TNF-α对负载种植体周骨髓间充质干细胞成骨分化和骨创伤修复影响
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摘要
研究背景和目的
种植学在国内起步时间晚,但是发展速度快。人们对其优缺点已耳熟能详,且己成为临床修复学的重要组成部分。在种植学发展中,学者们不断地对影响其成功率的重要因素之一—生物力学进行探索并取得了很大的进展：如在种植时机、负载时间等相关问题的不断提出探讨并将其成果付诸实践。
基于生物力学考虑,受到生理应力刺激的骨组织与无生理应力刺激相比,其成骨和抗应力能力是增强的。对种植体行早期负载或即刻负载可能有利于骨结合的形成。近年来相关研究逐步增多,在一定的生理条件下,避免过度的应力集中是可以获得较好骨结合的。相关的理论技术较为成熟,在临床上受到患者认可而已经广泛的应用。有学者甚至认为,主张对种植体延期负载仅仅是基于感性的经验考虑。种植义齿在受到咬合力的刺激后,种植体骨界面发生相应骨改建,这是与应力作用下的骨代谢相密切相关联的。
种植体倾斜一定角度后,其周围应力分布发生相应变化,这种应力变化必然对骨组织产生影响,其影响程度和研究目前知之甚少。在种植义齿行使功能过程中,由于解剖因素、咬合方向变化会导致咬合力方向发生不同变化。由此而会导致应力相同的情况下因角度变化产生种植体周围的应变则会不同。另一方面,对种植体骨界面所产生的骨结合不利之处在于种植体与骨组织应力传导过程中缺乏应有的缓冲而可能发生种植体骨界面的微小裂痕。这种现象主要是由于在应力传递在弹性模量差度较大两种材料——种植体与骨组织问,因为弹性模量相差越大,两者应力引起的形变差距越大。应力分布不同会相应的引起其作用部位骨组织的改建差异。应力的差异越大,相应的,改建的效果差异亦越大。而这种由不同角度引起的应力对种植体周围微环境是何种影响以及影响的程度如何的,骨组织在受这种角度引起的应力后在其引起微环境相关因子-TNF-α变化的影响下是如何进行改建的,相关研究较少。
TNF-α是多种生物学活性的炎症因子,在牙周病发展过程中对组织造成破坏的主要因子。通过研究得知,它在牙周炎的发病机理中扮演着起重要的作用。因为TNF-α是促炎症反应的一种免疫调节剂,可以促炎性细胞进入感染部位,使金属蛋白酶的释放。后者降解细胞外基质蛋白,促进胶原纤维的破坏和牙槽骨吸收,同时调节牙周膜与牙龈成纤维细胞的增殖。TNF-α在健康和治疗成功处于较低水平的位点,而在活动性破坏的位点却是较高。在多数细胞中,TNF-α与TNFR1的结合激发TNFR结合的死亡受体结构域蛋白(TRADD)、TNF受体结合因子2(TRAF2)、受体结合蛋白(RIP)复合物的形成。TNF-α促进破骨细胞的生成,提高破骨细胞活性,以此来促进骨吸收,同时亦有抑制成骨细胞骨形成。TNF-α可以抑制前成骨细胞分化成骨细胞,抑制骨钙蛋白、成骨分化基因RUNX2和I型胶原的表达。这说明TNF-α对骨吸收的影响至关重要,因此学者们不断对其表达阻断进行研究以期获得对其治疗的方法。
近年来,针对TNF-α的研究逐渐增多,涉及范围逐渐扩展至种植学科领域。主要是通过种植体周围炎的研究相对较多,研究方式通过测量种植体周围龈沟液的含量变化进行测定。但临床上的很多的种植体周围炎病因并不非常明确,针对其预防更是无从谈起。而目前针对由于生物力学因素引起的种植体周围微环境的变化研究相对较少,特别是针对TNF-α影响更少。通过临床可看出种植体植入时基本与原位自然牙的长轴存在一定的角度,这种角度所引起的种植体周围应力分布变化研究相对较少。在这种应力作用下,TNF-α的变化如何以及其相应变化与骨界面的改建影响如何,特别是对种植体周成骨细胞分化成骨的影响如何,未见相关研究。倾斜角度大小与种植体周围TNF-α变化关系如何,以及两者对成骨细胞分化成骨影响状况影响,目前尚未有研究见诸报道。
在临床上负载种植体周围骨组织改建无时不在进行中。为了明确以下问题：负载种植体周围微环境中的TNF-α的变化如何、对负载的种植体周围骨组织改建影响如何、TNF-α浓度变化的影响和种植体骨改建变化以及对其中的种植体周骨髓间质干细胞成骨分化的影响状况,本课题研究中通过不同角度负载的种植体周围应力变化不同而测定其微环境中TNF-α的变化,将此为依据进行种植体周骨髓间充质干细胞成骨分化和骨创伤修复影响研究。我们选择临床病例龈沟液量、TNF-α浓度测定；依据临床角度测定结果,通过动物实验观察龈沟液量、TNF-α浓度变化对骨结合情况和骨改建的影响；综合临床病例和动物实验结果,研究TNF-α对BMSCs增殖和成骨分化的影响,以探讨炎性微环境对干细胞增殖与分化的影响,可以进一步明确TNF-α对种植体骨界面骨组织变化的影响,有效地为临床检测TNF-α的水平变化提供预警,进行早预防、早治疗,为针对性的相关预防、治疗措施提供依据；为应用干细胞修复在炎性微环境中破坏的种植体周组织提供理论指导。
研究方法
1.自临床选择种植义齿病例,测定相应倾斜角度,按照术前设计模型测定与牙体长轴方向夹角归为三组：0°—10°、10°—20°及20°以上。在负载后一个月及六个月后行采集龈沟液进行定量分析和检测TNF-α,测定TNF-α浓度随种植体倾斜角变化；
2.通过在按照临床选择病例所测得倾斜角度,制作模板,常规方法将种植体植入成年犬颌骨内并于负载一个月及六个月后,用whatman42试纸行种植体周龈沟液的采集和定量检测TNF-α,同时采取骨标本进行骨结合、骨密度、骨新生等检测。对比分析不同角度倾斜种植体负载与测定的种植体龈沟TNF-α变化相关性；
