钛颗粒对破骨细胞骨吸收功能影响的研究

目的:研究钛颗粒对破骨细胞骨吸收功能的影响。方法:分别将含有和不含有钛颗粒的培养基与诱导的破骨细胞联合培养48h后,检测破骨细胞的骨吸收陷窝,破骨细胞对钛颗粒的吞噬及钛颗粒对破骨细胞骨架的影响。结果:钛颗粒培养基组份的骨吸收陷窝面积明显大于比不含钛颗粒培养基的组份,钛颗粒可被破骨细胞吞噬,但对其细胞骨架无显著影响。结论:钛颗粒可促进破骨细胞的骨吸收功能。     

丹参对骨髓培养中破骨细胞生成的影响

目的：研究丹参对体外骨髓细胞培养中破骨细胞生成的作用。方法：采用小鼠长骨骨髓体外培养方法,通过检测抗酒石酸磷酸酶（ＴＲＡＣＰ）阳性细胞生成数,反映破骨细胞的生成情况。结果：１．０ｇ／Ｌ丹参可明显抑制ＴＲＡＣＰ阳性细胞的生成,其生成数仅为对照组的２９．８％;ＰＧＥ２（１０－７ｍｏｌ／Ｌ）及１．２５（ＯＨ）２Ｄ３（１０－８ｍｏｌ／Ｌ）产生明显的促进作用,与对照组比较,ＴＲＡＣＰ阳性细胞生成数分别增加了６．０４和６．６８倍;丹参还可明显抑制ＰＧＥ２和１．２５（ＯＨ）２Ｄ３的作用,混合培养组ＴＲＡＣＰ阳性细胞生成数仅仅是单纯加ＰＧＥ２和１．２５（ＯＨ）２Ｄ３组的３５．２％和３９．３％,特别是多核破骨细胞数大量减少。结论：丹参可抑制骨髓细胞体外培养中破骨样细胞的生成,主要抑制破骨母细胞向成熟破骨细胞的转化,而成骨细胞可能在其中发挥了作用     

Ceramic hydroxyapatite coating on titanium implants drives selective bone marrow stromal cell adhesion

The aim of this study was to determine the cell characteristics that regulate implant osseointegration. The heterogeneity of bone marrow stromal cells obtained from 11 donors was assessed by measuring the expression of a large panel of adhesion molecules. Large differences in expression of adhesion molecules were detected depending on the culture conditions used. Cells cultured in fetal bovine serum induced the expression of different adhesion molecules from cells cultured in human serum. Donor-to-donor variation was determined by measuring the expression of adhesion molecules for stromal cells obtained from different donors that were processed identically. Fat adherent cells but also loose bone marrow cells showed large differences in expression of some but not all adhesion molecules. The flow cytometric data demonstrated large heterogeneity in expression of adhesion molecules, and this heterogeneity was influenced by culture conditions and varied from donor to donor. This demonstrates that the implant encounters different cell types, which could lead to different levels of integration. Surprisingly, in vitro only a subfraction of bone marrow stromal cells attached to titanium coated with ceramic hydroxyapatite. Adaptation of all cell types present in heterogeneous bone marrow to a coated surface is apparently not possible. Differential binding was not caused by aberrant staining of the stromal cells as the results were confirmed with bone marrow cells obtained from transgenic GFP mice. These results demonstrate that hydroxyapatite ceramics are selective in cell recruitment from the bone marrow, explaining the differences found in vivo for these coatings compared with titanium.     

周期性牵张力对骨髓破骨细胞形成的影响

目的 :通过大鼠骨髓破骨细胞的体外诱导培养 ,观察不同时段周期性牵张力对骨髓破骨细胞形成的影响。方法 :通过体外细胞培养加载系统 ,选择频率为 6周 /min ,弹性基底膜发生 12 %形变率的周期性牵张力。试验共分为 4组 ,A组 :为对照组 ,在整个培养期间不加力 ;B组 :从培养的第 2d开始加力 ,连续加力 3d ;C组 :从培养的第 2d开始加力 ,连续加力 5d ;D组 :从培养的第 5d开始加力 ,连续加力 2d。每组各 6孔 ,均于培养的第 7d进行多核细胞计数。结果 :B组、C组多核细胞数量与对照组相比明显减少 (P <0 .0 5 ) ,而D组多核细胞数量与对照组相比无显著性差异 (P >0 .0 5 )。B组与C组相比亦无显著性差异。结论 :周期性牵张力主要是在骨髓诱导培养的早期抑制破骨细胞的形成 ,后期施力对破骨细胞的形成无明显影响。     

