间充质干细胞的成骨细胞分化和免疫组织化学鉴定

目的 探讨脐带的间充质干细胞分化为成骨细胞的影响因素和免疫组织化学鉴定。 方法 收集306例冻存的健康胎儿脐带,采用华尔通胶组织块贴壁法从脐带组织中分离间充质干细胞,利用荧光显微镜观察原代细胞的细胞形态。脐带间充质干细胞的免疫表型和细胞周期采用免疫组织化学检测。复苏冻存的脐带间充质干细胞,并传代培养至10代。对第10代的脐带间充质干细胞进行成骨诱导,其成骨能力分别通过钙结节和骨涎蛋白的免疫荧光检测以及茜素红染色检测来确定。 结果 免疫组织化学结果显示,培养的细胞高表达间充质干细胞的表面标志物CD73、CD90和CD105,但是不表达造血细胞的表面标志物CD34和CD45。复苏后细胞的存活率是90%。细胞周期显示,第10代的细胞有80%处于G₀/G₁期,20%处于S+G₂/M期。成骨细胞刺激因子诱导干细胞的骨涎蛋白染色呈阳性,并形成矿化的钙结节。 结论 冻存的脐带间充质干细胞在成骨细胞刺激因子下能被诱导分化为成骨细胞,具有高度自我增殖和多向分化能力。     

降钙素基因相关肽联合重组人骨形态发生蛋白对兔成骨样细胞增殖和碱性磷酸酶活性的影响

目的研究降钙素基因相关肽(CGRP)及重组人骨形成蛋白-2(rhBMP-2)在促进兔骨髓来源成骨样细胞增殖和分化方面是否有协同作用。方法取经诱导第三代兔骨髓基质细胞获得的兔成骨样细胞以2×10~6/ml的浓度接种于96孔板,分为6组,用含有不同条件的培养基进行培养。包括①A组:空白对照组,②B组:0.05 ng/ml CGRP,③C组:5 ng/ml CGRP,④D组:100 ng/ml rhBMP-2,⑤E组:5 ng/ml CGRP+100ng/ml rhBMP- 2,⑥F组:0.05ng/ml CGRP+100 ng/ml rhBMP-2。用四唑盐比色法(MTT)法测定细胞增殖情况和碱性磷酸酶( ALP)染色法检测其对ALP活性的影响。结果①100ng/ml rhBMP-2组、0.05ng/ml CGRP+100ng/ml rhBMP-2组、5ng/ml CGRP+100ng/ml rhBMP-2组细胞增殖OD值比对照组显著增强(P<0.05),其中0.05ng/ml CGRP+100ng/ml rhBMP-2组最强(P<0.01)。②100ng/ml rhBMP-2组、0.05ng/ml CGRP+100ng/ml rhBMP-2组、5ng/ml CGRP+100ng/ml rhBMP-2组,3组之间的ALP表达无明显差异(P>0.05).但3组与对照组相比显著增强( P<0.05)。结论CGRP和rhBMP-2合用对兔骨髓来源成骨样细胞的增殖有一定的协同作用,但对ALP无协同作用,与单用rhBMP-2的效果相当。     

SARS-CoV-2和其突变毒株表面S蛋白修饰的生物信息学分析

目的通过生物信息学手段对SARS-CoV-2不同变异株的S蛋白进行研究, 分析不同病毒株S蛋白的氨基酸突变位点, 预测变异株的突变能否改变S蛋白修饰, 判断S蛋白修饰是否会对病毒结合并融合宿主细胞产生影响。方法从NCBI Virus的SARS-CoV-2数据库中下载病毒S蛋白的氨基酸序列, 并进行系统进化分析, 挑选其中代表不同进化分支的病毒毒株, 分别利用NetNGlyc-1.0-Services等软件对S蛋白的糖基化和磷酸化进行预测分析。结果 S蛋白上主要有17个潜在的N-糖基化位点, 受变异株序列改变影响的修饰位点是第17和654位点。S蛋白上潜在的O-糖基化位点有15个, 变异株中发生了第12、71、255和686位点的修饰改变。潜在的43个磷酸化修饰位点中受氨基酸突变影响的位点主要位于S1亚基上, 只有一个位点位于S2亚基。结论不同变异毒株的氨基酸序列改变会造成S蛋白糖基化和磷酸化修饰的明显改变, S蛋白的修饰改变可能与变异病毒的致病性和其在人群中传播感染密切相关, 今后需要通过实验研究和流行病分析进行进一步验证。     

转化生长因子β和骨形成蛋白2联合诱导小鼠成骨细胞系MC3T3-E1细胞的增殖与分化

背景:生长因子是骨组织形成、改建、修复的关键要素之一,多种生长因子联合诱导骨干细胞成骨分化相较于单一生长因子具有一定的优势,但是何种类型的联合、什么样的浓度组合才能发挥最佳的诱导作用?目前该方向研究较少.目的:探索转化生长因子β和骨形成蛋白2联合诱导对小鼠MC3T3-E1细胞增殖和分化的影响.方法:将不同质量浓度的转化...     

