In vivo monitoring of the bone healing process around different titanium alloy implant surfaces placed into fresh extraction sockets

Objectives

Increasing surface roughness and coating with tricalcium phosphate of titanium and titanium alloy implants has been proposed to provide better rates of osseointegration. However, how these changes in surface topography and chemistry influence the osseointegration process of immediate implants placed in fresh extraction sockets is unclear. This study investigated the influence of three clinically employed implant surfaces on the early bone healing events in vivo.

Methods

Machined smooth implants were milled from grade 5 Ti6Al4V titanium. Surfaces were moderately roughened by grit blasting, which were then coated with tricalcium phosphate. Implants were placed into freshly extracted incisor sockets of mandibles of normal Wistar rats and left for 1, 3 and 9 weeks. Healing bone tissue around the implants was examined by histochemistry and immunocytochemistry to localise PCNA proliferative cells, and osteoblast differentiation markers osteopontin and osteocalcin. Positive synthesising cells were counted using image analysis.

Results

Histology indicated no differences in the amount or pattern of bone formation within the healing tissue surrounding the different implant surfaces. Bone healing occurred predominantly on exposed bone surfaces (distance osteogenesis) and not on the implant surface (contact osteogenesis). No differences were observed in the number or timing of PCNA, osteopontin and osteocalcin positive cells within the bone healing tissue around each of the implant analysed.

Conclusion

For immediately placed implants, the surface modifications investigated appeared to have little influence on the activity of bone forming cells surrounding the implant, probably due to the high level of distance osteogenesis seen within this scenario.

Clinical significance

For immediate placement of implants into fresh extraction sockets, titanium implants with roughened surfaces and coating with tricalcium phosphate have negligible influence in accelerating the early bone healing events of osseointegration.     

骨母细胞性肿瘤的免疫组织化学研究

选择３２例骨母细胞性肿瘤，包括１４例良性骨母细胞瘤、２例侵袭性骨母细胞瘤和１６例骨母细胞型骨肉瘤，用ＡＢＣ法作多种抗血清标记（ｖｉｍｅｎｔｉｎ、Ｓ－１００、α－ＡＴ、ｌｙｓｏｚｙｍｅ、Ｌｅｕ－７、Ｋ１２和ＣＥＡ）。结果显示：３２例肿瘤性骨母细胞ｖｉｍｅｎｔｉｎ均呈不同程度的阳性反应；在６例骨母细胞型骨肉瘤和１例侵袭性骨母细胞瘤中散在的单个细胞Ｓ－１００蛋白呈阳性反应，表明肿瘤性的骨母细胞具有软骨分化的潜能１２４例肿瘤中的多核巨细胞α－ＡＴ和１５例肿瘤中的多核巨细胞ｌｙｓｏｚｙｍｅ均呈不同程度的阳性反应，进一步证实这些细胞可能是组织细胞起源；６例骨母细胞型骨肉瘤和４例良性骨母细胞瘤中的骨样基质Ｌｅｕ－７呈阳性反应。     

大豆异黄酮对成骨细胞增殖活性的测定和BGP mRNA的表达

目的研究不同浓度的大豆异黄酮对体外培养大鼠成骨细胞增殖活性和骨钙素(BGP)基因表达的影响。方法采用新生大鼠颅骨分离培养成骨细胞,加入不同浓度的大豆异黄酮(20、40、60、80μg/μL),另设空白对照组。MTT法测成骨细胞增殖活性。提取细胞总RNA,RT-PCR半定量分析细胞BGP mRNA表达。结果 MTT结果显示,60和80μg/μL组成骨细胞增殖活性与对照组相比显示增高,有统计学意义(P<0.05)。RT-PCR结果显示:60和80μg/μL组BGP mRNA表达增强,有统计学意义(P<0.01)。结论大豆异黄酮可以促进成骨细胞的增殖活性和BGP基因上调,并呈明显的剂量依赖关系。     

组织金属蛋白酶抑制因子3重组蛋白诱导小鼠成骨细胞MC3T3-E1凋亡

目的 研究组织金属蛋白酶抑制因子3（TIMP-3）重组蛋白对MC3T3-E1成骨细胞凋亡的影响。方法 细胞存活率和凋亡分别用MTT及ELISA检测。Fas，FasL，Bcl-2，Bax，caspase-3，caspase-8，细胞色素c的表达以及JNK，p38，细胞外信号调节激酶（ERK）1／2的磷酸化水平用Western印迹检测。结果 MTT及ELISA检测提示TIMP-3降低MC3T3-E1细胞存活率，诱导MC3T3-E1细胞凋亡。TIMP-1增加Fas、FasL蛋白表达，对Bax、Bcl-2蛋白表达无影响，但可诱导细胞色素c的释放及caspase-8、caspase-3的活化。TIMP-3促进p38和ERK磷酸化，而PD098059（ERK阻断剂）和SB203580（p38阻断剂）消除p38和ERK的促凋亡作用。结论 TIMP-3诱导MC3T3-E1细胞凋亡的信号途径为凋亡受体Fas介导，并有p38、ERK信号转导途径参与。     

