实验性骨折愈合中骨生成细胞的体视学分析及电镜观察

应用电子计算机图像分析处理系统和透射电镜技术对实验性骨折后早期不同时间的大鼠胫骨外骨痂内骨生成细胞进行体视学分析和超微结构观察。发现：骨折后早期外骨痂内骨生成细胞以向软骨细胞转化为主，其转化速率较快，软骨骨痂形成迅速；骨折３周以后，骨生成细胞则逐渐向成骨细胞转化，其转化速率较慢。提示：进一步探讨骨折愈合过程中，骨生成细胞分化方向性的生物学机制，加速其向成骨细胞转变，对促进骨折愈合、防止延迟性骨愈合或骨不连的发生，有重要意义。

骨形态生成蛋白-4与17β-雌二醇诱导MBA-1细胞分化的研究

目的探讨BMP-4与17β-雌二醇诱导MBA-1细胞分化的作用。方法用骨形态生成蛋白-4(BMP-4)诱导MBA-1细胞15～22d后进行HE染色观察细胞形态,用1型胶原染色、矿化结节染色鉴定成骨细胞功能;用BMP-4及17β-雌二醇干预MBA-1细胞后,用RT-PCR检测骨钙素的变化。结果①MBA-1细胞可向成骨细胞或脂肪细胞分化;②在无BMP-4或17β-雌二醇干预的情况下,MBA-1细胞在自身分化成熟过程中伴有骨钙素的表达增加;③经BMP-4与17β-雌二醇干预后,MBA-1细胞骨钙素的表达明显增加。结论MBA-1细胞可能具有多系分化潜能,BMP-4与17β-雌二醇可上调其骨钙素表达,诱导MBA-1细胞向成骨细胞方向分化。

人血源性间充质干细胞培养与体外成骨研究

目的建立分离、培养成人外周血来源的间充质干细胞(mesenchymalstemcells,MSCs)的方法,观察成人血源性MSCs体外成骨潜能。方法抽取成年志愿者外周静脉血30份,每份15ml。用淋巴细胞分离液(密度为1.077g/ml)梯度离心,取单个核细胞在含有20%胎牛血清的α-MEM培养液中培养扩增MSCs,流式细胞仪分析MSCs表型。第2次传代培养时在培养液中添加成骨诱导因子(地塞米松、β-磷酸甘油、维生素C和1,25二羟维生素D3)。第5代传代细胞检测碱性磷酸酶、型胶原(collagentype,Col)、骨钙素(osteocalcin,OC)和骨粘连蛋白(osteonectin,ON)表达,连续培养1个月后检测钙结节形成情况。结果成人外周血中存在MSCs,集落形成率为0.27±0.22/106单个核细胞,表达CD44、CD54、CD105和CD166,而不表达与造血系相关的CD14、CD34、CD45及内皮细胞特异的CD31。地塞米松、β-磷酸甘油、维生素C和1,25二羟维生素D3可作用于传代培养的血源性MSCs,表现为碱性磷酸酶染色阳性细胞增多,逆转录-聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)产物电泳条带显示ColI、OC和ON表达,盐酸四环素荧光标记证实有钙结节形成。结论建立的成人MSCs分离、培养条件可分选出外周血贴壁生长的细胞中一组独特的细胞群。成人血源性MSCs具有较强的体外成骨潜能,可望成为一种新的骨组织工程种子细胞。

小夹板外固定与钢板内固定材料置入对骨折断端成骨活性的影响(英文)

背景: AO技术存在许多的缺陷,如"应力遮挡"产生的负面效应等。近年来国内外学者认为弹性固定法最合理,是最有利于骨折愈合的治疗理念。目的:观察小夹板外固定对兔长管状骨骨折断端成骨活性的影响,并与钢板内固定材料置入方法比较。设计:随机对照动物实验。单位:湖北中医学院骨伤科研究所。材料:实验于 2006-04/2007-04 在湖北中医学院骨伤实验室完成。30只家兔随机分成小夹板固定组、钢板固定组,每组15只。自制小夹板,由具有较好弹性的杉树皮制成。分前后、内外侧四块夹板,夹板上宽下窄,在前后侧夹板靠近胫骨结节部刺一小孔。钢板由江苏金鹿集团医疗有限公司提供。方法:在左胫骨中下1/3处造成3mm骨缺损横行骨折模型,小夹板固定组用石膏固定5d后换成小夹板外固定,钢板固定组用4孔钢板内固定。术后 14,24,34 d 时分批处死各组动物,通过肉眼观察骨折处骨痂生长情况,并观察骨折愈合过程中骨痂组织形态学及骨生成细胞情况。主要观察指标:不同时期兔胫骨骨痂肉眼观察,骨痂组织形态学和骨生成细胞情况。结果:小夹板固定组骨痂形成早,早期成骨细胞丰富且活跃,34 d 时骨折全部骨性连接。钢板固定组:14d时骨折端见少量的纤维骨痂,仍有肉芽组织,24d时见少量的软骨连接,34 d 时骨痂已跨过骨折端,但未完全连接。小夹板外固定组与钢板固定组比较,骨折各期形成的骨痂量多,骨折愈合快结论:小夹板外固定能促进骨折处成骨细胞的分化增殖和血肿的吸收、骨痂的钙化、骨小梁的生长改建。

