Runx2、Osterix转录因子过表达驱动内皮细胞成骨分化的机制探讨

目的:探讨Runx2和Osterix (OSX)过表达对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)成骨分化的调控作用.方法:通过慢病毒载体,将Runx2和Osterix基因分别转染入HUVECs.通过碱性磷酸酶(ALP)染色、半定量活性检测,探讨过表达Runx2和Osterix对HUVECs成骨分化的影响.通过RT-PCR、蛋白免疫印迹、免疫荧光染色检测成骨相关标志物Runx2、OSX、ALP、骨涎蛋白(BSP)、骨桥蛋白(OPN)、骨钙蛋白(OCN)在HUVECs中的表达.采用GraphPad Prism 6.01软件包对数据进行统计学分析.结果:Runx2过表达有利于HUVECs的成骨分化,而Osterix过表达则无此作用.HUVECs转染Runx2过表达慢病毒后,成骨相关基因Runx2、OSX、ALP、BSP、OPN及OCN的转录水平上调,同时Runx2、OSX、OPN及OCN的蛋白表达水平亦有所上调.结论:Runx2过表达可促进HUVECs的成骨分化.     

钛表面形貌和亲水性表面对成骨细胞增殖、分化的影响

目的:通过体外实验研究普通喷砂酸蚀纯钛表面和亲水性喷砂酸蚀纯钛表面对成骨细胞增殖、分化等生物学行为的影响。方法:纯钛片表面分别采用光滑处理(smooth pretreated Ti,PT)、大颗粒喷砂酸蚀表面处理(sand-blasted,large-grit,acid-etched,SLA)及亲水性化学活化大颗粒喷砂酸蚀表面处理(chemically-modified SLA,modSLA/SLActive),在表面接种MC3T3-E1成骨细胞,采用MTT、碱性磷酸酶半定量测试以及茜素红染色检测其对成骨细胞增殖、分化的影响,并采用实时荧光定量PCR检测成骨细胞在不同材料表面骨功能基因表达的差异。应用SAS 9.0软件包对数据进行统计学分析。结果:与光滑钛表面相比,普通喷砂酸蚀钛表面能通过促进ALP、钙基质的分泌和成骨功能基因(Runx2、OSX、OCN和OPN)的表达而显著抑制成骨细胞增殖并促进其分化。在表面粗糙度的基础上增加亲水性,可使这一效应更加明显。结论:表面粗糙度和亲水性是影响成骨细胞生物学行为的重要因素,粗糙钛表面能显著抑制成骨细胞增殖,促进其分化,亲水性的粗糙钛表面促进成骨细胞分化的作用更加显著。     

牙髓卟啉单胞菌内毒素对成骨细胞分化的抑制作用

目的:研究牙髓卟啉单胞菌脂多糖(P.e-LPS)对成骨细胞分化的影响,探讨P.e-LPS在根尖周骨吸收疾病中的致病机制.方法:厌氧条件下培养P.e,应用热酚水法提取P.e-LPS,采用凝胶鲎试剂法对所提取的LPS进行定性分析.应用成骨细胞分化培养基(50μg/mL抗坏血酸、6 mmol/Lβ甘油磷酸钠)诱导前成骨细胞系...     

组蛋白去甲基化酶FBXL11抑制牙髓干细胞成骨和成牙本质分化

目的 研究组蛋白去甲基化酶FBXL11对牙髓干细胞定向分化能力的影响.方法 成骨分化诱导培养基诱导牙髓干细胞体外成骨/成牙本质分化.逆转录病毒转染构建过表达FBXL11的牙髓干细胞稳定转染细胞,进行FBXL11获得性功能研究.碱性磷酸酶活性实验及碱性磷酸酶染色检测成骨/成牙本质分化早期分化指标-碱性磷酸酶活性.茜素红染色及钙离子定量分析检测牙髓干细胞体外成骨/成牙本质分化能力.实时定量RT-PCR检测FBXL11及成骨/成牙本质分化相关基因-骨涎蛋白、骨桥蛋白和骨钙素的表达.结果 成骨诱导牙髓干细胞抑制FBXL11的表达.过表达FBXL11明显抑制牙髓干细胞的碱性磷酸酶活性、牙髓干细胞体外矿化能力以及骨涎蛋白和骨桥蛋白的表达.结论 组蛋白去甲基化酶FBXL11具有抑制牙髓干细胞成骨和成牙本质分化的潜能.     