3.小鼠骨髓间充质干细胞的取材、培养、分离纯化,建立成骨细胞负载模型,免疫细胞化学染色和矿化诱导初步鉴定其干细胞特性；MTT法检测不同浓度TNF-α对负载BMSCs增殖的影响；PNPP法检测矿化诱导条件下不同浓度TNF-α对负载BMSCs碱性磷酸酶活力的影响；实时定量PCR检测不同浓度TNF-α对负载BMSCs成骨分化基因的影响；矿化结节茜素红染色法检测不同浓度TNF-α对负载BMSCs矿化的影响。
结果
1.负载种植体角度测定和周围龈沟液的TNF-α检测与分析。
临床检测与动物实验均显示种植体周龈沟液量和TNF-α的浓度变化在种植体倾斜20°以上时,与种植体倾斜20°以下存在统计学差异(p<0.01)。
2.负载的倾斜种植体周TNF-α检测及其对骨修复的影响。
动物实验显示,骨结合指数和新骨生长率在种植体倾斜20。以上时,与种植体倾斜20°以下存在统计学差异(p<0.01)。
3. TNF-α对负载骨髓间充质干细胞成骨分化的影响。
(1) TNF-α对不同负载BMSCs增殖的影响无统计学意义(p>0.05)。
(2) TNF-α在1ng/ml时,与对照组(TNF-α为0ng/m1)相比,碱性磷酸酶活性和成骨分化基因(RUNX2、OC、OSX(?)ALP) mRNA随TNF-α浓度增加而增加；而在TNF-α高浓度(10,15,20ng/ml)时,碱性磷酸酶活性和成骨分化基因(RUNX2.0C、OSX和ALP) mRNA随TNF-α浓度增高而下降；高低浓度的TNF-α对BMSCs碱性磷酸酶活性和成骨分化基因(RUNX2、OC、OSX和ALP) mRNA的影响差异有统计学意义(p<0.05)。
(3) BMSCs碱性磷酸酶活力、成骨分化基因(RUNX2、OC、OSX和IALP) mRNA受应变负载在生理范围内则是无明显差异(p>0.05)；而超出生理应变时,对碱性磷酸酶活性和成骨分化基因(RUNX2、OC、OSX和ALP) mRNA的影响差异存在统计学意义(p<0.05)。
(4) TNF-α在1ng/ml时,与对照组(TNF-α为Ong/m1)相比,BMSCs所形成的矿化结节量无明显差异(p>0.05),在TNF-α浓度高于lOng/ml时,BMSCs所形成的矿化结节量显著降低(p<0.05)；与生理范围内应变对矿化结节量的影响相比,超出生理范围(5000u￡)时,应变对矿化结节量的影响有明显差异(p<0.05),在负载应变生理范围内矿化结节量变化则无统计学意义。
结论：
(1)种植体倾斜角度在小于20°时,龈沟液量和周围微环境TNF-α浓度变化较小；种植体倾斜角度大于20°时,种植体周围龈沟液量明显增多,而且TNF-a浓度较高,引起骨吸收,降低骨结合率和新骨形成；
(2)低浓度TNF-α对种植体周微环境影响较小,而较高浓度的TNF-α抑制种植体骨界面的碱性磷酸酶活性、成骨分化基因(RUNX2、OSX、ALP和IOC) mRNA以及并降低其矿化结节量；
(3)生理应力范围内时,应力变化对BMSCs增殖、碱性磷酸酶活性、成骨分化基因(RUNX2、OSX、ALP和OC) mRNA以及形成的矿化结节量的影响不明显,较高浓度TNF-α则产生抑制作用；超出生理范围的应力变化则对BMSCs增殖、碱性磷酸酶活性、成骨分化基因(RUNX2、OSX、ALP和OC)mRNA以及形成的矿化结节量影响较大。
Background and Objectives
Implantology has been made rapid progress while starting late demostic. Its advantage and disadvantage has been familiar with. In the course of the procession, the biomechanics has been studying and made great achievements put into practice, including the implanting time and loading time and so on.
The procession of the bone absorption, remodeling and new bone formation displayed and was stronger with the physiological stress stimulation in the interface between the bone and implant. Effection on the bone must be changing with variation of stresses when the different angles of inclination of implant. Increase of unbalanced distribution of stress lead to the absorption of bone around implant. While with the physiological stress stimulation, the osseointegration could come into being better in the condition of loss of excessive stress concentration. The bone remodeling was heavily associated with bone metabolism affected by stress in the implant and bone interface. The stress distribution was changing with the