TCP复合BMP-2促骨再生作用的研究

目的探讨TCP复合BMP-2促骨再生作用,为临床BMP-2的应用提供参考。方法利用16只雄性Wistar大鼠建立大鼠颅盖骨临界骨缺损模型,制备TCP支架及TCP复合BMP-2支架,并分别植入到动物模型的骨缺损处,术后12周测定新生骨形成面积并进行比较。结果所有实验动物无一死亡,全部存活,伤口愈合良好,未见感染症状。术后12周,TCP复合BMP-2组骨密度为(475.32±19.20)mg/cm2,而单纯TCP组明骨密度为(124.25±13.64)mg/cm2,两组比较TCP复合BMP-2组的成骨效果明显较TCP组为佳,差异显著有统计学意义(P0.05)。结论所制备的TCP复合BMP-2能改进单纯TCP的促成骨效果,具有良好的成骨活性,促进骨修复。     

肝素化丝素支架作为骨形态发生蛋白-2缓释载体的研究

骨形态发生蛋白-2(BMP-2)的缓释载体一直是骨组织工程中的研究热点.本研究通过化学改性制备了两种肝素化丝素支架,并浸渍吸附BMP-2,研究了BMP-2在不同丝素支架样品上的吸附能力、体外释放性能及其对人骨肉瘤细胞MG-63碱性磷酸酶活性(ALP)的影响.结果表明,肝素化丝素支架对BMP-2具有较强的吸附能力,并能保持其体外缓慢释放性能;MG-63细胞在肝素化支架上生长状态良好,并具有显著的增殖能力,负载BMP-2后的肝素化支架能显著促进MG-63细胞的分化.因此,肝素化丝素支架是一种较理想的BMP-2缓释载体.     

纳米羟基磷灰石/丝素蛋白复合支架材料的降解特性及生物相容性研究

应用共混法制备了纳米羟基磷灰石/丝素蛋白复合支架材料, 通过体外降解和细胞培养实验研究了复合支架材料的降解特性和生物相容性. 体外降解实验结果显示, 复合支架材料具有稳定的降解能力; 在降解过程中, 羟基磷灰石由于与降解液发生钙、磷等离子的交换, 使其结晶得到了进一步生长和完善. 利用细胞计数法、四甲基偶氮唑盐(MTT)比色法和碱性磷酸酶(ALP)活性测定等分析了复合支架材料的生物相容性, 结果表明, MG63细胞在复合支架材料上具有良好的粘附、增殖能力, 并可引起早期的骨分化. 因此, 纳米羟基磷灰石/丝素蛋白复合支架作为骨组织工程的支架材料具有良好的应用前景.     

稀土化合物TbCl3对成骨细胞系MC3T3-E1增殖、分化和矿化功能的影响

在细胞和分子水平上,研究了稀土化合物氯化铽(TbCl_3)对成骨细胞MC3T3-E1增殖、分化及矿化功能的影响。结果表明,细胞水平上,浓度为0.000 1、0.001、0.01、0.1、1和10μmol·L-1的TbCl_3均促进MC3T3-E1细胞的增殖、分化及其矿化功能,然而,当浓度升至为100和1 000μmol·L-1时,TbCl_3表现出抑制作用。分子水平上,浓度为0.000 1和0.1μmol·L-1的TbCl_3明显上调成骨分化相关基因骨形成蛋白2(BMP-2),碱性磷酸酶(ALP),骨涎蛋白(BSP),Ⅰ型胶原蛋白(ColⅠ),骨钙素(OCN)和runt相关转录因子2(Runx2)的表达。浓度为1 000μmol·L-1的TbCl_3则抑制上述成骨分化相关基因的表达。浓度为0.000 1、0.1和1μmol·L-1的TbCl_3促进成骨分化相关蛋白Runx2,BMP-2和OCN的表达;结果显示,低浓度的TbCl_3促进MC3T3-E1细胞的成骨分化及矿化功能,而高浓度TbCl_3则呈现出抑制作用。TbCl_3通过调控Runx2的表达刺激早期成骨分化相关基因BMP-2、ColⅠ和晚期成骨分化相关基因ALP、OCN的表达,从而诱导MC3T3-E1成骨分化。     