Preliminary Study on Biological Properties of Adult Human Bone Marrow-derived Mesenchymal Stem Cells

Objective： To establish a method of culture and expansion of adult human bone marrow-derived MSCs in vitro and to explore their biological properties. Methods： Mononuclear cells were obtained from 5 mL adult human bone marrow by density gradient centrifugation with Percoll solution. Adult human MSCs were cultured in Dulbecco＇s Modified Eagle＇s Medium with low glucose （LG-DMEM） containing 10% fetal calf serum at a density of 2×10^5 cell/cm^2. The morphocytology was observed under phase-contrast microscope. The cell growth was measured by MTT method. The flow cytometer was performed to examine the expression of cell surface molecules and cell cycle. The ultrastructure of MSCs was observed under transmission electron microscope. The immunomodulatory functions of MSCs were measured by MTT method. The effects of MSCs on the growth of K562 cells and the dynamic change of HA, IV-C, LN concentration in the culture supernatant of MSCs was also observed. Results： The MSCs harvested in this study were homogenous population and exhibited a spindle-shaped fibroblastic morphology. The cell growth curve showed that MSCs had a strong ability of proliferation. The cells were positive for CD44, while negative for hematopoietic cell surface marker such as CD3, CD4, CD7, CD13, CD14, CD15, CD19, CD22, CD33, CD34, CD45 and HLA-DR, which was closely related to graft versus host disease. Above 90% cells of MSCs were found at G0/G1 phase. The ultrastructure of MSCs indicated that there were plenty of cytoplasmic organelles. Allogeneic peripheral blood lymphocytes proliferation was suppressed by MSCs and the inhibition ratio was 60.68% （P〈0.01）. The suppressive effect was also existed in the culture supernatant of MSCs and the inhibition ratio was 9.00% （P〈0.05）. When lymphocytes were stimulated by PHA, the suppression effects of the culture supernatant were even stronger and the inhibition ratio was 20.91% （P〈0.01）. Compared with the cell growth curve of the K562 ceils alone, the K562 ceils cocultured with MSCs grew slowly and the exponential phase of growth wasn＇t significant. Seeing from the concentration curve, as time passed, the concentration of HA increased quickly, while those of IV-C and LN didn＇t change much. Conclusion： The method for culture and expansion of adult human bone marrow-derived MSCs in vitro has been successfully established in this study. MSCs were a homogenous population that had unique growth phenotype and multilineage potential. Preliminary study proved that it had the abilities of immunomodulatory function, antitumor, hematopoietic supporting and could act as seed cell of tissue engineering.

Study on biocompatibility of PDLLA/HA/DBM with co-cultured human osteoblasts in vitro

Objective: To evaluate the osteocompatibility of D, L-polylactic/hydroxyapatite/decalcifying bone matrix (PDLLA/HA/DBM), and compare with PDLLA and DBM. Methods: Human primary osteoblasts isolated from the femoral head of patients were inoculated onto PDLLA/HA/DBM, PLA and DBM respectively. The proliferation rate and collagen Ⅰ expression were detected. The interface between biomaterial and osteoblasts was investigated with phase contrast microscopy and electron scanning microscopy. Results: Best proliferation rate was observed with the PDLLA/HA/DBM and followed by DBM and PLA, suggesting that PDLLA/HA/DBM satisfying most requirements for the cultivation of human osteoblasts. Scanning electron microscopy showed the morphology of osteoblasts was correlated with the proliferation data. The cells, well spread and flattened, were attached closely on the surface of biomaterial with an arched structure and had normal morphology. The extracellular collagenous matrixs covered the surface of biomaterial and packed the granules of biomaterial. Conclusion: PDLLA/HA/DBM can form osteointerface early and have a good biocompability.