angle of implants. It is unknown that the extent of effection of stress on microenvironment around implant caused by angle of inclination of implant and the bone remodeling. There is seldom reported that the relationship between angular dimension and concentration of TNF-α and both effection on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implantation.
As an inflammatory factor with kinds of biological activity, tumor necrosis factor α (TNF-α) is known by researches to play the dominant role during the development of the periodontal disease. Because of an kind of immunoregulator in the inflammatory reaction, it can make the inflammatory cells enter the infection sites and release the metalloproteinase. The latter can degrade the extracellular matrix protein and promote absorption of alveolar and dstruction of collagenous fiber. At the same time, the proliferation of parodontium and gingiva cells is regulated. TNF-a is in the lower level locus in the controlled or healthy patients while it comes a higher level locus in the developing periodontal patients. The combination of TNF-α and TNFR1inspires the formation of compound of TNFR-associated death domain(TRADD), TNF receptor factor2(TRAF2), receptor integration protein (RIP). TNF-α can enhance osteoclast creation and stimulate its cytoactive to accelerate absorption of bone, simultaneously restraining differentiation of osteoblast. TNF-α could inhibit the preosteoblast differentiation, expression of the osteocalcin, Osteogenesis differentiation genes RUNX2and Type I collagen. Then many researches are concentrating on blocking the expression of TNF-α so as to attain the therapy of associated disease.
Recently, researches about TNF-α came to increasing trend and extended into implantology. They mainly concerned peri-implantitis with the way of detection quantity of gingival crevicular fluid. However, the factors of peri-implantitis in clinic was not clear. Preventive actions was hardly effectively prohibit the development of the disease. Some angle existed in the axis of implants and long axis in the clinic which leaded to the changes of press from masticatory muscle. Then what level concentration of TNF-α would be in the affection of such press and it was unclear that the effection on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation Seldom articles about this problem had been seen in the periodicals.