胰岛素样生长因子-1对甲状旁腺激素介导成骨细胞增殖及成骨活性的影响

目的：通过构建乳鼠颅盖骨成骨细胞分离培养与鉴定方法,研究胰岛素样生长因子－1（Insulin－like growth factor－1,IGF－1）及IGF－1联合重组人甲状旁腺激素1－34（recombinant human parathyroid hormone 1－34,rhPTH1－34）对成骨细胞增殖及I型胶原蛋白mRNA表达的影响。方法培养乳鼠成骨细胞,并观察其形态及功能,以原代培养乳鼠成骨细胞为实验模型,分空白组、PTH组、IGF－1组及不同浓度IGF－1作用的PTH介导的成骨细胞组（0、10、50、100 ng／L）。通过不同剂量的胰岛素样生长因子－1（0、10、50、100 ng／L）联合10－9 mol／L重组人甲状旁腺素刺激体外培养的成骨细胞,用噻唑蓝（ MTT）法检测细胞的增殖能力,RT－PCR法检测成骨细胞I型胶原蛋白mRNA的表达。结果 PTH和IGF－1均可促进成骨细胞增殖;IGF－1联合PTH可以促进成骨细胞增殖,且具有剂量依赖性。 PTH联合IGF－1使成骨细胞增殖能力增强、I型胶原蛋白mRNA表达增强。 IGF－1可以促进成骨细胞I型胶原蛋白mRNA的表达,且具有剂量依赖性。 IGF－1可以促进成骨细胞I型胶原蛋白mRNA的表达,且具有剂量依赖性。结论 PTH与IGF－1均可刺激成骨细胞增殖和分化,IGF－1可促进PTH对成骨细胞的分化、增殖。两者合用其作用增强,有协同促进作用。     

Effects of PEMF exposure at different pulses on osteogenesis of MC3T3-E1 cells

Objective

Pulsed electromagnetic fields (PEMFs) were considered to be a factor which may affect osteogenesis of osteoblasts, but the effects were diverse with different PEMF parameters. The aim of the current study is to explore the effects of exposure to PEMFs at different pulse number on osteogenesis of osteoblasts.

Design

The mouse osteoblast-like MC3T3-E1 cells were exposed to 0, 400 or 2800 pulses 400 kV/m PEMF and the proliferation, differentiation and mineralization of cells were observed after PEMF exposure by the methods of MTT, biochemical measurement, real-time PCR and Alizarin Red assay.

Results

Compared with 0 pulses groups, the growth curve, alkaline phosphatase (ALP) activity, mRNA level of osteocalcin (OCN) and mineralized nodule formation of MC3T3-E1 cells did not change after 400 pulses PEMF exposure, but decreased after 2800 pulses PEMF exposure. It suggested that under our experimental conditions, only 2800 pulses 400 kV/m PEMF exposure can suppress the proliferation, differentiation and mineralization of MC3T3-E1 cells, but 400 pulses 400 kV/m PEMF exposure cannot.

Conclusions

Pulse number is another involved parameter which may influence the effects of PEMF on osteogenesis of osteoblasts.     

Lipopolysaccharide derived from Aggregatibacter actinomycetemcomitans inhibits differentiation of osteoblasts

Background and objectiveAlveolar bone defects in aggressive periodontitis are generally caused by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) through osteoclast differentiation facilitated by the interaction between the host's immune system, stimulated by the bacterial outer components of A. actinomycetemcomitans, and osteoblasts. Recently, some reports on the direct effects of A. actinomycetemcomitans on osteoblast differentiation have been published. However, the mechanisms in detail have still remained unknown. We herein demonstrated that A. actinomycetemcomitans might inhibit differentiation of osteoblasts and identified a causative substance.Materials and methodsThe following culture supernatants of A. actinomycetemcomitans were added to mouse osteoblasts, MC3T3-E1: supernatants fractionated by the molecular weight, supernatant supplemented with proteinase K and then heated, and supernatant supplemented with polymyxin B. Subsequently, RNA was extracted to determine the expressions of bone differentiation markers, alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP). Additionally, lipopolysaccharide (LPS) was added to MC3T3-E1 culture to measure the markers.ResultsA. actinomycetemcomitans culture supernatant, added to the MC3T3-E1, inhibited the expressions of bone differentiation markers at all concentrations. Only the fraction with a molecular weight of >100 kDa inhibited the bone differentiation. Neither proteinase K nor heating had an effect. However, polymyxin B completely abrogated the differentiation inhibitory activity. A. actinomycetemcomitans LPS concentration dependently inhibited the expressions of the bone differentiation markers.ConclusionsA. actinomycetemcomitans LPS suppressed osteoblast differentiation. This suggests that suppressed bone differentiation is involved in the alveolar bone destruction.     

牙龈卟啉单胞菌脂多糖对成骨细胞EphA2表达的调控

目的：研究小鼠前成骨细胞系MC3T3-E1在成骨分化过程中,牙龈卟啉单胞菌脂多糖（Pg-LPS）对EphA2表达的影响。方法：使用终浓度1mg/L的Pg-LPS与MC3T3-E1共培养,分别在第3、7、14天3个时间点,采用RT-PCR和Western-Blot技术测定EphA2的表达和成骨相关基因的表达,并通过PNPP法测定碱性磷酸酶活性。结果：RT-PCR结果提示,在第3天和第7天,实验组比对照组EphA2基因表达分别增高4.5倍和1.3倍,两组之间存在显著差异（P〈0.05）。同时成骨标志物ALP和Sp7基因表达降低,与对照组相比差异有显著性,但Runx2和ColⅠ基因的表达则无明显差异。但是在第14天,实验组和对照组之间EphA2和成骨相关基因表达均无显著性差异。Western-Blot结果提示,在第7天实验组比对照组EphA2蛋白表达增加。结论：Pg-LPS对前成骨细胞系MC3T3-E1细胞,在成骨分化的早期和中期能够促进EphA2的表达,但是在成骨分化晚期对EphA2的表达则无明显作用。     

静磁场促进成骨作用机制的研究进展

相关研究表明,静磁场可以促进细胞成骨作用,并已经广泛应用于临床治疗,但关于其具体机制,至今仍然没有定论。大量学者通过实验试图明确这一机制。本文就此方面的研究进展进行系统阐述。