髓基质细胞,传代后分别观察其生长特性,绘制生长曲线,测定分裂指数和贴壁率。结果：大鼠骨髓基质细胞在第7代以前生长形状相对稳定,生长曲线相似;第4d分裂指数最高,为16％;传代后12h贴壁率最高,为90％。结论：本实验方法中,骨髓基质细胞在体外培养条件下,早期生长性状稳定,增殖速度快,适应性强,可作为骨组织工程学研究的种子细胞。     

激素诱导骨髓基质细胞成脂分化的实验研究

观察激素诱导骨髓基质细胞分化成脂肪细胞的作用,揭示激素性股骨头缺血性坏死的病理学机制。方法　采用原代骨髓基质细胞体外培养技术,取小鼠双侧股骨骨髓细胞,通过细胞贴壁、分离获取骨髓基质细胞。以地塞米松作为脂肪细胞分化诱导剂处理细胞,苏丹Ⅲ染色,光镜下脂肪细胞计数。结果以递增浓度（０、１× １０~－９、１× １０~－８、１× １０~－７、１× １０~－６ ｍｏｌ／Ｌ）地塞米松处理细胞并培养 ２１天后, １×１０~－６ ｍｏｌ／Ｌ组中诱导分化生成的脂肪细胞最多,对照组中脂肪细胞最少,各组脂肪细胞随地塞米松浓度增大而增多,具有一定的剂量依赖性。实验组细胞经高浓度（１×１０~－７ ｍｏｌ／Ｌ）地塞米松处理并培养１２天后,其细胞内碱性磷酸酶活性明显低于对照组,两者差异有非常显著性意义（Ｐ＜０．０１）。结论　地塞米松能够直接诱导骨髓基质细胞大量分化成脂肪细胞,其成骨分化减少,这可能是大剂量应用激素后股骨头骨髓内脂肪组织增多,骨内压升高,导致骨微循环障碍,同时骨修复过程缓慢,从而发生股骨头缺血性坏死的原因。     

bFGF Administration Lowers the Phosphate Threshold for Mineralization in Bone Marrow Stromal Cells

Basic fibroblast growth factor (bFGF) is a potent mitogen and acts as an autocrine/paracrine factor for osteoblasts. Long-term administration of bFGF in vivo increases osteoblast number and stimulates matrix formation, but induces hypophosphatemia and impairs matrix mineralization. The goal of this study was to examine the interaction between bFGF and low levels of organic phosphate in an effort to better understand the possible long-term therapeutic effects of bFGF. These data show that in vitro administration of bFGF accelerates the calcification process and lowers the phosphate threshold needed for successful bone nodule formation. This correlates well with the observed upregulation of mRNA production for alkaline phosphatase and osteocalcin at day 7. These findings help elucidate the mechanisms of bFGF action on bone marrow stromal cell differentiation and mineralization and indicate that the delay in mineralization observed in vivo may not be caused by decreased phosphate availability alone.     

Perfusion Enhances Functions of Bone Marrow Stromal Cells in Three-Dimensional Culture

Perfusion of medium through three-dimensional (3D) collagen sponges enhanced viability and function of cocultivated marrow stromal and hematopoietic cell lines. Cells of the murine bone marrow stromal cell line GPIa were cultured in novel 3D collagen sponges, made from pepsin-digested bovine skin. Static cultures of sponges were maintained in dishes with media changes every other day. Perfused sponges were contained in a glass column with medium flow set at 1.3 mL/min. In some sponges, the 32D cl3 c-fms^m (CRX-1) hematopoietic progenitor cell line was added 7 days after GPIa cells. At 7 and 16 days, light microscopic evaluation showed poor viability of cells in static sponge cultures. In perfused sponge cultures, there was greater cellularity throughout the sponge and abundant accumulation of metachromatic extracellular matrix surrounding GPIa cells. Chondroitin 6-sulfate and heparan sulfate were identified as components of the matrix by immunohistochemical methods. DNA synthesis was evaluated by 15-h exposure of cultures to bromodeoxyuridine (BrdU), with subsequent immunohistochemical localization with monoclonal anti-BrdU antibody. Cells positive for BrdU were identified at the outer surfaces of both static and perfused sponges; however, positive cells were also seen throughout the internal areas of the sponges that were perfused. These results suggest that better nutrient exchange occurred in perfused sponges. In static cocultures of GPIa and CRX-1 cells, there was no detectable viability of the IL-3–dependent CRX-1 cells; however, under perfused conditions, CRX-1 cells flourished within the sponges as documented by BrdU incorporation. Thus, medium perfusion enhanced GPIa stromal cell line viability and function in 3D collagen sponge cultures, as demonstrated by BrdU incorporation, matrix production, and support of CRX-1 cells. This novel culture system may be useful for examining the interactions of bone marrow stromal cells with extracellular matrix molecules, soluble and matrix-bound factors, and with other cell types.     