拔牙后富血小板血纤蛋白作为牙槽嵴位点保留材料可显著减少牙槽骨高度及宽度的吸收:一项Meta分析

目的:目前富血小板血纤蛋白在拔牙后位点保留中的应用更加广泛,而与其他材料相比富血小板血纤蛋白保存牙槽嵴的效果不是很明确。文章评估富血小板血纤蛋白在拔牙后牙槽嵴保存中的应用效果,为临床应用富血小板血纤蛋白减少牙槽骨吸收提供理论依据。方法:截止到2020年6月,在The Cochrane Library、PubMed、Web of Science核心合集、EMbase、中国知网、维普和万方数据库进行文献检索,收集所有关于富血小板血纤蛋白用于牙槽嵴保存的临床随机对照研究,包括单纯应用富血小板血纤蛋白和富血小板血纤蛋白复合异质骨。由2位研究人员从检索的文献中提取数据,使用Cochrane偏倚风险评估工具评价纳入研究的偏倚风险。由RevMan 5.3软件对结果指标进行Meta分析。结果:(1)共检索出159篇文献,最终纳入11个随机对照试验,共531例患者,575个拔牙位点;(2)Meta分析结果显示:单纯应用富血小板血纤蛋白时牙槽骨高度吸收量(SMD=-0.38,95%CI:-0.83至-0.06,P> 0.05)差异无显著性意义;富血小板血纤蛋白复合异质骨与单纯异种骨在拔牙后3,6个月时牙槽骨吸收高度(3个月:SMD=-1.40,95%CI:-1.79至-1.01,P <0.05;6个月:SMD=-1.37,95%CI:-1.68至-1.06,P <0.05)、牙槽骨吸收宽度(SMD=-0.18,95%CI:-0.25至-0.11,P <0.05)及拔牙2周后术区黏膜愈合率(SMD=9.90,95%CI:8.61-11.19,P <0.05)方面差异均有显著性意义。结论:拔牙后应用富血小板血纤蛋复合异质骨作为牙槽嵴位点保留的材料时可以明显减少牙槽骨高度及宽度的吸收,也可以加快软组织愈合,但由于纳入研究的样本量较少,还需要更大的样本量临床研究来验证以上结论。     

雌激素受体β基因沉默对人成骨细胞转化生长因子β1和骨形态发生蛋白2表达的影响

BACKGROUND: There are few studies concerning estrogen receptor β gene, and its mechanism of regulating the bone metabolism is still unclear now.  OBJECTIVE: To analyze the effect of estrogen receptor β (ER β) silencing on the expressions of transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2) in human osteoblasts METHODS: There were three groups: blank control group (hFOB 1.19 uninfected with any retrovirus); negative control group (containing invalid interference fragment ER β-shRNA-nc); optimal RNAi group (ER β-shRNA-3). ER β-shRNA retroviral vectors in the optimal RNAi group were used to transfect human osteoblasts followed by resistance screening and cell expansion. MTT assay was used to detect the proliferative activity of ER β-silenced osteoblasts. Then under estrogen intervention, the stable inhibition rate of ER β was determined using western blot assay, and the expressions of TGF-β1 and BMP-2 in human osteoblasts after ER β silencing were detected by RT-PCR technology and western blot assay. RESULTS AND CONCLUSION: Human osteoblasts that were stably transfected by ER β-shRNA-3 retroviral vector was selected successfully, and ER β silencing had no significant influence on the cell proliferation (P > 0.05). Under the interference of estrogen, the silencing efficiency of ER β protein was (93.11±0.57)% (P < 0.05), and after ER β silencing, the expressions of TGF-β1 and BMP-2 were increased by (26.65±3.81)% and (16.62±1.71)% at mRNA level, and increased by (23.79±3.76)% and (18.08±3.20)% at protein level (both P < 0.05). In conclusion, ER β may play an important role in bone metabolism by regulating the expressions of TGF-β1 and BMP-2. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