Effect of gamma irradiation on nuclear factor-kappa B in cultured bone marrow stromal cells

Objective: To explore the effect of gamma irradiation on nuclear factor-kappa B in cultured bone marrow stromal cells. Methods: Immunocytochemistry, Western blot and electrophoretic mobility shift assay (EMSA) were used. Results: The expression of NF-kB in cultured mouse bone marrow stromal cells (BM-SCs) on the level of protein was elevated after exposure to 60Co in the dosage of 8. 0 Gy with the use of im-munocytochemistry and Western blot. The activity of nuclear factor-kappa B in cultured BMSCs was significantly increased after exposure to gamma irradiation by using EMSA. The activity peak was at the 4th h after irradiation. Conclusion: Our results suggest that the activation of nuclear factor-kappa B in the BMSCs after irradiation may be involved in the protection of BMSCs against apoptosis and in the recovery of hematopoiesis after radiation.

Effect of BMPs on hematopoietic injury of acute radiation sickness in mice

Objective: To investigate the effect of bone morphogenetic proteins (BMPs) on hematopoietic injury of acute radiation sickness in mice. Methods: Mice were subjected to whole-body 60Co γ ray irradiation, then bpBMP was put into spatium intermusculare or rhBMP-2m, PBK/ hBMP-2 -NIH3T3 cells were injected into abdominal cavity. The effect of BMPs on hematopoiesis including some hematological parameters, the survival rate of 30 d and formation of bone marrow CFU-GM colony were detected at postradiation. Results: pbBMP (purified bovine bone morphogenetic protein) increased the formation of bone marrow CFU-GM colony (P<0. 05) on d 10 after irradiation. rhBMP-2m increased the survival rate of mice irradiated by 7. 5 Gys Mice in control group died in 30 days, while 10%, 15% and 35% mice survived when they were injected i. p. with 0. 5 mg, 1. 0 mg and 2. 0 mg of rhBMP-2m respectively. All hematological parameters of treated mice were significantly higher than those of control group (P<0. 01). PBK/ hBMP-2 -NIH3T3 cells were established and transplanted into mice irradiated by 7. 0 Gy γ ray with i. p. . The survival ratio of treated mice was higher than that of negative control group (P<0. 01), and all hematopoietic parameters were increased statistically significantly (P<0. 01). Conclusion: Results indicate that in adult mice, BMPs can recover or treat the hematopoietic injury of acute radiation sickness, the mechanism may be related with repairing of hematopoietic injury.

Microtubular elements of cortex in Euplotes Woodruffi

The cortical ciliature microtubular organelles of the ciliated protozoan Euplotes Woodruffi were analyzed with FLUTAX and anti-a-tubulin antibody. It showed that the cortical cytoskeleton was composed of non-ciliature structure, ciliature structure including adoral zone of membranelles （AZM）, undulating membranes （UM）, frontal-ventral-transverse cirri （FVTC）, caudal cirri （CC）, dorsal kineties, as well as base-associated microtubules. The silverline system is composed of longitudinal and transverse microtubules, concave structure appeared on the cell dorsal side, the base of DK containing rosette-like skeleton structure. All these suggested that the non-ciliature structure, ciliature structure of Euplotes be quite different from that of other species of ciliate, the silverline-system be true pare of cortical microtubute cytoskeleton in Euplotes, rosette structure be part of the basecytoskeleton.

Morphological Evaluation of Adhesion and Proliferation of Osteoblast Like Cells Grown on Gelatin/Genipin Scaffold

This work focusing on studying the biocompatibility and the effect of gelatin porous scaffold on the characteristics of human osteoblast like cells, including proliferation, adhesion, scaffold-cell interaction and its potential to induce bone regeneration. Osteoblast like cells were seeded on gelatin/genipin scaffolds for 7, 14 and 21 days. Cell proliferation assay, light microscopy, transmission electron microscopy and high resolution scanning electron microscopy were carried to evaluate cell viability, cell adhesion and the production of extracellular matrix. Cell proliferation assay showed a high biocompatibility of the material. High resolution scanning electron microscopy and light microscopy showed a strong adhesion of MG63 ceils on the surface of gelatin scaffold and high penetration in the macroporosities of the material. TEM analysis showed an intense production of extracellular matrix protein. In vitro analysis indicated a good biocompatibility of the scaffold and presents it as a potential candidate material for tissue engineering.