小鼠口腔癌淋巴道转移模型骨髓播散肿瘤细胞的检测

目的：检测小鼠口腔癌淋巴道转移模型癌变过程不同时期的骨髓播散肿瘤细胞（disseminated tumor cell,DTC）,探讨小鼠口腔癌变过程与骨髓DTC的关系.方法：病理检查明确诊断为正常舌黏膜、舌单纯性增生的小鼠各10只,舌轻中度异常增生、舌重度异常增生、舌鳞癌的小鼠各20只.采用Ficoll密度梯度离心法提取小鼠股骨骨髓里单个核细胞并制成细胞涂片,免疫细胞化学（Immunocytochemistry,ICC）检测骨髓DTC,比较其数目差异.结果：舌重度异常增生8只、舌鳞癌组13只发现DTC,阳性率分别为40％（8/20）和65 ％（13/20）,每100个单核细胞中DTC数分别为3.03±0.75个和5.20±0.74个,差异均有统计学意义（P＜0.05）.其余各组均未发现DTC（0/10）.结论：小鼠舌黏膜恶变过程中,舌重度异常增生时已出现DTC,并随病变程度加重,骨髓DTC的发生率及其细胞数量增加.     

骨形态发生蛋白-2与碱性成纤维细胞生长因子在异位和原位成骨中的作用

目的通过骨髓基质细胞（BMSCs）的体外增殖和分化、异位成骨和原位成骨实验来观察骨形态发生蛋白-2（BMP-2）和碱性成纤维细胞生长因子（bFGF）在成骨过程中的作用。方法分别用含BMP-2、bFGF和BMP-2+bFGF的培养液体外培养Beagle犬的BMSCs,通过甲基噻唑基四唑（MTT）比色法测定细胞增殖水平,通过测定碱性磷酸酶（ALP）活性观察细胞的分化情况。将BMSCs与多孔磷酸钙（CPC）分别在含BMP-2、bFGF和BMP-2+bFGF的培养液中复合培养,制成复合材料,一部分植入裸鼠皮下,观察异位成骨情况,另一部分植入Beagle犬的种植体周围骨缺损区,经过荧光标记观察原位成骨情况。结果含有BMP-2+bFGF的培养液促进BMSCs增殖和分化的能力最强。异位成骨情况：BMP-2+bFGF组的成骨量较其他组明显增加,其新骨形成百分比为48.79%±11.31%,高于单一BMP-2组（30.71%±10.85%）和bFGF组（27.33%±9.67%）以及对照组（10.65%±6.05%）。原位成骨术后12周,BMP-2+bFGF组的矿化沉积率高于其他组,其差异有统计学意义（P<0.01）。结论在促进成骨方面,BMP-2和bFGF共同作用优于单一因子。     

BMSCs复合PRF修复牙槽骨缺损中OPG和RANKL的表达及意义

目的：探讨骨髓间充质干细胞（BMSCs）复合富血小板纤维蛋白（PRF）修复牙槽骨缺损中骨保护素（OPG）和核因子B受体活化因子配体（RANKL）的表达及意义。方法：取健康雄性2月龄新西兰兔36只,随机分为A、B、C、D 4组,均在全麻下微创拔除下颌左侧中切牙。 A组植入BMSCs与PRF复合物,B组植入PRF,C组植入BMSCs,D组为空白对照组。按术后4,8,12周3个时间点（每个时间点9只）处死动物并立即于骨缺损部位取材,免疫组织化学方法检测OPG和RANKL的表达。结果：4,8,12周时A组、B组、C组OPG的表达高于D组（P＜0．05）;4,8,12周时A组、B组、C组RANKL的表达高于D组（P＜0．05）;析因分析显示自体BMSCs复合PRF修复牙槽骨缺损中OPG,RANKL的表达高于单独使用BMSCs或PRF（P＜0．05）。结论：自体BMSCs复合PRF修复牙槽骨缺损会增加OPG,RANKL的表达,有助于牙槽骨改建。     