BMSCs is recognized the most fully researched and the most easy to separation and the most widely applied stem cells of all the stem cells. Then BMSCs was separated, cultivated and purified in our research. The aim of the study is to dicuss effects of TNF-α on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation. We study the effection of the TNF-α on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation to discuss the effection of inflammatory microenvironment on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells in order to provide the theoretical guidance of application of stem cells in the restoration of inflammatory microenvironment around implants.
Methods:
1. Patients with implantation was selected and angles of inclination of their implants was caculated. Three teams was divided by0-10°、10°-20°and above20°.After3and6months, the concentration of TNF-α and the PICF was tested with different angles of inclination of implants.
2. According to the data from the patients with angles of inclination of implants, the model was made. Implants were placed into the dog alveolar and loded. After1and6months the concentration of TNF-α and the PICF were tested.The osseointegration index(OI) and bone ingrowth fraction,(BIF) were calculated.
3. BMSCs were cultured and loaded by strain. Cells were treated with murine TNF-α at concentrations of0,1,10,15and20ng/ml, respectively. At24,48and72h after the TNF-α treatment, the proliferation/survival of the cells was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) test;The changes in the expression levels of osteogenic transcription factors (Runx2and Osx) and bone matrix proteins (0C and ALP) during osteogenic differentiation were observed by real-time PCR; The change in ALP activity was determined using PNPP assay; In vitro mineralization of BMSCs treated with TNF-α at concentrations of0,1,15and20ng/ml for four weeks under osteogenic induction was monitored using Alizarin red staining.
Results:
1. Test and analysis of TNF-a and PCIF around loaded implants in patients.
It did not reveal any statistically significant changes about PCIF and the concentrations of TNF-a in the angles of inclination of implants from0to10°while there did show statistically significant changes about PCIF and the concentrations of TNF-α compareing the below-20°angle to the above-20°angle of implants.
2. Test and analysis of TNF-α and PCIF around loaded implants and effects of TNF-α on bone regeneration in dogs
There were statistically significance between the the below-20°angle to the above-20°angle of implants in OI and BIF, PCIF and the concentrations of TNF-α. It showed coherence significant result about data from patients and dogs.
3.Effects of TNF-α on osteogenic differentiation of BMSCs revealed some statistically significant changes as below:
(1) The stem cells were successfully cultured. The ability of colony formation was high, and stem cells had formed mineralized nodules when cultured in osteogenic medium.
(2) Real time PCR analysis showed that the mRNA levels of the potent osteogenic transcription factors (Runx2and Osx) and some bone matrix proteins (OC and ALP) were up-regulated in cell cultures treated with TNF-a at lower concentrations (0and1ng/ml). In contrast, the mRNA levels of Runx2, Osx, OC and ALP were dose-dependently down-regulated in cell cultures treated with TNF-a at concentrations above1ng/ml.
(3)48h treatment of the BMSCs with TNF-a at lower concentrations (1ng/ml) enhanced ALP activity in these cells when compared with the control cells (treated with Ong/ml TNF-a). Cells treated with TNF-a at higher concentrations (10,15and20ng/ml) showed decreased ALP activity when compared with the control cells.
(4) Long term treatment with TNF-α resulted in a dose-dependent decrease in mineral nodule formation after the4-week osteogenic induction.
(5) MTT assay did not reveal any statistically significant changes in the proliferation/survival of BMSCs treated with TNF-a at concentrations ranging from0to20ng/ml.
Conclusions:
(1) Effects of TNF-a on microenvironment around the implants revealed little effect on implant-bone interface when the angle inclination of implants was below-20°with TNF-α at lower concentrations. While the angle was over20°, effects of TNF-a on microenvironment was that the bone around implants had been absorbed.
(2) TNF-α treatment at lower concentrations and short time moderately enhances expression of osteogenic genes (RUNX2、OSX、ALP和OC) and ALP activity in BMSCs while TNF-α treatment at higher concentrations level and long time displays inhibitory effect on osteogenic differentiation and mineralization in BMSCs. at higher concentrations it lower expression of these osteogenic genes and reduce mineralized nodule amount.
(3) In the physiology strain extent, it did not reveal changes in the proliferation of BMSCs. While manifest changes are revealed when the strain out of physiology range.