与胶原特异性结合的BMP2模拟肽/PLGA 3D打印复合支架的制备及成骨诱导活性

将胶原绑定结构域(CBD)多肽序列与骨形态发生蛋白2模拟肽(BMP2-MP)序列连接制备具有胶原绑定能力的CBD-BMP2-MP, 再将CBD-BMP2-MP与聚丙交酯-乙交酯/胶原(PLGA/COL)3D打印支架相结合, 以支架表面的胶原成分为媒介, 将CBD-BMP2-MP更有效地固定于骨修复材料上, 达到对其进行改性的目的. 利用扫描电子显微镜(SEM)、 电子万能试验机和接触角测量仪对复合支架表面形貌、 力学强度和亲水性等材料学性能进行评价. 用荧光成像法评测 CBD-BMP2-MP及BMP2-MP与支架材料的结合能力. 在各组支架材料表面接种MC3T3-E1细胞进行体外培养, 采用CCK-8、 鬼笔环肽荧光染色、 茜素红染色及qPCR综合评价细胞在材料表面的黏附、 增殖和成骨分化等细胞行为, 研究CBD-BMP2-MP修饰的3D多孔PLGA/COL复合支架的生物学性能. 研究结果表明, 利用3D打印技术制备的多孔支架具有形貌可控的孔隙结构, 为细胞生长创造更有利的细胞微环境, 支架表面胶原成分的加入提高了支架材料的亲水性, 同时对支架材料本身的力学性能无任何影响, 提高了复合支架本身的生物相容性. 与普通BMP2-MP相比, CBD-BMP2-MP具有更好的胶原绑定能力, 与复合支架的结合更稳定, 提高了PLGA/COL复合支架对BMP2-MP的负载能力. 支架表面负载CBD-BMP2-MP后具有极强的促细胞成骨分化能力. MC3T3-E1细胞表现出更高的钙沉积能力, 并且成骨分化相关基因Runx2, ALP, COL-I及OPN等水平也有了明显提升. 表明CBD-BMP2-MP多孔复合支架具有良好的生物相容性和成骨诱导活性, 在骨组织修复领域具有良好的应用前景.     

β-HCG在早期异位妊娠治疗中的应用

目的:探讨人绒毛膜促性腺激素β亚单位(β-HCG)在早期异位妊娠药物治疗中的监测价值及药物治疗的疗效.方法:60例宫外孕行保守治疗的患者为研究对象,采用氨甲喋呤(MTX)联合米非司酮治疗,7 d为1疗程,连续治疗2～3个疗程.监测治疗前及治疗后第7、14、21、27天时患者血清β-HCG水平.结果:治疗后第7、14、21、27天,患者血清β-HCG水平与治疗前比较均显著下降(P<0.05),且随着治疗时间的延长而逐渐降低(P<0.05);治疗后,β-HCG≤2 000 mIU/mL组成功率最高(91.2%),2 000<β-HCG<5 000 mIU/mL组成功率为80.0%,β-HCG≥5 000mIU/mL组无1例成功.不同β-HCG水平成功率比较有缔计学差异(P<0.05).结论:以血清β-HCG为监测指标,采用MTX联合米非司酮治疗早期异位妊娠临床疗效好,对于β-HCG值≤2000 mIU/mL的患者推荐采用保守治疗.     