钛颗粒对小鼠骨髓间充质干细胞生物学行为影响的体外研究

目的：研究钛颗粒对小鼠骨髓间充质干细胞生物学行为的影响。方法：将钛颗粒脱内毒素处理后,与小鼠BMSC共培养,检测钛颗粒对它们存活率、增殖率的影响,并通过透射电镜检测细胞对钛颗粒的吞噬情况。此外,对细胞骨架F-actin进行荧光染色,观察钛颗粒对BMSC细胞骨架的影响。结果：钛颗粒降低了BMSC的生存率并抑制其增殖活性,该抑制作用随钛颗粒浓度的增加而升高。BMSC可吞噬钛颗粒,而且钛颗粒可影响BMSC的正常细胞骨架。结论：钛颗粒对BMSC具有抑制作用,促进了种植体周无菌性骨吸收。     

Dynamics of bone marrow pressure with tapping of titanium and hydroxyapatite implants in rabbits

The purpose of the present study was to evaluate the difference in stress transfer between titanium (Ti) and hydroxyapatite (HA) by the measurement of bone marrow pressure using a catheter pressure transducer. Ti and HA implants were inserted in the tibiae of rabbits. A hole of 1 mm in diameter was drilled in the bone and a fine catheter pressure transducer was placed in the bone marrow through a tube. The top of the abutment was vertically tapped with an impulse hammer, and the acceleration signal from the hammer and pressure signal from the catheter pressure transducer were examined. The time of contact (impulse duration) recorded in the impulse with Ti and HA was 166+/-17 micro sec and 164 +/- 17 micro sec, respectively. Maximum bone marrow pressure (BMP) with Ti and HA was 54.2 +/- 32.6 and 47.5 +/- 10.0 mmHg, respectively. Variation of the BMP with Ti was significantly larger than that with HA (P < 0.05). A negative correlation coefficient between impulse duration and BMP was found. The results of the present study suggest that the stress transfer is different between Ti and HA implants using dynamics of the bone marrow pressure.     

白介素-6与1,25(OH)_2维生素D_3联合作用下大鼠骨髓破骨细胞的体外诱导培养

目的观察联合应用白介素(IL)-6和1,25(OH)2D3对大鼠骨髓破骨细胞生成诱导的影响。方法采用4周龄SD大鼠无菌条件下取出股骨,剪去两骺端,以α-MEM培养液将骨髓细胞冲出,然后将细胞悬液接种于预置盖玻片或牙本质片的24孔培养板内,24 h后换液。试验分为4组,A组:不加任何诱导因子;B组:加入IL-6(10 U/ml);C组:加入1,25(OH)2D3(1×10-8mol/L);D组:加入IL-6(10 U/ml)和1,25(OH)2D3(1×10-8mol/L)。每组各6孔,于培养的第7天进行多核细胞计数。结果培养1周左右IL-6与1,25(OH)2D3在体外均可单独诱导骨髓破骨细胞的形成,但D组多核细胞数与B、C组相比差异有显著性(P<0.05)。结论IL-6与1,25(OH)2D3联合作用下对骨髓破骨细胞生成的诱导效果明显高于单因素诱导效果。     

Biostimulation of bone marrow cells with a diode soft laser

In recent years, the use of low-intensity red light in regeneration of soft tissue has been increasingly pursued. As far as hard tissue is concerned, the biostimulating effect of laser has already been demonstrated successfully in more rapid healing of tibial bone fractures in mice at a dosage of 2.4 J. However, the effect of light of a low dose laser directly on osteoblasts has not been investigated yet. The aim of this study was to determine the effect of continuous wave diode laser irradiation on osteoblasts derived mesenchymal cells. Three groups of 10 cultures each were irradiated 3 times (days 3, 5, 7) with a pulsed diode soft laser with a wavelength of 690 nm for 60 s. Another 3 groups of 10 cultures each were used as control groups. A newly developed method employing the fluorescent antibiotic tetracycline was used to compare bone growth on these culture substrates after a period of 8, 12 and 16 days, respectively. It was found that all lased cultures demonstrated significantly more fluorescent bone deposits than the non-lased cultures. The difference was significant, as tested by the Tukey Test (P < 0.0001) in the cultures examined after 16 days. Hence it is concluded that irradiation with a pulsed diode soft laser has a biostimulating effect on osteoblasts in vitro, which might be used in osseointegration of dental implants.     