种植学在国内起步时间晚,但是发展速度快。人们对其优缺点已耳熟能详,且己成为临床修复学的重要组成部分。在种植学发展中,学者们不断地对影响其成功率的重要因素之一—生物力学进行探索并取得了很大的进展：如在种植时机、负载时间等相关问题的不断提出探讨并将其成果付诸实践。
基于生物力学考虑,受到生理应力刺激的骨组织与无生理应力刺激相比,其成骨和抗应力能力是增强的。对种植体行早期负载或即刻负载可能有利于骨结合的形成。近年来相关研究逐步增多,在一定的生理条件下,避免过度的应力集中是可以获得较好骨结合的。相关的理论技术较为成熟,在临床上受到患者认可而已经广泛的应用。有学者甚至认为,主张对种植体延期负载仅仅是基于感性的经验考虑。种植义齿在受到咬合力的刺激后,种植体骨界面发生相应骨改建,这是与应力作用下的骨代谢相密切相关联的。
种植体倾斜一定角度后,其周围应力分布发生相应变化,这种应力变化必然对骨组织产生影响,其影响程度和研究目前知之甚少。在种植义齿行使功能过程中,由于解剖因素、咬合方向变化会导致咬合力方向发生不同变化。由此而会导致应力相同的情况下因角度变化产生种植体周围的应变则会不同。另一方面,对种植体骨界面所产生的骨结合不利之处在于种植体与骨组织应力传导过程中缺乏应有的缓冲而可能发生种植体骨界面的微小裂痕。这种现象主要是由于在应力传递在弹性模量差度较大两种材料——种植体与骨组织问,因为弹性模量相差越大,两者应力引起的形变差距越大。应力分布不同会相应的引起其作用部位骨组织的改建差异。应力的差异越大,相应的,改建的效果差异亦越大。而这种由不同角度引起的应力对种植体周围微环境是何种影响以及影响的程度如何的,骨组织在受这种角度引起的应力后在其引起微环境相关因子-TNF-α变化的影响下是如何进行改建的,相关研究较少。
TNF-α是多种生物学活性的炎症因子,在牙周病发展过程中对组织造成破坏的主要因子。通过研究得知,它在牙周炎的发病机理中扮演着起重要的作用。因为TNF-α是促炎症反应的一种免疫调节剂,可以促炎性细胞进入感染部位,使金属蛋白酶的释放。后者降解细胞外基质蛋白,促进胶原纤维的破坏和牙槽骨吸收,同时调节牙周膜与牙龈成纤维细胞的增殖。TNF-α在健康和治疗成功处于较低水平的位点,而在活动性破坏的位点却是较高。在多数细胞中,TNF-α与TNFR1的结合激发TNFR结合的死亡受体结构域蛋白(TRADD)、TNF受体结合因子2(TRAF2)、受体结合蛋白(RIP)复合物的形成。TNF-α促进破骨细胞的生成,提高破骨细胞活性,以此来促进骨吸收,同时亦有抑制成骨细胞骨形成。TNF-α可以抑制前成骨细胞分化成骨细胞,抑制骨钙蛋白、成骨分化基因RUNX2和I型胶原的表达。这说明TNF-α对骨吸收的影响至关重要,因此学者们不断对其表达阻断进行研究以期获得对其治疗的方法。
近年来,针对TNF-α的研究逐渐增多,涉及范围逐渐扩展至种植学科领域。主要是通过种植体周围炎的研究相对较多,研究方式通过测量种植体周围龈沟液的含量变化进行测定。但临床上的很多的种植体周围炎病因并不非常明确,针对其预防更是无从谈起。而目前针对由于生物力学因素引起的种植体周围微环境的变化研究相对较少,特别是针对TNF-α影响更少。通过临床可看出种植体植入时基本与原位自然牙的长轴存在一定的角度,这种角度所引起的种植体周围应力分布变化研究相对较少。在这种应力作用下,TNF-α的变化如何以及其相应变化与骨界面的改建影响如何,特别是对种植体周成骨细胞分化成骨的影响如何,未见相关研究。倾斜角度大小与种植体周围TNF-α变化关系如何,以及两者对成骨细胞分化成骨影响状况影响,目前尚未有研究见诸报道。
在临床上负载种植体周围骨组织改建无时不在进行中。为了明确以下问题：负载种植体周围微环境中的TNF-α的变化如何、对负载的种植体周围骨组织改建影响如何、TNF-α浓度变化的影响和种植体骨改建变化以及对其中的种植体周骨髓间质干细胞成骨分化的影响状况,本课题研究中通过不同角度负载的种植体周围应力变化不同而测定其微环境中TNF-α的变化,将此为依据进行种植体周骨髓间充质干细胞成骨分化和骨创伤修复影响研究。我们选择临床病例龈沟液量、TNF-α浓度测定；依据临床角度测定结果,通过动物实验观察龈沟液量、TNF-α浓度变化对骨结合情况和骨改建的影响；综合临床病例和动物实验结果,研究TNF-α对BMSCs增殖和成骨分化的影响,以探讨炎性微环境对干细胞增殖与分化的影响,可以进一步明确TNF-α对种植体骨界面骨组织变化的影响,有效地为临床检测TNF-α的水平变化提供预警,进行早预防、早治疗,为针对性的相关预防、治疗措施提供依据；为应用干细胞修复在炎性微环境中破坏的种植体周组织提供理论指导。
研究方法
1.自临床选择种植义齿病例,测定相应倾斜角度,按照术前设计模型测定与牙体长轴方向夹角归为三组：0°—10°、10°—20°及20°以上。在负载后一个月及六个月后行采集龈沟液进行定量分析和检测TNF-α,测定TNF-α浓度随种植体倾斜角变化；
2.通过在按照临床选择病例所测得倾斜角度,制作模板,常规方法将种植体植入成年犬颌骨内并于负载一个月及六个月后,用whatman42试纸行种植体周龈沟液的采集和定量检测TNF-α,同时采取骨标本进行骨结合、骨密度、骨新生等检测。对比分析不同角度倾斜种植体负载与测定的种植体龈沟TNF-α变化相关性；
3.小鼠骨髓间充质干细胞的取材、培养、分离纯化,建立成骨细胞负载模型,免疫细胞化学染色和矿化诱导初步鉴定其干细胞特性；MTT法检测不同浓度TNF-α对负载BMSCs增殖的影响；PNPP法检测矿化诱导条件下不同浓度TNF-α对负载BMSCs碱性磷酸酶活力的影响；实时定量PCR检测不同浓度TNF-α对负载BMSCs成骨分化基因的影响；矿化结节茜素红染色法检测不同浓度TNF-α对负载BMSCs矿化的影响。