人牙髓细胞条件培养液对人牙囊细胞成骨分化作用的体外研究

目的:探索人牙髓细胞条件培养液(HDPSCs-CM)对人牙囊细胞(HDFSCs)向成骨分化的作用.方法:利用胶原酶消化法获得HDFSCs,经纯化鉴定后培养于HDPSCs-CM中;CCK-8检测HDFSCs增殖活性,观察细胞形态改变;碱性磷酸酶(ALP)染色、茜素红S染色定性分析细胞成骨能力的改变.qRT-PCR检测HDFSCs中骨膜蛋白(POSTN)、Ⅰ型胶原(Col-Ⅰ)、碱性磷酸酶(ALP)、骨涎蛋白(BSP)以及骨桥蛋白(OPN)的mRNA表达情况.结果:经HDPSCs-CM诱导后,HDFSCs形态发生成牙骨质或成骨样改变;诱导组HDFSCs增殖活性受到明显抑制(P<0.05或P<0.01);诱导组ALP染色强于对照组,茜素红S染色矿化结节多于对照组;诱导组POSTN、Col-Ⅰ、ALP、BSP、OPN的表达量明显增高(P<0.05或P<0.01).结论:HDPSCs-CM能促进HDFSCs成骨分化.     

The osteogenic activity of human mandibular fracture haematoma-derived cells is stimulated by low-intensity pulsed ultrasound in vitro

Low intensity pulsed ultrasound (LIPUS) stimulation is a clinically established treatment method used to accelerate long bone fracture healing; however, this method is currently not applied to mandibular fractures. In this study, we investigated the effects of LIPUS on human mandibular fracture haematoma-derived cells (MHCs) in order to explore the possibility of applying LIPUS treatment to mandibular fractures. MHCs were isolated from five patients. The cells were divided into two groups: (1) LIPUS (+) group: MHCs cultured in osteogenic medium with LIPUS treatment; and (2) LIPUS (−) group: MHCs cultured in osteogenic medium without LIPUS treatment. The osteogenic differentiation potential and proliferation of the MHCs were compared between the two groups. The waveform used was equal to the wave conditions of a clinical fracture healing system. The gene expression levels of ALP, OC, Runx2, OSX, OPN, and PTH-R1 and mineralization were increased in the LIPUS (+) group compared to the LIPUS (−) group. There were no significant differences in cell proliferation between the two groups. These findings demonstrate the significant effects of LIPUS on the osteogenic differentiation of MHCs. This study provides significant evidence for the potential usefulness of the clinical application of LIPUS to accelerate mandibular fracture healing.     

Isolation of murine cementoblasts: unique cells or uniquely-positioned osteoblasts?

While cementoblasts express a number of mineral-related proteins, including bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OC), these proteins do not appear to be expressed by cells of the intermediate dental follicle/periodontal ligament (PDL). This information was utilized in an experimental strategy to isolate presumptive cementoblasts from the root surface of day 24 murine mandibular first molars. Using microscopic dissection techniques, molars were carefully extracted from their alveolar crypts and subjected to trypsin-collagenase digestion to remove adherent cells. Primary cultures were established and assayed for expression of proteins known to be expressed by cementoblasts at this timepoint in vivo (i.e. BSP, OPN, OC) and also an odontoblast-specific protein (i.e. DSP) to rule out contamination by pulpal cells. A subgroup of cells were found to express Type I collagen (89% of cells), BSP (46%), OPN (23%) and OC (30%); DSP was not detected within these cultures. We propose that cells within this heterogeneous population, which express this profile of osteogenic proteins, represent cementoblasts. The availability of a cementoblast cell line will make possible rigorous and controlled in vitro analysis of these cells and allow for determination of the unique characteristics of these cells not shared with other cells, particularly osteoblasts.     

抑菌浓度米诺环素对成骨细胞增殖、分化和矿化的影响

目的 研究抑菌浓度的米诺环素对成骨细胞增殖、分化和矿化及Runt相关转录因子2（Runx2）、碱性磷酸酶（ALP）、骨桥蛋白（OPN）mRNA表达的影响。方法 将米诺环素溶液（0、0.1、0.5、1、10 μg·mL^-1）与原代成骨细胞共培养,CCK-8检测增殖活性,通过ALP活性检测、茜素红染色、荧光定量聚合酶链反应检测探讨米诺环素对成骨细胞分化、矿化的影响。结果 0.1、0.5、1 μg·mL^-1的米诺环素可以促进细胞的增殖,上调ALP、Runx2 mRNA的表达水平,增加钙含量及钙化结节的形成,其中1 μg·mL^-1具有最大促进作用（P<0.05）;当浓度为10 μg·mL^-1时这种促进作用开始下降,并对ALP活性和OPN表达有显著抑制作用（P<0.01）。结论 适宜抑菌浓度的米诺环素能促进成骨细胞增殖,上调Runx2、ALP、OPN的表达水平,促进成骨细胞分化和矿化。     