Effect of prostaglandin E2 on recombinant human bone morphogenetic protein-2-stimulated osteoblastic differentiation in human periodontal ligament cells

Recombinant human (rh) bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic differentiation in cells isolated from human periodontal ligament (HPLC), and this action of rhBMP-2 may be modulated by prostaglandins (PGs), which are local regulatory factors in the bone metabolism. In the present study, we investigated the effect of prostaglandin E2 (PGE2) on rhBMP-2-stimulated osteoblastic differentiation in cultured HPLC. rhBMP-2 (500 ng/ml)-stimulated alkaline phosphatase (ALPase) activity was enhanced by simultaneous treatment with low concentrations (10(-10)-10(-8) M) of PGE2, whereas a high concentration (10(-6) M) of PGE2 suppressed it. rhBMP-2 did not induce cyclo-oxygenase-2 (COX-2) mRNA expression or subsequent PGE2 production, whereas it remarkably suppressed rhIL-1 beta-induced COX-2 mRNA expression and PGE2 production. The rhBMP-2 action on osteoblastic differentiation in HPLC was also enhanced by co-treatment with 0.25 to 25 ng/ml of rh interleukin-1 beta (IL-1 beta). The ALPase activity stimulated by simultaneous treatment with rhBMP-2 and rhIL-1 beta was partially inhibited by addition of 10(-6) M of indomethacin, which completely inhibited rhIL-1 beta-induced PGE2 production. These results reveal that PGE2 at different concentrations exerts a biphasic effect on BMP-2-stimulated osteoblastic differentiation in HPLC, BMP-2 inhibits IL-1 beta-induced PGE2 production through suppressing COX-2 expression, and the BMP-2-stimulated osteoblastic differentiation may be enhanced by the endogenous PGE2 induced by BMP-2 and IL-1 beta. These suggest that BMP-2 action on osteoblastic differentiation in HPLC may be modulated by PGE2 in autocrine and paracrine fashions.     

三维打印含铜钛合金对骨髓间充质干细胞成骨分化的影响

目的: 探讨三维打印含铜钛合金的体外生物相容性和对骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）成骨分化的影响。方法: 以Ti-6Al-4V-5Cu合金粉末为原料,通过选择性激光熔化（selective laser melting, SLM）技术制备三维打印含铜钛合金（3D Ti-5Cu）;将BMSCs与3D Ti-5Cu合金共同培养,观察其对细胞活性、黏附、增殖、碱性磷酸酶（ALP）活性及成骨分化相关基因表达的影响,以医用钛合金（Ti-6Al-4V）作为对照组。采用 GraphPad Prism 9.3.1软件包对数据进行统计学分析。结果: 成功制备3D Ti-5Cu合金,该新型合金无细胞毒性,与对照组相比无统计学差异（P>0.05）。3D Ti-5Cu合金能促进BMSCs的早期黏附、增殖,并显著提高ALP活性和Runx2、Alp、Bmp2、Opn及Col1a1等成骨分化基因的表达水平（P<0.05）。结论: 3D Ti-5Cu合金具备出色的生物相容性,能促进BMSCs的成骨分化,有利于改善钛合金的生物惰性,为实现钛合金的生物功能化提供了理论依据。     