结果
1.负载种植体角度测定和周围龈沟液的TNF-α检测与分析。
临床检测与动物实验均显示种植体周龈沟液量和TNF-α的浓度变化在种植体倾斜20°以上时,与种植体倾斜20°以下存在统计学差异(p<0.01)。
2.负载的倾斜种植体周TNF-α检测及其对骨修复的影响。
动物实验显示,骨结合指数和新骨生长率在种植体倾斜20。以上时,与种植体倾斜20°以下存在统计学差异(p<0.01)。
3. TNF-α对负载骨髓间充质干细胞成骨分化的影响。
(1) TNF-α对不同负载BMSCs增殖的影响无统计学意义(p>0.05)。
(2) TNF-α在1ng/ml时,与对照组(TNF-α为0ng/m1)相比,碱性磷酸酶活性和成骨分化基因(RUNX2、OC、OSX(?)ALP) mRNA随TNF-α浓度增加而增加；而在TNF-α高浓度(10,15,20ng/ml)时,碱性磷酸酶活性和成骨分化基因(RUNX2.0C、OSX和ALP) mRNA随TNF-α浓度增高而下降；高低浓度的TNF-α对BMSCs碱性磷酸酶活性和成骨分化基因(RUNX2、OC、OSX和ALP) mRNA的影响差异有统计学意义(p<0.05)。
(3) BMSCs碱性磷酸酶活力、成骨分化基因(RUNX2、OC、OSX和IALP) mRNA受应变负载在生理范围内则是无明显差异(p>0.05)；而超出生理应变时,对碱性磷酸酶活性和成骨分化基因(RUNX2、OC、OSX和ALP) mRNA的影响差异存在统计学意义(p<0.05)。
(4) TNF-α在1ng/ml时,与对照组(TNF-α为Ong/m1)相比,BMSCs所形成的矿化结节量无明显差异(p>0.05),在TNF-α浓度高于lOng/ml时,BMSCs所形成的矿化结节量显著降低(p<0.05)；与生理范围内应变对矿化结节量的影响相比,超出生理范围(5000u￡)时,应变对矿化结节量的影响有明显差异(p<0.05),在负载应变生理范围内矿化结节量变化则无统计学意义。
结论：
(1)种植体倾斜角度在小于20°时,龈沟液量和周围微环境TNF-α浓度变化较小；种植体倾斜角度大于20°时,种植体周围龈沟液量明显增多,而且TNF-a浓度较高,引起骨吸收,降低骨结合率和新骨形成；
(2)低浓度TNF-α对种植体周微环境影响较小,而较高浓度的TNF-α抑制种植体骨界面的碱性磷酸酶活性、成骨分化基因(RUNX2、OSX、ALP和IOC) mRNA以及并降低其矿化结节量；
(3)生理应力范围内时,应力变化对BMSCs增殖、碱性磷酸酶活性、成骨分化基因(RUNX2、OSX、ALP和OC) mRNA以及形成的矿化结节量的影响不明显,较高浓度TNF-α则产生抑制作用；超出生理范围的应力变化则对BMSCs增殖、碱性磷酸酶活性、成骨分化基因(RUNX2、OSX、ALP和OC)mRNA以及形成的矿化结节量影响较大。
Background and Objectives
Implantology has been made rapid progress while starting late demostic. Its advantage and disadvantage has been familiar with. In the course of the procession, the biomechanics has been studying and made great achievements put into practice, including the implanting time and loading time and so on.
The procession of the bone absorption, remodeling and new bone formation displayed and was stronger with the physiological stress stimulation in the interface between the bone and implant. Effection on the bone must be changing with variation of stresses when the different angles of inclination of implant. Increase of unbalanced distribution of stress lead to the absorption of bone around implant. While with the physiological stress stimulation, the osseointegration could come into being better in the condition of loss of excessive stress concentration. The bone remodeling was heavily associated with bone metabolism affected by stress in the implant and bone interface. The stress distribution was changing with the angle of implants. It is unknown that the extent of effection of stress on microenvironment around implant caused by angle of inclination of implant and the bone remodeling. There is seldom reported that the relationship between angular dimension and concentration of TNF-α and both effection on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implantation.