The Role of Cytokines in the Changes in Bone Turnover Following Bone Marrow Transplantation

Osteoporosis is a common disease among patients undergoing transplantation and a loss of bone mass is usually detected after bone marrow transplantation (BMT), particularly during the immediate post-BMT period. Post-BMT bone loss is primarily related to gonadal dysfunction and immunosuppression. Cytokines, especially interleukin 6, play an important role in the pathogenesis of postmenopausal osteoporosis. However, the pathogenetic role of cytokines in post-BMT bone loss is unknown and data on the changes of cytokines in accordance with bone turnover markers are scarce. The aim of this study was to assess the relationship between bone turnover markers and cytokines, which are regularly sampled at peripheral blood and bone marrow before and after allogeneic BMT. This prospective study included two analyses. The first was a study of 46 BMT recipients (M/F 28/18), examining the relationship between bone turnover markers and serum cytokines that were measured before and at 1 week, 2 weeks, 3 weeks, 4 weeks and 3 months after BMT. Serum intact parathyroid hormone was measured before BMT and at 3 weeks after BMT and its relation to other cytokines and bone turnover markers was evaluated. The second analysis was a study of 14 (M/F 9/5) of 46 patients in whom bone marrow plasma cytokines [interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α)] were measured at 3 weeks after BMT. The relationship between bone marrow plasma cytokines and bone turnover markers was studied because bone marrow is the microenvironment where the real changes in bone turnover occur. Serum type I collagen carboxyterminal telopeptide (ICTP), a bone resorption marker, increased progressively until 4 weeks (peak) after BMT and then decreased thereafter. Serum osteocalcin, a bone formation marker, decreased progressively until 3 weeks after BMT and then increased thereafter. Serum IL-6 increased until 2 weeks after BMT and declined thereafter. Serum TNF-a increased until 3 weeks after BMT and declined thereafter. There was a significant positive correlation between serum ICTP and bone marrow IL-6 levels at 3 weeks after BMT, when a marked change in bone metabolism occurs following BMT. However, a correlation between bone turnover markers and bone marrow TNF-aor peripheral blood cytokines was not found. At 3 months after BMT, there was a significant negative correlation between the mean daily steroid dose and the serum osteocalcin level (r=−0.43, p<0.05). The correlation between the mean daily steroid dose and serum ICTP was also significant (r= 0.41, p<0.05). Our data suggest that the progressive increase in bone resorption during the immediate post-BMT period is related to both steroid dose and the increase in bone marrow IL-6, which is a potent stimulator of bone resorption in vivo. Received: 29 November 2000 / Accepted: 1 August 2001     