Effects of basic fibroblast growth factor on biological characteristics of osteoblasts

Objective: To elucidate the effects of exogenous basic fibroblast growth factor ( bFGF ) on biological characteristics of rat osteoblasts cultured in vitro.Methods: The osteoblasts isolated from a Sprague-Dawley rat and cultured in vitro were treated with different concentrations of bFGF (5-50 ng/ml) respectively. At 24 hours after treatment, the proliferating cell nuclear antigen was measured with immunocytochemistry, alkaline phosphatase ( ALP) activity was determined and the expression of transforming growth factor beta 1 (TGF-β1) was detected to observe the effects of bFGF on growth and differentiation of osteoblasts. Results: bFGF ( 5-50 ng/ml ) could obviously promote the growth of osteoblasts. The intracellular expression of TGF-β, mRNA increased significantly, but the intracellular ALP content decreased.Conclusions: bFGF can obviously stimulate the proliferation of osteoblasts and promote the synthesis of TGF-β1, but cannot promote the differentiation of osteoblasts.

Effects of recombinant human basic fibroblast growth factor on cell proliferation during mandibular fracture healing in rabbits

Objective:: To investigate the effects of recombinant human basic fibroblast growth factor (rhbFGF) on the cell proliferation during mandibular fracture healing in rabbits. Methods: The complex of rhbFGF and bovine type I collagen was implanted into the mandibular fracture site under periosteum of the animal. The whole mandible was harvested at 7, 14, 28, 56 and 84 days respectively after operation. The expression of proliferating cell nuclear antigen (PCNA) in callus was examined with immunohistochemical staining. Results: PCNA-positive cells in callus in the rhbFGF-treated group on days 7 and 14 were more than that in the control group (P< 0.01 ). Conclusions: It indicates that rhbFGF can stimulate cell proliferation during mandibular fracture healing in rabbits.

Combinatorial effects of NaomaiYihao Capsules(脑脉一号胶囊) and vascular endothelial growth factor gene-transfected bone marrow mesenchymal stem cells on angiogenesis in cerebral ischemic tis sues in rats

OBJECTIVE:To investigate the combinatorial effects of Naomai Yihao(NMYH) Capsules(脑脉一号胶囊) and vascular endothelial growth factor(VEGF) gene-transfected bone marrow mesenchymal stem cells(BMSCs) on angiogenesis in cerebral ischemic tissues in rats and the mechanism.METHOD:BMSCs were isolated and cultured from bone marrow by an adherence method.Then,BMSCs were transfected with the eukaryotic expression plasmid pEGFP-VEGF 165 by positive ionic liposome transfection.A rat model of middle cerebral artery occlusion(MCAO) was established.Rats were allocated to six groups:model,BMSC,VEGF gene-transfected BMSC transplantation(BMSC/VEGF),NMYH,combined NMYH and BMSC/VEGF(combined treatment group) and sham operation groups.The behavioral rating score(BRS) of rat and the expression of CD34 and VEGF in brain tis sue were measured by immunohistochemistry on days 7,14 and 21 after reperfusion.Angiogenesi was observed and evaluated with laser scanning confocal microscopy.RESULTS:The BRS of rats in NMYH,BMSC transplan tation and combined treatment groups was significantly lower than that of the model group(P< 0.001),with no significant difference between NMYH and transplantation groups(P=0.619).The expression of CD34 andVEGF in NMYH,transplanta tion and combined treatment groups increased(P< 0.001),with a significant difference between NMYH and transplantation groups(P<0.001).The blood vessel area in NMYH,transplantation and com bined treatment groups was significantly increased(P<0.05),without a significant difference between NMYH and transplantation groups(P=0.873).CONCLUSIONS:VEGF gene-transfected BMSCs im prove angiogenesis in the cerebral ischemic area NMYH Capsules promote angiogenesis in MCAO rats treated with BMSC transplantation,which show an improved BRS.The mechanism of angio genesis may be related to up-regulation ofVEGF ex pression.

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

Objective:Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques.The aim of this study is to investigate the effect of connective tissue growth factor(CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells(MSCs).Methods:A CTGF-expressing plasmid(pCTGF) was constructed and transfected into MSCs.Then expressions of bone morphogenesis-related genes,proliferation rate,alkaline phosphatase activity,and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs.Results:Overexpression of CTGF was confirmed in pCTGF-MSCs.pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs(P<0.05).CTGF induced a 7.5-fold increase in cell migration over control(P<0.05).pCTGF transfection enhanced the expression of bone matrix proteins,such as bone sialo-protein,osteocalcin,and collagen type I in MSCs.The levels of alkaline phosphatase(ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0-and 3.0-fold higher than those of MSCs cultured in OS-medium,significantly higher than those of mock-MSCs and normal control MSCs(P<0.05).Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules.Conclusion:Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs,and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.