p38信号通路参与调控上颌突间充质细胞的成骨分化

目的探索p38信号通路在上颌突间充质细胞体外成骨分化中的调控作用。方法取第1代E12.5 d的小鼠上颌突间充质细胞进行成骨诱导培养1周,实验组加入SB203580 （p38磷酸化抑制剂）。通过免疫荧光检测磷酸化p38的表达,通过Brdu标记和免疫荧光检测细胞的增殖能力,通过ALP染色和定量PCR检测成骨标志物的表达。采用SPSS18.0软件包对数据进行统计学分析。结果成骨诱导可促进上颌突间充质细胞中p38的磷酸化（p-p38）。抑制p38的磷酸化,可抑制上颌突间充质细胞增殖,降低成骨标志物ALP、Runx2、OCN和OPN的表达,使ALP染色减弱。结论p38信号通路参与调控体外培养的上颌突间充质细胞的成骨分化。     

微小RNA-29a-3p调节卷曲蛋白4表达影响高脂血症大鼠种植体骨整合的实验研究

目的 研究microRNA-29a-3p（miR-29a-3p）对高脂环境下大鼠骨髓间充质干细胞（BMSCs）成骨分化和高脂血症大鼠种植体骨整合的影响及其作用位点。方法 1）体外实验：对BMSCs分别进行普通和高脂成骨诱导,通过逆转录实时定量聚合酶链反应和Western blot检测miR-29a-3p及成骨相关因子碱性磷酸酶（ALP）、Runt相关基因2（Runx2）的基因和蛋白质表达;高脂培养的BMSCs分别转染miR-29a-3p模拟物、抑制物及阴性对照（NC）质粒,RT-qPCR检测miR-29a-3p、ALP及Runx2基因表达情况,Western blot检测ALP、Runx2蛋白表达情况。通过靶基因预测软件（Target Scan、MiRNA.org等）预测miR-29a-3p与成骨相关的靶基因为卷曲蛋白4（Fzd4）,双荧光素酶报告检测miR-29a-3p与Fzd4的相互作用关系。2）体内实验：高脂血症大鼠为实验组,普通大鼠为对照组,两组分别植入种植体,检测种植体周围骨组织中miR-29a-3p、ALP、Runx2的表达差异;行种植体-骨组织的硬组织切片,亚甲基蓝-酸性品红染色,进行组织学观察。分别将miR-29a-3p过表达慢病毒载体及空白对照慢病毒载体注射入高脂血症大鼠,种植体植入后3、10 d检测种植体周围骨组织中ALP、Runx2的表达变化以研究miR-29a-3p对高脂血症大鼠成骨的作用。结果 高脂组与普通组相比,ALP、Runx2和miR-29a-3p表达下调,BMSCs成骨分化能力下降。miR-29a-3p模拟物组与抑制物组相比,ALP、Runx2表达升高,BMSCs成骨分化能力增强。体内实验也得到了相似结果。双荧光素酶报告基因分析证实miR29a-3p通过直接结合3’-UTR抑制Fzd4表达。结论 miR-29a-3p对高脂环境下大鼠BMSCs成骨分化起正向调节作用,可直接与Fzd4结合调节成骨分化;miR-29a-3p能够促进高脂血症大鼠种植体周围成骨标志基因的表达,有利于骨整合。     

Complications in the use of the mandibular body,ramus and symphysis as donor sites in bone graft surgery. A systematic review

Background

To develop a systematic review by assessing and comparing the different complications that occurs in bone graft surgery using the mandibular body, ramus and symphysis as donor sites.

Material and Methods

In order to respond to the following question, a systematic review was developed: does the use of intraoral mandibular body and ramus as donor sites in bone graft surgery, produce fewer and less severe complications in comparison to the use of the mandibular symphysis in patients that present bone resorption that needs augmentation using autologous grafts? The review was carried out between January 1990 and 2015, during which only clinical essays with a minimum follow-up period of six months were included.