聚酯/磷酸三钙人工骨载体的表面修饰及不同骨移植材料的比较研究

采用热致分相法(TIPS)与熔铸颗粒沥取法(SCPL),实验室条件下制备适宜组分配比(7:3)的聚乳酸-聚羟乙酸/磷酸三钙[PLGA/TCP]复合人工骨载体[PLGA/TCP(L)];另外采用先进快速成形技术(RP)制备相同组分配比(7:3)的PLGA/TCP(RP)复合材料.扫描电镜(SEM)观察人工骨载体的超微结构,并用I型胶原(Col I)对载体材料进行表面修饰.进而复合诱骨生长因子-牛骨形态发生蛋白(bBMP)以制备出仿生活性人工骨.将PLGA/TCP(L)、PLGA/TCP(RP)、牛松质骨脱钙骨基质(DBM)、仿生活性人工骨及OsteoSet(R)人工骨进行多种比较.结果发现,先进RP技术制备的PLGA/TCP(RP)大段人工骨载体结构明显优于实验室常规方法所制备的PLGA/TCP(L)载体.PLGA/TCP(L)载体、DBM、PLGA/TCP(RP)载体及OsteoSet(R)人工骨的孔隙率分别为21.5%、70.4%、58.6%和0%,其中DBM和PLGA/TCP(RP)载体的孔隙率最高(P＜0.01).此外,RP制备的PLGA/TCP(RP)人工骨载体孔径大(350 μm),并与天然DBM的孔径最为接近.PLGA/TCP(L)、PLGA/TCP(RP)人工骨载体经I型胶原表面修饰后[PLGA/TCP(L)-Col I、PLGA/TCP(RP)-Col I],明显改善其对bBMP的亲和力,人工骨载体表面及孔隙内对bBMP的复合能力明显增强,仿生活性人工骨[PLGA/TCP(L)-Col I-bBMP、PLGA/TCP(RP)-Col I-bBMP]的制备效率亦显著提高.其中采用先进RP技术结合载体材料表面修饰新工艺研制的PLGA/TCP(RP)-Col I-bBMP仿生活性人工骨具有与天然骨最接近的空间三维结构,并含有关键的成骨活性因子,因此具有更为广阔的研究与应用前景.     

ATMFS对犬骨盆弓状线骨折愈合及骨钙蛋白和骨唾液酸蛋白表达的影响

目的探索骨盆髋臼三维记忆内固定系统(acetabular tridimensional memory fixation system,ATMFS)对犬弓状线骨折愈合及骨钙蛋白和骨唾液酸蛋白表达的影响。方法选用15只成年杂种家犬,双侧髋臼臼顶上方1.5cm处横形截骨,分别采用ATMFS前柱固定器和6孔重建钢板内固定,于术后2、4、6、8、12周行影像学检查、大体观察、骨钙蛋白及骨唾液酸蛋白原位杂交、骨钙蛋白实时定量PCR(RT-PCR)检测,评价骨盆弓状线的骨愈合特征及骨钙蛋白和骨唾液酸蛋白的表达特征。结果 ATMFS侧骨折端无凌乱骨痂,骨折愈合时间明显快于钢板侧。双侧骨折端组织中骨钙蛋白和骨唾液酸蛋白表达与正常比均明显增强。术后4～8周,ATMFS侧骨钙蛋白和骨唾液酸蛋白表达量明显大于钢板侧,两侧比较具有显著性差异(P0.05),其中术后6周双侧差异最为显著(P0.01)。结论 ATMFS固定后于骨折端产生的持续顺应生理力线的压应力能够刺激骨钙蛋白及骨唾液酸蛋白的表达,从而促进骨折早期愈合。     

3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration

Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1–2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.     

Characteristics of high-density lipoprotein binding sites in cultured parenchymal,endothelial, and Kupffer's cells from rat liver

Liver endothelial cells posses surface high-affinity binding sites for HDL₃, whose affinity is 4 times higher than that of the sites on hepatocytes and Kupffer's cells. The maximal number of high-affinity binding sites on endothelial cells is 437 ng/mg protein, which surpasses this parameter on the hepatocyte surface several times. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 3, pp. 268–270, March, 1994     

A self-assembling peptide matrix used to control stiffness and binding site density supports the formation of microvascular networks in three dimensions