As an inflammatory factor with kinds of biological activity, tumor necrosis factor α (TNF-α) is known by researches to play the dominant role during the development of the periodontal disease. Because of an kind of immunoregulator in the inflammatory reaction, it can make the inflammatory cells enter the infection sites and release the metalloproteinase. The latter can degrade the extracellular matrix protein and promote absorption of alveolar and dstruction of collagenous fiber. At the same time, the proliferation of parodontium and gingiva cells is regulated. TNF-a is in the lower level locus in the controlled or healthy patients while it comes a higher level locus in the developing periodontal patients. The combination of TNF-α and TNFR1inspires the formation of compound of TNFR-associated death domain(TRADD), TNF receptor factor2(TRAF2), receptor integration protein (RIP). TNF-α can enhance osteoclast creation and stimulate its cytoactive to accelerate absorption of bone, simultaneously restraining differentiation of osteoblast. TNF-α could inhibit the preosteoblast differentiation, expression of the osteocalcin, Osteogenesis differentiation genes RUNX2and Type I collagen. Then many researches are concentrating on blocking the expression of TNF-α so as to attain the therapy of associated disease.
Recently, researches about TNF-α came to increasing trend and extended into implantology. They mainly concerned peri-implantitis with the way of detection quantity of gingival crevicular fluid. However, the factors of peri-implantitis in clinic was not clear. Preventive actions was hardly effectively prohibit the development of the disease. Some angle existed in the axis of implants and long axis in the clinic which leaded to the changes of press from masticatory muscle. Then what level concentration of TNF-α would be in the affection of such press and it was unclear that the effection on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation Seldom articles about this problem had been seen in the periodicals.