间接共培养诱导大鼠骨髓间充质干细胞向牙周膜成纤维样细胞分化

目的：探讨通过间接共培养诱导骨髓间充质干细胞（Bone marrow mesenchymal stem cells,BMSCs）向牙周膜成纤维细胞（Periodontal ligament fibroblasts,PDLFs）分化的可行性。方法：将大鼠BMSCs与PDLFs间接共培养,分别于不同时间段检测碱性磷酸酶（Alkaline phosphatase,ALP）活性以及骨钙素（Osteocalcin,OCN）、Ⅲ型胶原（Collagen typeIII,COLⅢ）mRNA表达的变化。结果：间接共培养后的BMSCs表现出类似PDLFs的性质,各检测指标与单独培养的PDLFs无统计学差异,与单独培养的BMSCs有统计学差异。结论：牙周膜成纤维细胞通过旁分泌机制可以诱导骨髓间充质干细胞向其分化。     

降钙素基因相关肽通过Hippo通路调控小鼠骨髓间充质干细胞成骨分化的实验研究

目的 前期研究发现降钙素基因相关肽（CGRP）能够促进成骨细胞的生物学活性,为了进一步揭示CGRP在骨修复中的作用,检测CGRP对小鼠骨髓间充质干细胞（BMSCs）成骨分化的影响,并对Hippo通路在这个过程中的作用进行了初步探讨。方法 在体外诱导培养的BMSCs中加入不同浓度的CGRP,处理48 h后测试碱性磷酸酶（ALP）活性,以筛选的优势浓度;处理7 d后进行茜素红染色,分别检测细胞的分化情况。应用Western blot检测CGRP作用于BMSCs后,Hippo通路核心分子Mst1/2蛋白磷酸化的表达水平;利用Hippo通路抑制剂维替泊芬（Verteporfin）阻断下游Yap信号,逆转录聚合酶链反应检测其对成骨相关因子Ⅰ型胶原蛋白（ColⅠ）、Runt相关转录因子2（Runx2）mRNA的表达影响。结果 ALP活性检测结果显示,与空白对照组相比,10^-9、10^-8、10^-7 mol·L^-1浓度范围的CGRP都能显著促进小鼠ALP活性的增加（P<0.05）,以10^-8 mol·L^-1浓度的CGRP为最佳刺激浓度。用10^-8mol·L^-1浓度的CGRP处理后,茜素红染色显示钙化结节明显增多。CGRP能够显著上调p-Mst1/2蛋白的表达（P<0.05）;当运用了抑制剂Verteporfin时,显著降低了CGRP诱导的Runx2、ColⅠ mRNA的表达（P<0.05）。结论 CGRP能够促进小鼠BMSCs的成骨分化,且Hippo信号通路介导了CGRP作用于小鼠BMSCs成骨分化的过程。     

钛网加强的BMSC/松质骨基质修复下颌骨缺损的实验研究

目的：观察钛网加强的骨髓基质细胞(BMSC)／松质骨基质复合物修复下颌骨缺损的能力。方法：体外培养兔BMSC经扩增,诱导分化后复合同种异体松质骨基质,植入自体下颌骨缺损区,修复骨缺损,钛网固位和加强,植入6周、12周后经X线,组织学检查,观察骨形成情况。结果：骨髓基质细胞(BMSC)/松质骨基质复合物有很强的成骨作用,实验组X线观察有骨形成,组织学染色证实有新骨形成,骨磨片显示钛网和新骨获得良好的愈合。结论：钛网加强的BMSC／松质骨基质可诱导修复兔下颌骨缺损,为组织工程方法修复骨缺损提供了新的思路。     

新型多孔磷酸钙复合骨髓基质干细胞组织工程实验研究

目的观察新型生物材料多孔磷酸钙的生物相容性及复合骨髓基质干细胞（BMSCs）异位成骨情况。方法体外培养第2代Beagle犬BMSCs,转染绿色荧光蛋白（GFP）后与多孔磷酸钙（CPC）复合培养,获得最佳复合浓度,倒置和荧光显微镜、扫描电镜下观察BMSCs黏附和生长情况,复合体植入裸鼠皮下8周观察异位成骨。结果BMSCs转染GFP与多孔CPC复合培养1 d,细胞从材料中爬出,形态正常,7 d可见细胞伸出伪足,分泌基质;复合体可异位成骨。结论多孔CPC生物相容性好,是一种较理想的骨组织工程支架材料。