Expression of BMP-2 by Rat Bone Marrow Stromal Cells in Culture

To investigate the role of bone morphogenetic protein (BMP-2) in ossifying rat bone marrow stromal cell cultures, we determined the population of fibroblast-like stromal cells that expressed BMP-2 immunocytochemically (anti-rhBMP-2 monoclonal antibody), and compared that to alkaline phosphatase (AP) and collagen synthesis formed in culture over a 4-week period in control and dexamethasone-supplemented mineralizing media. In control media, the percentage of BMP-2-positive stromal cells (BMP-2⁺) increased from 12 to 25% within the first 4 days of culture. In mineralizing media, the level of BMP-2⁺ cells was significantly increased (43–44%). The intensity of immunostaining gradually increased with time. The levels of AP were undetectable at 1 week in both control and mineralizing media, but increased gradually over the next 2 weeks and peaked at 3 weeks. ALP levels were significantly greater in cultures grown in mineralizing medium (P < 0.05 at 3 weeks, P < 0.01 at 4 weeks). Collagen synthesis peaked and was significantly greater at 3 weeks (P < 0.05) in cultures grown in mineralizing medium. The levels of AP and collagen synthesis most closely reflected the changes in the percentage of BMP-2⁺ cells from 7 to 28 days. Though these changes may reflect a primary action of BMP-2 on marrow osteoprogenitor-like stromal cells, they do not exclude a mechanism that involves the induction of other members of the BMP family known to stimulate AP and collagen synthesis. We conclude that BMP-2 expression in cultures of fibroblast-like marrow stromal cells is enhanced when those cells are induced to become osteoblasts by exposure to dexamethasone. Received: 30 October 1997 / Accepted: 24 June 1998     

低氧状态下联合培养骨髓基质干细胞及其诱导来源的内皮细胞的实验研究

目的在低氧条件下联合培养兔骨髓基质干细胞(Bone Marrow Stromal Cell,BMSC)及由其诱导而来的内皮细胞(Endothelial Cell,EC),观察并探讨低氧状态及EC对BMSC增殖的影响。方法从兔骨髓中分离获得BMSC,体外培养扩增后取部分向EC诱导。实验分4组:①BMSC常氧培养组;②BMSC低氧培养组;③BMSC+EC常氧联合培养组;④BMSC+EC低氧联合培养组。通过观察细胞的形态、增殖能力等了解低氧条件及EC对BMSC生长的影响。结果 BMSC可诱导为EC,EC与BMSC混合生长良好。MTT法检测显示,低氧培养组的细胞增殖能力明显高于常氧培养组(P<0.01),但相同条件下的BMSC单独培养组和BMSC+EC联合培养组间的细胞增殖能力没有显著性差异(P>0.05)。结论在一定时间内,低氧培养可以促进BMSC的增殖;BMSC来源的EC不能促进BMSC的增殖。     

可注射型生物蛋白胶包埋骨髓基质细胞工程化组织的体外实验

目的构建可注射型生物蛋白胶包埋骨髓基质细胞的工程化组织,体外培养并研究其生物学特性,探讨将可注射型生物蛋白胶作为组织工程支架用于临床的实验基础。方法体外培养浇铸有骨髓基质细胞的生物蛋白胶,通过倒置相差显微镜、激光共聚焦显微镜观察载体内细胞生长及载体降解情况,5-溴脱氧尿苷(5-Bromodeocyuridine,BrdU)掺入标记后免疫组化等方法研究可注射型载体内包埋细胞的增殖情况。结果骨髓基质细胞包埋于生物蛋白胶内能很好地存活并增殖,2d后细胞呈典型的成纤维细胞形态;6d后生物蛋白胶边缘部分开始降解,细胞脱落至培养板;体外培养14d,细胞生长良好,大部分生物胶降解,脱落的细胞增多,贴壁生长的细胞形态正常;3周后生物蛋白胶完全降解。结论生物蛋白胶聚合后包埋的种子细胞能够正常增殖,生物蛋白胶是一种理想的适用于微创方法修复组织的可注射型组织工程培养和移植的支架。     

Repair of the damaged heart by bone marrow cells: from experimental evidence to clinical hope

Heart failure remains the leading cause of death in developed countries in spite of improvements in medical and surgical treatments. However, recent observations in experimental studies that bone marrow cells may repair cardiac tissue have offered renewed hopes for the treatment of heart failure. This optimism is further supported by encouraging results from some clinical trials, although the degree of benefits, the underlying mechanisms, and the cell types involved remain to be elucidated. This review summarizes the relevant experimental and clinical studies supporting the use of bone marrow cells in myocardial repair, as well as the controversies and challenges encountered.     

