【作者】 颜世果；

【导师】 杨丕山；

【作者基本信息】 山东大学 ， 口腔临床医学， 2010， 博士

【摘要】 种植体在临床的应用越来越广泛,除了修复和替代缺失牙齿外,在上下颌骨的整形修复中也被大量使用,种植体治疗已经成为一个标准和常规的程序。种植体成功的一个重要标志就是形成骨结合,骨结合是在光学显微镜水平观察到的种植体和宿主骨的直接接触形式,此状态下种植体可以承受压力并行使功能。骨结合一个重要的前提条件就是种植体周围新骨的生成。但是在种植体植入后,种植体周围骨再生相对比较慢。而且在初始愈合期以后,由于种植体周围新形成骨的骨密度比较低,种植体周围的骨组织对种植体的支持有限,因此对种植体骨结合的研究依然面临很大的挑战。很多的基础和临床研究致力于如何通过缩短种植体骨结合的时间来缩短治疗周期,提高治疗的效率。为了实现这个目标,最常见的方法就是通过改变种植体的设计参数来实现,这些改变包括种植体的材料,种植体外形的修改以及种植体表面的修饰。目前认为,种植体的骨结合是通过成骨细胞的活动来完成的,大部分对种植体表面的修饰都是为了促进或者改善种植体周围组织中细胞对种植体的反应。除了通过对种植体表面的修改来促进种植体的骨结合,也有一些通过系统或者局部应用与成骨相关的转录因子或者生长因子增加骨-种植体的结合。Satb2是近几年来发现的在成骨细胞的分化以及牙和颅颌面发育中起重要重要作用的转录因子。Satb2可以与成骨分化相关的重要调控转录因子Runx2、Osx以及Atf4等协同作用,提高其在骨骼发育及成骨细胞分化中的调节作用,并可以直接结合于成骨细胞特异性基因BSP和OCN的启动子促进BSP和OCN的表达,另外,Satb2可以下调成骨细胞中抑制骨形成的Hoxa2的表达而促进成骨细胞的分化和成骨。Satb2基因敲除鼠的胚胎显示多发的颅颌面缺陷,包括下颌骨明显的短小,鼻骨和上颌骨的发育不足,舌骨的畸形以及腭裂,且基因敲除小鼠一般在出生后立即死亡,在对Satb2基因敲除鼠的成骨细胞的研究中发现：其成骨细胞的分化和功能存在缺陷,骨的形成和钙化延迟,基质中胶原的表达显著降低,在细胞外基质的沉积中存在明显的缺陷。研究者认为,Satb2在成骨细胞的分化以及颅颌面的发育中是一个重要的骨发育性调节分子,是骨骼发育以及成骨细胞分化的基因调控系统中的分子枢纽。因此有理由相信,Satb2在骨形成及骨重建中起到重要的调节作用,是未来骨修复和骨组织工程中理想的候选因子。因此本实验纳入Satb2作为研究对象,采用RCAS或pBABE-Hygro两种不同的逆转录病毒系统,以基因转染的方法将Satb2过表达于植入BSP-TVA转基因小鼠中的种植体周围,观察Satb2局部高表达在种植体周围成骨及骨结合中的作用。这两种不同的病毒表达载体感染BSP-TVA转基因小鼠的细胞不同。其中RCAS逆转录病毒表达系统可以使编码Satb2的病毒RCAS-Satb2特异性感染种植体周围组织中BSP阳性的细胞,而pBABE-Hygro-Satb2逆转录病毒表达系统可以感染种植体周围所有分裂期细胞。通过检测两组结果的异同,可进一步了解不同细胞参与种植体周围组织的成骨是否存在差异。同时,采用局部使用高表达Satb2骨髓基质细胞的办法,检测局部使用Satb2基因转染的骨髓基质细胞在种植体骨结合中的作用。从而探讨Satb2基因治疗促进种植体骨结合的效果及基因转染方式。材料和方法：第一部分：本实验纳入了研究成骨的理想的动物模型：BSP-TVA转基因小鼠。该转基因小鼠利用独特的RCAS-TVA系统,通过4.9kb的BSP启动子控制TVA基因的表达,使TVA基因选择性表达在成骨细胞中,表达TVA的分裂期哺乳细胞可以很容易被RCAS感染。成功构建RCAS-Satb2逆转录表达载体。在种植体植入BSP-TVA小鼠的股骨的远心端前,将含有108cfu/ml的编码Satb2或者RCAS空载体的逆转录病毒3μl注入种植体窝。术后1周及3周取材,通过H&E染色观察种植体周围组织的成骨情况,采用H&E染色、BSP免疫组化染色和组织学测量评价Satb2高表达在种植体的愈合及骨结合中的作用。同时对术后1周及3周种植体上下1mm范围的骨组织提取RNA后,通过Real-time RT-PCR检测组织中Satb2及成骨相关基因Osx、Runx2、BSP、COLI及OC的表达情况,观察Satb2在实验组和对照组组织中的变化。第二部分：实验采用了另外一个不同的病毒表达系统,pBABE-Hygro逆转录病毒表达载体系统。在成功包装pBABE-Hygro-Satb2及pBABE-Hygro病毒并将病毒滴度调整到108cfu/ml后,我们采用与第一部分相同的手术方法及检测方法对pBABE-Hygro-Satb2促进种植体骨结合的作用进行了检测。第三部分：应用pBABE-Hygro-Satb2及pBABE-Hygro逆转录病毒体外感染EGFP标记的BMSCs,检测感染后2天,9天及23天的BMSCs中Satb2及成骨相关基因Osx、Runx2、BSP、COLI及OC的表达。将感染后48小时的BMSCs细胞悬液局部应用于B6D2F1小鼠股骨的种植体部位,采用与第一部分相同的实验方法对超表达Satb2的BMSCs在种植体的骨结合中的作用进行检测。同时用GFP免疫组化染色的方法追踪外源性的BMSCs在组织中的去向及功能。结果：第一部分：局部使用RCAS及RCAS-Satb2病毒,术后1周,H&E染色结果显示：种植体周围有新形成的编织骨；与种植体直接接触的宿主组织中,活化的成骨样细胞排列规律,呈复层围绕种植体；周围组织中还有一些炎症细胞的浸润。实验组中种植体周围的骨面积百分比(% bone area)高于对照组,但差异无显著性。实验组中,与种植体直接接触的宿主组织已有30%左右与种植体形成骨结合,可见处于活跃期的成骨细胞,实验组中宿主与种植体发生骨结合的区域是对照组2倍。采用Real-time RT-PCR技术对种植体周围1mm范围内成骨相关基因的检测结果发现：实验组中Satb2的表达增加约3倍,Osx, Runx2, BSP, COLI和OC的表达也显著增加。而BSP免疫组化结果与实时定量检测BSP的结果一致,实验组中BSP的阳性细胞数和间质中BSP的信号都高于对照组。术后3周,种植体被大量的骨组织包绕,与种植体直接接触的部位,有编织骨及板层骨,周围的组织中已经没有明显的炎症。H&E染色组织测量结果显示：3周时,实验组中种植体周围的骨面积百分比高于对照组,宿主组织与种植体之间的结合主要为骨结合,实验组中与种植体直接接触的宿主组织已有80%左右与种植体形成骨结合,实验组中骨结合区域的比例是对照组的1.4倍。同时检测了3周时,种植体周围1mm范围内Satb2及成骨相关基因的表达,实验组中Satb2, Osx, Runx2, BSP, COLI和OC的表达均略高于对照组,但差异均无显著性。而BSP免疫组化的计数结果在实验组和对照组也无显著差异。第二部分：局部使用pBABE-Hygro及pBABE-Hygro-Satb2逆转录病毒,术后1周,H&E染色结果显示：种植体周围出现新形成的编织骨,与种植体直接接触的部位,活化的成骨样细胞排列规律整齐,呈复层围绕种植体,周围的组织中可见少量的炎症细胞浸润。实验组中种植体周围的骨面积百分比高于对照组。与种植体直接接触的宿主组织已有10%左右与种植体形成骨结合,实验组中宿主与种植体发生骨结合的区域大于对照组,但差异无显著性。Real-time RT-PCR结果显示：术后1周,实验组中Satb2的表达增加约2倍,成骨相关的转录因子Osx和Runx2,及成骨特异性基因BSP及COLI表达显著增加；而OC的表达虽然有增加,但与对照组相比差异没有显著性。实验组BSP阳性细胞数高于对照组。