Results

The initial search yielded a total of 2912 articles, of which 6 were finally selected. In total, 259 graft surgeries were performed; 118 using the mandibular body and ramus as donor sites, and 141, the symphysis. The most frequent complications that arose when using the mandibular symphysis were temporary sensory alterations in the anterior teeth (33.87%), followed by sensory alterations of the skin and mucosa (18.57%). As for the mandibular body and ramus donor sites, the most frequent complications relate to temporary sensory alterations of the mucosa (8.19%) and to minor postoperative bleeding (6.55%).

Conclusions

The analyzed results show a higher prevalence and severity of complications when using mandibular symphysis bone grafts, producing more discomfort for the patient. Therefore, it would be advisable to perform further clinical essays due to the lack of studies found. Key words:Alveolar ridge augmentation, autogenous bone, mandibular bone grafts, chin, mandibular symphysis, mandibular ramus.     

高糖对人骨髓间充质干细胞成骨能力影响的体外研究

目的:体外模拟糖尿病病人高血糖状态,观察高糖对人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)成骨能力的影响。方法 :将使用不同含糖量培养液体外培养的hMSCs分为5.5mmol/L生理糖浓度组、25mmol/L高糖组、44 mmol/L高糖组。CCK-8试剂盒检测各组hMSCs的增殖速度;骨诱导7、14、21 d时分别观察高糖对hMSCs骨向分化相关基因骨钙素(OCN)、骨桥蛋白(OPN)、Runx相关因子2(Runx-2)的表达,碱性磷酸酶(ALP)活性,钙沉积量的影响。结果:两个高糖组相对生理糖组,有抑制hMSCs的增殖能力(P<0.05),且抑制ALP活性及细胞外钙基质沉积,下调了骨向分化相关基因OCN、OPN、Runx-2表达水平(P<0.01),该抑制作用随糖的浓度升高而加强。结论:高糖显著抑制了hMSCs生物矿化过程,并以浓度依赖的方式降低hMSCs的增殖及成骨能力。     

miR-199a调控IGF1表达对机械刺激下成骨细胞分化的影响

目的： 探讨微小RNA（miR）-199a在机械牵张力刺激下MC3T3-E1细胞中的表达变化及其对牵张力刺激MC3T3-E1细胞成骨分化的作用机制。方法： 对体外培养的MC3T3-E1细胞加载12%牵张力0、3、6、12和24 h后,利用碱性磷酸酶（ALP）活性检测试剂盒检测ALP活性,实时荧光定量PCR检测骨钙素（OCN）、成骨细胞特异性转录因子Osterix（OSX）、Runt相关转录因子2（Runx2） mRNA和miR-199a的表达。将MC3T3-E1细胞分为对照组、牵张力组、牵张力+miR-NC组和牵张力+miR-199a组,加载12%牵张力和转染miR-199a模拟物后,观察miR-199a和OCN、OSX、Runx2 mRNA及蛋白表达以及ALP活性。茜素红S（ARS）染色观察钙结节形成能力。采用双荧光素酶报告基因实验检测miR-199a与胰岛素样生长因子1（IGF1）的靶向关系,实时荧光定量PCR法和免疫印迹法检测miR-199a模拟物对IGF1 mRNA和蛋白表达的影响。采用SPSS 24.0软件包对数据进行统计学分析。结果： 与0 h时间点相比,以机械牵张力刺激3、6、12和24 h后,MC3T3-E1细胞ALP活性和OCN、OSX、Runx2 mRNA表达水平均显著升高,而miR-199a表达水平显著降低（P<0.05）,12 h时变化最为显著。与对照组相比,牵张力组细胞中miR-199a表达水平显著降低,而细胞ALP活性、OCN、OSX、Runx2 mRNA及蛋白表达水平、钙结节形成水平均显著升高（P<0.05）;与牵张力组相比,牵张力+miR-NC组细胞中上述各指标差异均无统计学意义（P>0.05）;与牵张力+miR-NC组相比,牵张力+miR-199a组细胞中miR-199a表达水平显著升高,而细胞ALP活性、OCN、OSX、Runx2 mRNA及蛋白表达水平、钙结节形成水平均显著降低（P<0.05）。miR-199a可与IGF1靶向结合,miR-199a模拟物可使MC3T3-E1细胞中IGF1 mRNA和蛋白表达水平显著降低（P<0.05）。结论： miR-199a可抑制机械牵张力刺激诱导的MC3T3-E1细胞成骨分化,其作用机制可能与靶向调控IGF1表达有关。     

Effects of ultrasound on the proliferation and differentiation of cementoblast lineage cells