肺康复联合无创通气对慢阻肺急性加重患者生活质量的影响

目的观察肺康复治疗在无创通气基础上对慢阻肺急性加重期患者肺功能指标、运动耐力、生活质量的影响。方法选择2014年10月—2016年10月于广东医科大学附属厚街医院住院的71例慢阻肺急性加重期患者作为研究对象,随机分为两组,对照组28例,观察组43例,对照组给予有效抗感染、吸入糖皮质激素+长效β2受体激动剂(ICS/LABA)及氧疗,无创辅助通气,观察组在对照组基础上给予肺康复治疗,比较两者患者在肺功能指标、运动耐力、生活质量的变化。结果 (1)两组患者治疗前肺功能指标(FEV1、FEV1/FVC(%)、FEV1%Pred(%))6MWD、CAT评分差异无统计学意义。(2)两组患者治疗后观察组FEV1高于对照组,但差异无统计学意义(P0.05)。但治疗后第7天FEV1、FEV1/FVC(%)、FEV1%Pred(%)观察组高于对照组,差异有统计学意义;两组患者治疗后较治疗前6 min步行距离(6 MWD)明显升高,差异有统计学意义(P0.05);CAT评分明显下降,差异有统计学意义(P0.05)。(3)治疗后观察组6 MWD对照组,差异有统计学意义(P0.05);治疗后观察组CAT评分对照组,差异有统计学意义(P0.05),结论肺康复治疗联合无创通气能够明显改善COPD急性加重期患者运动耐力及生活质量,但肺通气功能指标FEV1无明显优于单用无创通气治疗。     

益生菌联合谷氨酰胺的肠内营养对重症急性胰腺炎肠黏膜屏障恢复的作用

目的研究了益生菌联合谷氨酰胺的肠内营养治疗对恢复重症急性胰腺炎（SAP）的肠道屏障功能的作用。方法将74例重症急性胰腺炎患者随机分为3组：常规肠内营养组（EN组）,肠内营养＋谷氨酰胺组（Gln组）,肠内营养＋谷氨酰胺＋益生菌制剂组（益生菌组）。治疗第1、7、14、21天进行APACHEⅡ评分,并取血测定血清中的谷氨酰胺、内毒素、CRP、D-乳酸水平。结果三组病人APACHE11评分、CRP均逐渐下降,在第7天,Gln组和益生菌组CRP水平下降,并且与EN组对比,差异有统计学意义,而益生菌组和Gln组在第14天的APACHEⅡ评分与EN组差异有统计学意义。三组患者血清Gln浓度在第7天测定均成不同程度下降,Gln组与益生菌组分别与EN组比较,差异无统计学意义。在第14天,三组Gln水平均上升,益生菌组与EN组对比,差异有统计学意义（P〈0.05）。在第7天,益生菌组的血清内毒素、D-乳酸水平低于EN组,差异有统计学意义。在第14天,Gln组和益生菌组的内毒素水平和D-乳酸水平均低于EN组,差异有统计学意义,益生菌组的DAO水平低于EN组,差异有统计学意义（P〈0.05）。结论应用益生菌制剂和Gln的肠内营养能更好地维护肠道黏膜屏障的完整,应用前景广阔。但益生菌制剂和Gln免疫制剂问的协同作用需要进一步论证。     

稀土化合物TbCl3对成骨细胞系MC3T3-E1增殖、分化和矿化功能的影响

在细胞和分子水平上,研究了稀土化合物氯化铽(TbCl₃)对成骨细胞MC3T3-E1增殖、分化及矿化功能的影响。结果表明,细胞水平上,浓度为0.0001、0.001、0.01、0.1、1和10 μmol·L^-1的TbCl₃均促进MC3T3-E1细胞的增殖、分化及其矿化功能,然而,当浓度升至为100和1000 μmol·L^-1时,TbCl₃表现出抑制作用。分子水平上,浓度为0.0001和0.1 μmol·L^-1的TbCl₃明显上调成骨分化相关基因骨形成蛋白2(BMP-2),碱性磷酸酶(ALP),骨涎蛋白(BSP),Ⅰ型胶原蛋白(Col Ⅰ),骨钙素(OCN)和runt 相关转录因子2(Runx2)的表达。浓度为1 000 μmol·L^-1的TbCl₃则抑制上述成骨分化相关基因的表达。浓度为0.000 1、0.1和1 μmol·L^-1的TbCl₃促进成骨分化相关蛋白Runx2,BMP-2和OCN的表达;结果显示,低浓度的TbCl₃促进MC3T3-E1细胞的成骨分化及矿化功能,而高浓度TbCl₃则呈现出抑制作用。TbCl₃通过调控Runx2的表达刺激早期成骨分化相关基因BMP-2、Col Ⅰ和晚期成骨分化相关基因ALP、OCN的表达,从而诱导MC3T3-E1成骨分化。     