术后3周,H&E染色结果显示,种植体主要被骨组织包绕,与种植体直接接触的部位,主要为不同形态的骨组织,实验组中种植体周围的骨组织是排列规则的板层骨及少量的编织骨,与种植体直接接触的区域也是矿化良好的骨组织；对照组中,与种植体直接接触的骨组织中,局部有未完全矿化的细胞成分存在。3周时,实验组中种植体周围骨面积百分比高于对照组,宿主组织与种植体之间的结合主要为骨结合,实验组中宿主与种植体发生骨结合的区域是对照组的约1.6倍。种植体植入3周时,实验组中Satb2的表达仍显著高于对照组,实验组中成骨相关的转录因子Osx, Runx2及与成骨密切相关的BSP, COLI, OC的基因表达均明显增加,与对照组相比差异具有显著性。实验组中BSP的阳性细胞数高于对照组。第三部分：1周时,GFP阳性的BMSCs分散位于种植体周围的组织中,分布不均匀；3周时,在种植体周围仍然可以检测到GFP阳性的BMSCs存在；Satb2过表达的BMSCs在3周时促进了种植体的骨结合,并促进了种植体周围新骨的形成。体内外Real-time RT-PCR结果基本一致,在3周时,种植体周围组织中Satb2及成骨相关基因的表达仍然高于对照组,与第二部分3周时的结果相一致。结论：1.局部使用RCAS或pBABE-Hygro两种不同的逆转录病毒表达系统,均可以以基因转染的方法将Satb2转入BSP-TVA转基因小鼠股骨种植体周围组织中,并在种植体的局部高表达Satb2。2. Satb2在种植体局部的高表达能够促进种植体部位组织中成骨相关转录因子Osx和Runx2的表达,同时提高了成骨特异性基因BSP, COLI及OC的表达；使用RCAS-Satb2逆转录病毒时,能够显著提高种植体1周时的骨结合；3周时,Satb2促进了种植体的骨结合并促进了种植体周围新骨的形成。3.通过RCAS及pBABE-Hygro两种不同的逆转录病毒表达系统将Satb2应用于种植体的局部在种植体周围组织的成骨机制中存在明显的差异。两组在种植体周围组织中成骨机制的差异,可能与RCAS逆转录病毒表达系统只能感染表达TVA的细胞,而pBABE-Hygro逆转录表达系统能够感染所有分裂期细胞,从而使感染了pBABE-Hygro-Satb2的细胞尤其是具有多向分化潜能的干细胞更积极地参与到种植体周围的成骨和骨重建中有关。4.体外感染pBABE-Hygro-Satb2的BMSCs在第2、9及23天高表达Satb2,高表达Satb2的BMSCs局部应用于种植体部位能够促进种植体周围新骨的形成并促进种植体的骨结合。更多还原

【Abstract】 Titanium dental implants are widely used in dentistry and orthopaedic surgery for replacement of teeth and restoration of the maxillary and mandibular bone defects. Dental implants have become a standard and routine procedure in oral health care.Osseointegration plays a key role in the fixation and anchoring of the implants. It is a phenomenon in which intimate contact between bone and implant at the optical microscopy level and enables dental implant to restore intraoral function. Nevertheless, bone regeneration around implants is relatively slow after placement. The bone support to the implants after initial healing time is limited due to the low density of the newly formed bone tissue around the implants. So, osseointegration at the dental implant surface still remains a challenge. Much effort has been done to reduce the healing time of the dental implants osseointegration and decrease the treatment time by scientists and clinicians. To gain this purpose, implant design parameters are modified including implant material, macrogeometry and surface modification. It is generally accepted that osseointegration of dental implants is achieved by the activity of osteoblasts. Most of these researchs were aimed at host response. The recruitment and differentiation of osteoprogenitor cells are the key in bone formation. It is generally accepted that the bone forming cells at the dental implants surface are from the neighboring host bone. Besides these modifications of the design parameters, application of some bone-related molecules and factors have been adopted to increase bone-to-implant contact (BIC) systemically or locally. It was recently found that special AT-rich sequence-binding protein 2 (SATB2) plays a pivotal role in craniofacial development and osteolbast differentiation. It can regulate skeletal development and osteoblast differentiation by synergizing, amplifying and thus exponentially augmenting the activity of multiple osteogenic factors including Runx2, Osx and Atf4. Satb2 can bind to the promoters of BSP and OC and up-regulate their expression. Satb2 represses Hoxa2 gene expression and regulates multiple genes in osteoblasts. SATB2 gene knockout mouse shows multiple craniofacial defects that include a significant