BACKGROUND: The purpose of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) stimulation on the proliferation and differentiation of cementoblast lineage cells. METHODS: An immortalized human periodontal ligament cell line (HPL) showing immature cementoblastic differentiation was used. Cultured HPL cells were subjected to LIPUS exposure (frequency = 1 MHz; pulsed 1:4; intensity = 30 mW/cm(2)) or sham exposure for 15 minutes per day. Expression levels of alkaline phosphatase (ALP), type I collagen (Col-I), runt-related gene 2 (Runx2), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OPN) mRNA were analyzed with real-time polymerase chain reaction analysis. Furthermore, ALP activity, collagen synthesis, and protein level of Runx2 were examined after 6 days of LIPUS exposure. RESULTS: mRNA and protein levels of ALP, Col-I, and Runx2 were significantly increased by LIPUS exposure compared to controls, whereas BSP, OCN, and OPN mRNA expression could not be detected in HPL cells, irrespective of LIPUS exposure. CONCLUSION: LIPUS enhanced ALP activity, collagen synthesis, and Runx2 expression of HPL cells, which provides important insight into the promotion of early cementoblastic differentiation of immature cementoblasts.     

MicroRNA-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响

目的:探讨miR-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响。方法:以小鼠前成骨细胞MC3T3-E1为实验对象,对MC3T3-E1细胞进行成骨诱导,分别在0、3、5、7 d应用实时荧光定量PCR（real-time PCR）检测细胞中Runx2、Osx、ALP、miR-103-3p表达水平,Western免疫印迹（Western blotting）检测Runx2、Osx蛋白表达并进行碱性磷酸酶（ALP）染色。通过脂质体lipofectamine2000瞬时转染miR-103-3p模拟物 （miR-103-3p mimics）及模拟物阴性对照进入MC3T3-E1细胞内,Real-time PCR检测2组细胞miR-103-3p的表达水平,CCK-8试剂盒检测细胞增殖。分别对2组细胞进行成骨诱导,在成骨诱导后0、3、7 d,分别使用Real-time PCR和Western免疫印迹检测2组细胞Runx2、Osx等成骨相关基因mRNA和蛋白的表达变化,并对2组细胞进行ALP染色。实验数据采用SPSS19.0软件包进行统计学分析。结果:MC3T3-E1经成骨诱导0、3、5、7 d后,细胞内Runx2、Osx、ALP转录水平持续显著升高;Runx2、Osx蛋白表达升高。ALP染色逐渐加深。在成骨诱导3、5、7 d的MC3T3-E1细胞中,miR-103-3p水平较诱导前受到持续显著抑制(P<0.05)。瞬时转染miR-103-3p mimics后,MC3T3-E1细胞中的miR-103-3p表达水平较对照组显著上调(P<0.05),细胞增殖受到抑制,Runx2、ALP转录水平显著抑制（P<0.05）,Runx2蛋白表达显著抑制。对转染后的细胞进行成骨诱导3、7 d后,miR-103-3p转染组细胞Runx2、Osx、ALP在转录水平的表达较对照组显著降低,Runx2、Osx在蛋白水平的表达较对照组显著降低,且miR-103-3p转染组细胞ALP活性较对照组显著降低。结论:miR-103-3p可能对小鼠前成骨细胞MC3T3-E1的成骨分化早期起抑制作用。     

人脐带Wharton's Jelly来源间充质干细胞与人牙周膜干细胞成骨分化能力的对比研究

目的:比较人脐带Wharton's Jelly来源间充质干细胞(human umbilical cord Wharton's Jelly-derived mesechymal stem ceils,hUCWJMSCs)与人牙周膜干细胞(periodontal mesenchymal stem cells,hPDLSCs)成骨分化能力.方法:体外培养hUC-WJMSCs和hPDLSCs.MTT法检测细胞增殖情况;成骨诱导后测定细胞的ALP活性,茜素红染色检测细胞矿化能力,Real-timePCR分析OPN和Runx2基因的表达.结果:hUCWJMSCs增殖能力高于hPDLSCs;经矿化诱导后hPDLSCs ALP表达、矿化结节形成高于hUCWJMSCs(P＜0.05);Runx2在hPDLSCs中表达高于hUCWJMSCs(P ＜0.05);而hUCWJMSCs中OPN表达高于hPDLSCs(P＜0.05).结论:hUCWJMSCs、hPDLSCs均具有成骨分化能力,hPDLSCs成骨分化能力较强.