Assessment of Multipotent Mesenchymal Stromal Cells in Bone Marrow Aspirate From Human Calcaneus

Bone marrow aspirates (BMAs), owing to their innate osteogenic potential, are well-documented supplements to osteoconductive and/or osteoinductive materials. The calcaneal body provides foot and ankle surgeons a convenient harvest site with low morbidity and minimal cost. In the present study, we sought to identify and characterize multipotent mesenchymal stromal cells (MSCs) in BMAs harvested from the human calcaneal body. Ten healthy patients aged 18 to 65 years were enrolled in the present study. BMAs were harvested from the patients without any reported postoperative complications related to the harvest. Cells isolated from all the aspirates were adherent to culture plates and expressed positive MSC surface markers (CD105, CD90, and CD73) and a low level of negative MSC markers (CD34 and CD45). The cells maintained the ability to proliferate and differentiate into cells of mesenchymal lineages. The BMAs from the human calcaneal body offer a healthy source of multipotent MSCs.     

Serum-Mediated Osteogenic Effects of Head Injury on Cultured Rat Marrow Stromal Cells

Central nervous system (CNS) injuries in humans are frequently associated with heterotopic ossification (HO) and with enhanced fracture healing. In search for an experimental HO model we tested sera, from an established rat model of closed head injury (CHI), for their osteogenic effects on rat marrow stromal cells. Most normal rat sera increased cell proliferation not discriminating between osteoprogenitors and other stromal cells. Rats followed longitudinally by sequential blood sampling were bled 24 hours before and 24 hours, 48 hours, and 7 days after CHI. Sera obtained 24 and 48 hours after CHI progressively decreased cell proliferation and specific alkaline phosphatase (ALP) activity compared with pre-CHI sera of the same rats. Sera obtained from these rats, 7 days post-CHI, partially recovered proliferation induction, more than recovering induction of specific ALP activity. A positive correlation between day 11 ALP activity and day 21 mineralization was found under stimulation by pre-CHI sera. However, no correlation was found on stimulation with sera obtained 48 hours after CHI. Correlation between ALP and mineralization partially recovered in cultures exposed to sera obtained 7 days after CHI. In cross-sectional experiments where rats were subjected to single blood sampling, sera of 24 hours and 7 days post-CHI induced proliferation, whereas sera of 48 hours and 14 days post-CHI did not. The results indicate that 48 hours post-CHI, the mitogenicity of sera decreased in both cross-sectional and longitudinal blood sampling. In addition, 48 hours post-CHI, the specific ALP activity increases in cultured marrow stromal cells. Thus, changes in bone-seeking factors, causing serum-mediated osteogenic activity in rats, are expected close on 48 hour post-CNS injury. Received: 25 March 1998 / Accepted: 12 March 1999     

骨髓基质干细胞扩增体系研究

目的 比较三种不同培养体系对人骨髓基质干细胞(h BMSCs)增殖能力、表面标志和分化潜能的影响,探索h BMSCs适合的培养体系。方法 获取h BMSCs,分别用DMEM、DMEM+碱性成纤维细胞生长因子(b FGF)和无动物组份的间充质干细胞培养基(Mesenchymal Stem Cell Medium-animal component free,MSCM-acf)进行培养,反复传代至第6代,分别计数每种培养体系每个代次的细胞得率。取第4~6代细胞,用流式细胞仪对细胞表面标志进行检测,同时对各组第6代细胞分别向成软骨、成脂和成骨方向进行诱导分化。结果 培养至第2代后,MSCM-acf培养体系细胞得率显著大于其他两组;流式细胞分析结果表明,MSCM-acf培养体系表面阳性标志CD73、CD90和CD105均大于99%,总的阴性标志表达小于2%,优于其他两组,尤其是DMEM+b FGF培养体系;三组均保留了多向分化潜能,添加b FGF的培养体系成软骨分化明显优于其他两组,MSCM-acf培养体系成脂和成骨分化优于其他两组。结论 MSCM-acf培养体系可以实现h BMSCs干性维持下的大量扩增,是h BMSCs较为理想的培养体系。