truncation of the mandible, a shortening of the oral maxillofacial bones, malformations of the hyoid bone. The Satb2-/-mice die immediately after birth. The defects in osteoblast differentiation and function were found, and it showed delayed bone formation and mineralization in Satb2-/-mice embryos. The expression level of the collagen in bone matrix decreased dramatically, and the deposition defects of extracellular matrix were also detected in Satb2-/-mice embryos. The expression level of Nanog can be upregulated by Satb2 and play an important role in the regulatory network of embryonic stem cell differentiation. While binding to AT-rich DNA elements, SATB2 modifies chromatin structure and regulates multiple genes. Based on the researches on Satb2, it can tell that Satb2 has a pivotal role in craniofacial development and skeletal patterning, and it is a master gene in osteoblast differentiation. It is suggested that Satb2 can be a robust osteoinductive molecule regulating other transcription factors to form a molecular node for a transcriptional network.Based on researches above, the function of Satb2 on implant osseointegration was studied in this research. The retrovirus RCAS and pBABE-Hygro were used in this study to deliver Satb2 into the cells in the tissue surrounding the implant. And the roles of Satb2 in implant osseointegration were evaluated by the detection of the bone-related genes and bone formation around the implants. The cells of BSP-TVA transgenic mouse infected by RCAS and pBABE-Hygro virus stocks were different from each other. Mammalian cells engineered to express TVA are highly susceptible to be infected by RCAS. While all the dividing mammalian cells can be infected by pBABE-Hygro virus. So the function of the different cells infected on implant osseointegration by these two retrovirus systems was analyzed according to the results. Meanwhile, BMSCs with forced Satb2 expression were used by local administration to detect its function on implant osseointegration.Materials and Methods: Part I:BSP-TVA transgenic mice provided an excellent in vivo model for achieving the purpose of this study. The strategy was based on the receptor for subgroup-A avian aleukosis virus (RCAS), TVA. The retroviral receptor TVA was driven by a 4.9 kb BSP promoter and was selectively expressed in skeleton tissues. The retrovirus construct, RCAS-Satb2, was confirmed by sequencing. The implantation sites were prepared on anterior-distal surface of the femurs.3μl of RCAS or RCAS-Satb2 virus stock of about 108 cfu/ml was applied to the bone defects before the SLA surface implants were press-fitted into slightly undersized holes. The femurs were isolated 1 week and 3 weeks after the surgery. H&E staining was performed to detect the bone formation around the implants. H&E staining, BSP immunohistochemical staining and histomorphometric evaluation were performed to investigate the roles of Satb2 in osseointegration of the implants. Total RNA was extracted from the bone tissues (1 mm mesial and distal to the implantation site) with TRIzol reagent. Quantitative real-time reverse transcription-PCR (qRT-PCR) assay was performed to detect the expression levels of Satb2, Osx, Runx2, BSP, OC and COLI.PartⅡ:To better understand the results from Part I, pBABE-Hygro