重金属铅胁迫下镧对螺旋藻生长及生理特性的影响

以鄂尔多斯高原碱湖钝顶螺旋藻为实验材料,在Pb(NO3)2(70 mg·L-1)胁迫下,采用生理学方法研究了不同浓度La(NO3)3对螺旋藻生长速率、叶绿素a含量、硝酸还原酶及谷氨酸脱氢酶活性的影响。结果表明:各处理组在培养初期生长速率差异不大(P0.05),6 d后添加La(NO3)3的各处理组螺旋藻的生长速率显著高于对照(P0.05);当La(NO3)3处理浓度为8μg·L-1时可显著促进螺旋藻叶绿素a的合成,有效缓解Pb(NO3)2对硝酸还原酶及谷氨酸脱氢酶活性的影响(P0.05),高浓度La(NO3)3则会与Pb(NO3)2协同、抑制螺旋藻的生长。     

DOPA-IGF-1 Coated HA/PLGA Microspheres Promoting Proliferation and Osteoclastic Differentiation of Rabbit Bone Mesenchymal Stem Cells

To explore the ability of dihydroxyphenylalanine-insulin-like growth factor-1 (DOPA-IGF-1) coated hydroxyapatite/poly(lactic-co-glycolic acid)(HA/PLGA) microspheres to promote the proliferation and osteoclastic differentiation of rabbit bone mesenchymal stem cells(rBMSCs), HA/PLGA microspheres with different HA content (10%, 30%, 50%, mass fraction) were prepared by electrospinning method and HA/PLGA microspheres with 50% HA were coated with IGF-1 and DOPA-IGF-1, respectively. They were co-cultured with rBMSCs, respctively. Cell counting kit-8(CCK-8) detection, confocal laser scanning microscopy(CLSM), alkaline phosphatase(ALP) detection and osteogenesis related genes COL IA1, Runx2 and bone morphogenetic protein-2(BMP-2) detection were conducted to detect the proliferation activity, cell morphology, differentiation ability and the expression level of osteogenesis-related genes of cells cultured on all microspheres groups. The results showed that rBMSCs proliferation increased in an HA content dependent manner, and cells proliferated more in the IGF-1 coated and DOPA-IGF-1 coated groups, in particular in DOPA-IGF-1 coated group, and the differences were more remarkable over time (P<0.05). HA/PLGA microspheres promoted the proliferation and osteogenic differentiation of rBMSCs, and DOPA-IGF-1 coating enhanced the proliferation and osteogenic differentiation of rBMSCs.     

A pneumatic micro cell chip for the differentiation of human mesenchymal stem cells under mechanical stimulation

A new micro cell chip which can induce stem cells to differentiate into specific body cell types has been designed and fabricated for tissue engineering. This paper presents the test results of a micro cell stimulator which can provide a new miniaturized tool in cell stimulation, culture and analysis for stem cell research. The micro cell stimulator is designed to apply compressive pressure to the hMSCs (human mesenchymal stem cells) for inducing osteogenesis. The micro cell stimulator is based on the pneumatic actuator with a flexible diaphragm which consists of an air chamber and cell chambers. The hMSCs under cyclic compressive stimulation for one week were observed and assessed by monitoring CD90 (Thy-1), actin, alkaline phosphatase (ALP) and alizarin red expression. The results suggest that cyclic mechanical stimulation is attributed to the different phenomenon of cultured hMSCs in cell proliferation and differentiation. These results are important for the feasibility of the micro cell stimulator to provide the reduction of the necessary quantity of cells, process cost and the increase of the throughput.     