BMSCs is recognized the most fully researched and the most easy to separation and the most widely applied stem cells of all the stem cells. Then BMSCs was separated, cultivated and purified in our research. The aim of the study is to dicuss effects of TNF-α on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation. We study the effection of the TNF-α on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells and their effects on bone regeneration around the loaded implementation to discuss the effection of inflammatory microenvironment on cell proliferation and osteogenic differentiation of bone mesenchymal stem cells in order to provide the theoretical guidance of application of stem cells in the restoration of inflammatory microenvironment around implants.
Methods:
1. Patients with implantation was selected and angles of inclination of their implants was caculated. Three teams was divided by0-10°、10°-20°and above20°.After3and6months, the concentration of TNF-α and the PICF was tested with different angles of inclination of implants.
2. According to the data from the patients with angles of inclination of implants, the model was made. Implants were placed into the dog alveolar and loded. After1and6months the concentration of TNF-α and the PICF were tested.The osseointegration index(OI) and bone ingrowth fraction,(BIF) were calculated.
3. BMSCs were cultured and loaded by strain. Cells were treated with murine TNF-α at concentrations of0,1,10,15and20ng/ml, respectively. At24,48and72h after the TNF-α treatment, the proliferation/survival of the cells was evaluated using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) test;The changes in the expression levels of osteogenic transcription factors (Runx2and Osx) and bone matrix proteins (0C and ALP) during osteogenic differentiation were observed by real-time PCR; The change in ALP activity was determined using PNPP assay; In vitro mineralization of BMSCs treated with TNF-α at concentrations of0,1,15and20ng/ml for four weeks under osteogenic induction was monitored using Alizarin red staining.
Results:
1. Test and analysis of TNF-a and PCIF around loaded implants in patients.
It did not reveal any statistically significant changes about PCIF and the concentrations of TNF-a in the angles of inclination of implants from0to10°while there did show statistically significant changes about PCIF and the concentrations of TNF-α compareing the below-20°angle to the above-20°angle of implants.
2. Test and analysis of TNF-α and PCIF around loaded implants and effects of TNF-α on bone regeneration in dogs
There were statistically significance between the the below-20°angle to the above-20°angle of implants in OI and BIF, PCIF and the concentrations of TNF-α. It showed coherence significant result about data from patients and dogs.
3.Effects of TNF-α on osteogenic differentiation of BMSCs revealed some statistically significant changes as below:
(1) The stem cells were successfully cultured. The ability of colony formation was high, and stem cells had formed mineralized nodules when cultured in osteogenic medium.
(2) Real time PCR analysis showed that the mRNA levels of the potent osteogenic transcription factors (Runx2and Osx) and some bone matrix proteins (OC and ALP) were up-regulated in cell cultures treated with TNF-a at lower concentrations (0and1ng/ml). In contrast, the mRNA levels of Runx2, Osx, OC and ALP were dose-dependently down-regulated in cell cultures treated with TNF-a at concentrations above1ng/ml.
(3)48h treatment of the BMSCs with TNF-a at lower concentrations (1ng/ml) enhanced ALP activity in these cells when compared with the control cells (treated with Ong/ml TNF-a). Cells treated with TNF-a at higher concentrations (10,15and20ng/ml) showed decreased ALP activity when compared with the control cells.
(4) Long term treatment with TNF-α resulted in a dose-dependent decrease in mineral nodule formation after the4-week osteogenic induction.
(5) MTT assay did not reveal any statistically significant changes in the proliferation/survival of BMSCs treated with TNF-a at concentrations ranging from0to20ng/ml.
Conclusions:
(1) Effects of TNF-a on microenvironment around the implants revealed little effect on implant-bone interface when the angle inclination of implants was below-20°with TNF-α at lower concentrations. While the angle was over20°, effects of TNF-a on microenvironment was that the bone around implants had been absorbed.
(2) TNF-α treatment at lower concentrations and short time moderately enhances expression of osteogenic genes (RUNX2、OSX、ALP和OC) and ALP activity in BMSCs while TNF-α treatment at higher concentrations level and long time displays inhibitory effect on osteogenic differentiation and mineralization in BMSCs. at higher concentrations it lower expression of these osteogenic genes and reduce mineralized nodule amount.
(3) In the physiology strain extent, it did not reveal changes in the proliferation of BMSCs. While manifest changes are revealed when the strain out of physiology range.
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