retrovirus system was used in this part. After produce of the virus stocks of pBABE-Hygro and pBABE-Hygro-Satb2, the viral stocks were used as RCAS and RCAS-Satb2 virus stock in PartⅠ. The same procedure and analysis were performed like what have been done in PartⅠI.PartⅢ:BMSCs labeled by EGFP were infected by pBABE-Hygro-Satb2and pBABE-Hygro viral stocks. The gene expression levels of Satb2, Osx, Runx2, BSP, COLI and OC were detected 2 days,9 days, and 23 days after infection.2 days after infection, BMSCs were locally administrated in implant sites of B6D2F1 mice. The same procedure and analysis were performed like that in PartⅠ. Immunohistochemical staining of GFP was performed to trace labeled BMSCs.Results:PartⅠ:1 week after the surgery, the formation of woven bone could be detected. It was found that the cells in contact with the implants were well organized. The cells lined in several layers surrounding the implants. The inflammatory cells could be detected in the tissue around the implants. The bone area/total implant area was higher in the test group than that in the control group, but there was no significant difference. About 30%of the tissue in contact with the implants was bone relative to 70%soft tissue in test group. And the%BIC in Satb2 group was significantly higher than that in control group. The real-time RT-PCR results showed that the expression level of SATB2 was up-regulated about 3 times in RCAS-Satb2 group compared with control group. The expression levels of OSX, Runx2, BSP, COLI and OC were significantly up-regulated in RCAS-Satb2 group than that in control group. The results of BSP immunohistochemical staining were in coincidence with the results of H&E staining. There were more BSP positive cells in test group than that in control group.3 weeks after implantation, the implants were surrounded by woven bone and lamella bone. There were no detectable inflammatory cells in the tissue surrounding the implants. The bone area/total implant area was higher in the test group than that in the control group 3 weeks after implantation. About 80%of the tissue in contact with the implants was bone. And the%BIC in Satb2 group was significantly higher in the test group than that in control group. It showed in real-time RT-PCR results that 3 weeks after the operation, there were no significant difference in the expression levels of Satb2, Osx, Runx2, BSP, COLI and OC in RCAS-Satb2 group and control group. Immunohistochemical staining of BSP showed that there was no difference between control group and test group.PartⅡ:H&E staining, immunohistochemical staining and histomorphometric analysis of the pBABE-Hygro and pBABE-Hygro-Satb2 group 1 week after the implantation were almost the same with that in RCAS and RCAS-Satb2 group 1 week after the operation. Quantitative real-time RT-PCR analysis results implied that the expression levels of Satb2, Osx, Runx2, BSP and COLI were significantly higher in pBABE-Hygro-SATB2 group than that in control group 1 week after implantation. The expression level of OC was upregulated, but there was no significant difference between test group and control group. 