A three-dimensional (3-D) cell culture system that allows control of both substrate stiffness and integrin binding density was created and characterized. This system consisted of two self-assembling peptide (SAP) sequences that were mixed in different ratios to achieve the desired gel stiffness and adhesiveness. The specific peptides used were KFE ((acetyl)-FKFEFKFE-CONH2), which has previously been reported not to support cell adhesion or MVN formation, and KFE-RGD ((acetyl)-GRGDSP-GG-FKFEFKFE-CONH2), which is a similar sequence that incorporates the RGD integrin binding site. Storage modulus for these gels ranged from ～60 to 6000 Pa, depending on their composition and concentration. Atomic force microscopy revealed ECM-like fiber microarchitecture of gels consisting of both pure KFE and pure KFE-RGD as well as mixtures of the two peptides. This system was used to study the contributions of both matrix stiffness and adhesiveness on microvascular network (MVN) formation of endothelial cells and the morphology of human mesenchymal stem cells (hMSC). When endothelial cells were encapsulated within 3-D gel matrices without binding sites, little cell elongation and no network formation occurred, regardless of the stiffness. In contrast, matrices containing the RGD binding site facilitated robust MVN formation, and the extent of this MVN formation was inversely proportional to matrix stiffness. Compared with a matrix of the same stiffness with no binding sites, a matrix containing RGD-functionalized peptides resulted in a ～2.5-fold increase in the average length of network structure, which was used as a quantitative measure of MVN formation. Matrices with hMSC facilitated an increased number and length of cellular projections at higher stiffness when RGD was present, but induced a round morphology at every stiffness when RGD was absent. Taken together, these results demonstrate the ability to control both substrate stiffness and binding site density within 3-D cell-populated gels and reveal an important role for both stiffness and adhesion on cellular behavior that is cell-type specific.     

间歇牵拉应变对大鼠骨髓间充质干细胞增殖与成骨分化的影响

目的探讨体外间歇拉伸应变对大鼠骨髓间充质干细胞(rat bone mesenchymal stem cell,rBMSC)的增殖与成骨分化效应。方法应用Flexcell-4 000应力系统,将间歇拉伸应变(振幅10%,频率0.5 Hz,每天作用2次,4 h/次)作用于体外分离培养的正常rBMSC,加力1、3、5、7 d后检测应力对细胞形态改变、细胞增殖及骨形成核心结合因子Cbfα1、碱性磷酸酶(ALP)和I型胶原等成骨基因和Cbfα1蛋白的影响。结果 rBMSC在间歇牵拉应力作用1 d后即出现增殖速度减缓,一直延续到加力第7 d,ALP和I型胶原等成骨基因的表达自加力3 d后升高了3～6倍(P<0.05),并且Cbfα1的基因和蛋白表达在力学刺激下都出现上调。结论力学刺激在rBMSC的增殖与分化中起重要作用,适宜的间歇拉伸应变可以减缓BMSC的增殖,促进其成骨分化。     

通过骨支架的数字化建模分析其力学性能与内部流场分布

目的研究不同骨支架结构的力学性能与内部流场分布,为模型结构的优劣提供直观上的比较和评判,为骨支架结构设计提供有效的指导方法。方法利用Pro/Engineer和MIMICS等软件重建自然结构骨支架、编织状骨支架和球形孔骨支架,并通过有限元方法分析3种支架的有效弹性模量、应力分布和三维灌注培养下支架内部流场分布。结果采用相同材料设计得到的自然结构骨支架表现出更小的有效弹性模量;当3种支架受到相同压力时,自然结构骨支架内部应力峰值更小且应力分布更均匀;初始流速和流体黏度相同时,自然结构骨支架表现出更小的内部流速、壁面剪切应力和壁面压力。结论自然结构骨支架模型具有相对较好的生物力学性能,在3种骨支架中最适合用于骨组织工程中骨支架结构选型。     

Analysis of the activating receptors and cytolytic function of human natural killer cells undergoing in vivo differentiation after allogeneic bone marrow transplantation

Patients undergoing allogeneic bone marrow transplantation offer a unique system to analyze NK cell development in vivo. We analyzed NK cells from 23 such patients to assess the acquisition of activating receptors. Four patients displayed an immature NK cell surface phenotype at engraftment, as their cells were CD16(-)KIR(-) and NKG2D(-) but expressed low levels of NKp46, NKp30, 2B4 and NKG2A. These NK cells had particularly low cytolytic activity against the HLA-class-I(-) melanoma F01 cell line and the 721-221 EBV-infected B cell line. Moreover, cytoxicity was inhibited upon mAb-mediated crosslinking of 2B4. Analysis of NK cells at day 30 after bone marrow transplantation revealed the occurrence of both phenotypic and functional maturation. These data are in agreement with a previous in vitro study showing that immature NK cell precursors express CD16, NKG2D and KIR only at a late stage of differentiation and also express inhibitory 2B4. Our present study allows a better understanding of the NK cell differentiation in vivo.