Electrochemical Monitoring of Saos‐2 Cell Differentiation on Pyrolytic Carbon Electrodes

In this study we establish an electrochemical platform based on two dimensional (2D) pyrolytic carbon electrodes for in vitro analysis of osteoblast differentiation. Electrochemical impedance spectroscopy (EIS) was used to monitor cell adhesion and proliferation, while an electrochemical assay based on square wave voltammetry (SWV) was applied to measure the activity of the differentiation marker alkaline phosphatase (ALP). 2D pyrolytic carbon electrodes were fabricated and used to monitor Saos‐2 cell differentiation for a period of up to 21 days. With this method it was possible to detect a faster increase of ALP activity for cells cultured in medium supplemented with differentiation factors compared to cells cultured in growth medium. This was confirmed by the results obtained with Alizarin Red staining, showing that cells subjected to osteogenic medium went through the entire differentiation process, from proliferation to mineralization. Finally, for the first time, real‐time monitoring of ALP activity combined with continuous EIS monitoring of the same cell culture was achieved using the pyrolytic carbon electrodes.     

5-azacytidine induces cardiac differentiation of P19 embryonic stem cells

The P19 embryonal carcinoma cell line is a useful model cells for studies on cardiac differentiation. However, its low efficacy of differentiation hampers its usefulness. We investigated the effect of 5-azacytidine (5-aza) on P19 cells to differentiate into a high-efficacy cardiomyocytes. Embryoid-body-like structures were formed after 6 days with 1 mM of 5-aza in a P19 cell monolayer culture, beating cell clusters first observed on day 12, and, the production of beating cell clusters increased by 80.1% (29 of 36-wells) after 18 days. In comparison, the spontaneous beating cells was 33.3% (12 of 36-wells) for the untreated control cells. In response to 1 mM of 5-aza, P19 cells expressed bone morphogenetic protein-2 (BMP-2), BMP-4, Bmpr1a and Smad1 at day 6 or 9, and also cardiac markers such as GATA-4, Nkx2.5, cardiac troponin I, and desmin were up-regulated in a time-dependent manner after induction of BMP signaling molecules. Immunocytochemistry revealed the expression of smooth muscle a-actin, sarcomeric a-actinin, cardiac myosin heavy chain, cardiac troponin T and desmin, respectively. The proportion of sarcomeric a-actinin positive cells accounted for 6.48% on day 15 after 5-aza exposure as measured by flow cytometry. This study has demonstrated that 5-aza induces differentiation of P19 cells into cardiomyocytes in a confluent monolayer culture in the absence of prior embryoid formation and dimethyl sulfoxide exposure, depending in part on alteration of BMP signaling molecules. These results suggest that 5-aza treatment could be used as a new method for cardiac differentiation in P19 cells.     

An Electrochemical Assay for Monitoring Differentiation of the Osteoblastic Cell Line (MBA‐15) on the Sensor Chip

An electrochemical assay for the indication of the activity of the cell bound differentiation marker alkaline phosphatase (ALP) is proposed using voltammetry on an in‐vitro cell culture. The basis of the assay is cultivation of cells on gold microelectrodes in wells of a microplate, catalytic hydrolysis of p‐aminophenyl phosphate by ALP and indication of p‐aminophenol oxidation by square wave voltammetry (SWV) with the sensors onto which the cells attached. The morphology of the bone marrow stromal cell line (MBA‐15) on the electrode surface was investigated and it exhibited in vitro osteogenic characteristics. Since ALP is expressed on the cell surface in early differentiation stage of osteoblastic cells, its activity was followed after different culture times over a period of 144 h by recording repetitive voltammograms at different time points upon addition of the substrate p‐aminophenyl phosphate. The ALP activity was estimated from the signal increase related to formation rate of p‐aminophenol and the number of cells. The highest value was measured at 120 h, when the cells reached confluence. The results of the electrochemical activity assay are consistent with the colorimetric acquired value from p‐nitrophenol formation rate.     