3 weeks after implantation, organized bone around the implants could be detected in pBABE-Hygro group and PBABE-Hygro-Satb2 group. In pBABE-Hygro-Satb2 group, the mature and organized lamellar bone in contact with the implant could be seen. While in pBABE-Hygro group, there was immature bone in contact with the implant site. Histomorphometric analysis showed that the percentage of the bone at implantation site was significantly higher in pBABE-Hygro-Satb2 group than that in the control group 3 weeks after implantation. And the percentage of bone in contact with the implant was significantly higher in pBABE-Hygro-Satb2 group than that in the control group. The percentage of bone in direct contact with implant was 1.6 times in Satb2 group than that in control group.21 days after surgery, the expression levels of Satb2, Osx, Runx2, BSP, COLI and OC were significantly higher in pBABE-SATB2 group than that in control group. Cell counting results showed that there were more BSP positive cells in pBABE-Hygro-Satb2 group than that in pBABE-Hygro group at 3 weeks after surgery.PartⅢ:EGFP labeled BMSCs scattered in the tissues surrounding the implants 1 week after implantation. The GFP positive BMSCs were still detectable 3 weeks after the surgery. Local administration of BMSCs with forced expression of Satb2 enhanced osseointegration of the implants 3 weeks after the implantation and promoted bone formation. The real-time RT-PCR results from in vivo smples coincided with that of in vitro results. The expression levels of Satb2, Osx, Runx2, BSP, COLI, and OC were significantly higher in the Satb2 group than that of control group, which was the same pattern with the 3-week results of PartⅡ.Conclusions:1. In this presented study, it can be found that Satb2 can be delivered into the cells of the tissue surrounding the implant successfully by both pBABE-Hygro and RCAS retrovirus system when virus stocks are used locally. And the expression level of Satb2 in the tissue surrounding the implant can be up-regulated.2. Overexpression of Satb2 upregulates the expression levels of transcriptional factors Osx and Runx2 in implantation site. Satb2 overexpression enhances the expression levels of bone matrix proteins and osteogenic factors, such as BSP, COLI, and OC. While RCAS-Satb2 viral stocks are locally used, implant osseointegration can be enhanced 1 week after implantation.3 weeks after operation, Satb2 overexpression enhances osseointegration of the implants and promotes bone formation around the implants.3. The mechanism on bone formation between pBABE-Hygro-Satb2 and RCAS-Satb2 is different from each other after local administration. Local use of RCAS-Satb2 viral stocks can improve bone-to-implant contact significantly 1 week after implantation. The different effects on bone formation between RCAS-Satb2 and pBABE-Hygro-Satb2 may lie in the different cells infected when the viral stocks were locally used. Only BSP positive cells can be infected by RCAS retrovirus. While all mammalian dividing cells can be infected by pBABE-Hygro retrovirus, and the cells infected by pBABE-Hygro-Satb2 virus get involved in bone formation and remodeling of the tissue surrounding the implants.4. Overexpression of Satb2 can be detected in BMSCs infected with pBABE-Hygro-Satb2 viral stocks at 2,9 and 23 days after infection. Satb2 overexpressed BMSCs enhance osseointegration of implants and promote bone formation around the implants.更多还原