Scanning electron microscopy and roughness study of dental composite degradation

Our aim was to test the hypothesis that the use of mouthwashes, consumption of soft drinks, as well as the type of light curing unit (LCU), would change the surface roughness (Ra) and morphology of a nanofilled composite resin (Z350? 3M ESPE). Samples (80) were divided into eight groups: Halogen LCU, group 1, saliva (control); group 2, Pepsi Twist?; group 3, Listerine?; group 4, Colgate Plax?; LED LCU, group 5, saliva; group 6, Pepsi Twist?; group 7, Listerine?; group 8, Colgate Plax?. Ra values were measured at baseline, and after 7 and 14 days. One specimen of each group was prepared for scanning electron microscopy analysis after 14 days. The data were subjected to multifactor analysis of variance at a 95% confidence followed by Tukey's honestly significant difference post-hoc test. All the treatments resulted in morphological changes in composite resin surface, and the most significant change was in Pepsi Twist? groups. The samples of G6 had the greatest increase in Ra. The immersion of nanofilled resin in mouthwashes with alcohol and soft drink increases the surface roughness. Polymerization by halogen LCU (reduced light intensity) associated with alcohol contained mouthwash resulted in significant roughness on the composite.     

Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening

In this work, we demonstrated that biological cells could be cultured in a continuous-perfusion glass microchip system for drug screening. We used mouse Col1a1GFP MC-3T3 E1 osteoblastic cells, which have a marker gene system expressing green fluorescent protein (GFP) under the control of osteoblast-specific promoters. With our microchip-based cell culture system, we realized automated long-term monitoring of cells and sampling of the culture supernatant system for osteoblast differentiation assay using a small number of cells. The system successfully monitored cells for 10 days. Under the 3D microchannel condition, shear stress (0.07 dyne/cm² at a flow rate of 0.2 μL/min) was applied to the cells and it enhanced the GFP expression and differentiation of the osteoblasts. Analysis of alkaline phosphatase (ALP), which is an enzyme marker of osteoblasts, supported the results of GFP expression. In the case of differentiation medium containing bone morphogenetic protein 2, we found that ALP activity in the culture supernatant was enhanced 10 times in the microchannel compared with the static condition in 48-well dishes. A combined system of a microchip and a cell-based sensor might allow us to monitor osteogenic differentiation easily, precisely, and noninvasively. Our system can be applied in high-throughput drug screening assay for discovering osteogenic compounds.     

Raman spectroscopic monitoring of the osteogenic differentiation of human mesenchymal stem cells

The differentiation of stem cells into multi-lineages is essential to aid the development of tissue engineered materials that replicate the functionality of their tissue of origin. For this study, Raman spectroscopy was used to monitor the formation of a bone-like apatite mineral during the differentiation of human mesenchymal stem cells (hMSCs) towards an osteogenic lineage. Raman spectroscopy observed dramatic changes in the region dominated by the stretching of phosphate groups (950-970 cm(-1)) during the period of 7-28 days. Changes were also seen at 1030 cm(-1) and 1070 cm(-1), which are associated with the P-O symmetric stretch of PO(4)(3-) and the C-O vibration in the plane stretch of CO(3)(2-). Multivariate factor analysis revealed the presence of various mineral species throughout the 28 day culture period. Bone mineral formation was observed first at day 14 and was identified as a crystalline, non-substituted apatite. During the later stages of culture, different mineral species were observed, namely an amorphous apatite and a carbonate, substituted apatite, all of which are known to be Raman markers for a bone-like material. Band area ratios revealed that both the carbonate-to-phosphate and mineral-to-matrix ratios increased with age. When taken together, these findings suggest that the osteogenic differentiation of hMSCs at early stages resembles endochondral ossification. Due to the various mineral species observed, namely a disordered amorphous apatite, a B-type carbonate-substituted apatite and a crystalline non-substituted hydroxyapatite, it is suggested that the bone-like mineral observed here can be compared to native bone. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analyses for monitoring the various mineral species, degree of mineralisation and the crystallinity of hMSCs as they differentiate into osteoblasts.