骨化三醇局部应用对骨缺损修复影响的实验研究

发布时间：2018-05-18 14:33

本文选题：骨化三醇 + 局部应用　；参考：《南方医科大学》2016年硕士论文

【摘要】：研究背景骨缺损的修复一直以来是临床工作中关注的焦点,口腔种植修复中,也常常会面临骨量不足的问题,而获得和长期维持种植体骨结合最重要的先决条件是种植位点存在充足的健康骨量,可用骨高度和厚度允许植入适当长度和直径的种植体。但是,半数以上的种植位点存在骨量不足,必须进行与种植体植入同期或分阶段的骨量扩增。自体骨被认为是骨修复重建的“金标准”,但由于其来源的局限性,供骨区术后出现的失血、疼痛及感染等并发症,使用具有骨引导能力的人工植骨材料成为临床修复骨缺损的主要方式。人工植骨材料在骨缺损修复过程中主要起维持成骨空间,稳定血凝块,促进愈合的作用,其促进新骨生长的作用有限,成骨缓慢,成骨效能较差,不能满足临床上对早期负重的诉求。为了获得更好的成骨效果,尝试复合不同的骨生长因子以提高成骨质量,加快新骨的形成,一直是骨修复研究的热点。目前应用的骨生长因子多为蛋白质或多肽类分子,如骨形态发生蛋白(BMP)、转化生长因子(TGF-β)等,它们在体内会被蛋白酶降解,或被体液迅速稀释,半衰期极短,难以维持有效的治疗浓度,且均具有一定的免疫原性,可能引起免疫排斥反应。另外,此类大分子药物无论从何种途径获取,都存在价格昂贵的问题,这使得骨生长因子的临床应用受到了极大的限制。运用非蛋白小分子激活胞内信号传导通路,促进成骨,是骨缺损修复研究的新方向。近年来,有一些学者注意到一种之前未曾局部应用小分子药物一骨化三醇[1,25(OH)2D3]。在许多体外实验中发现,它可以促进成骨细胞及骨细胞的成熟,且与BⅧ-2有协同作用,促进矿化,进而促进成骨,同时,它能通过下调c-Fos和RANK的表达,抑制破骨细胞的生成。目前,骨化三醇及其类似物的合成已十分成熟,且骨化三醇没有免疫原性,不会引起机体的排斥反应,是一种理想的骨生长因子。骨化三醇是维生素D生物活性最高的形式,是维生素D在经过肝脏和肾脏的一系列代谢作用后产生的活性成分,最终进入血液循环,在调控骨形成以及骨发育中起重要作用∞]。目前,有许多动物模型(鸡、老鼠、兔子)支持系统性补充维生素D与骨愈合之间的正相关作用,在这些实验中维生素D的代谢物促进了骨折的愈合,并且增加了骨痂的强度。然而,通过全身循环到达靶器官的骨化三醇已经寥寥无几,仅能维持机体基本的骨代谢平衡,尤其在绝经期女性,骨代谢异常或骨质疏松患者中,体内维生素D的含量更会大大下降,促进局部骨缺损修复的效能有限；而大量口服骨化三醇又会产生药物毒性,引起高血钙、食欲不振、呕吐、腹泻,甚至软组织异位骨化等。因此,学者们开始研究局部应用骨化三醇类药物的效果。Salomo等在比格犬下颌骨植入种植体的同时局部应用维生素D2,发现其可以减少种植体冠方骨丧失并且增加骨结合的面积。但其所用药物为骨化三醇的前体,且直接对此类药物进行局部应用,存在易被体液稀释发生快速降解,很难在局部达到有效浓度的问题,同时还会因缺乏支架而变得杂乱无序,其促进成骨细胞成熟和促进骨基质矿化的作用无法有效发挥。而Hsiang-Hsi Hong等学者在以比格犬为动物模型的研究中,为克服直接局部应用骨化三醇易被体液迅速稀释的问题,采用了每周在下颌骨环状骨缺损周围注射骨化三醇含量较低的成品药物溉纯的方式,研究骨化三醇局部应用的成骨效能。但这种方法仍存在一定问题。首先,这种每周注射一次的给药方式使到达靶器官的药物量仍有所损耗,并且增加了手术次数,反复对缺损部位产生刺激,增加了成骨过程中的干扰因素；其次,实验中所制备的骨缺损并非标准骨缺损,不利于检验药物的成骨效能。为克服以上问题,我们需要寻找另一种局部应用骨化三醇的方式,并制备标准骨缺损以检验其成骨效能。大量文献报道,羟基磷灰石基团如：-OH-NH2,-COOH-,具有非共价吸附生长因子的功能,是良好的生长因子缓释载体,开放连通的不规则多孔结构能增加支架的生长因子吸附表面积,有利于生长因子的吸附和缓释。因此,羟基磷灰石常常作为支架材料携带PDGF-BB、 VEGF、BMP-2等生长因子修复各类骨缺损。而无机脱矿小牛骨的主要成分就是羟基磷灰石,它也是目前应用最广泛、研究最多的异种骨材料。因此,本研究拟以无机脱矿小牛骨粉为载体,复合骨化三醇后修复兔颅骨标准骨缺损,研究局部应用骨化三醇对骨缺损修复中早中期成骨的影响。目的研究局部应用骨化三醇对于早期及中期成骨的影响。方法1.动物模型及分组18只雄性新西兰大白兔,15-16周龄,体重2.9-3.2kg,由广东省医学动物实验中心提供,实验过程中和术后处理均符合动物伦理学标准。适应性喂养一周后,在广东省动物实验检测所进行实验。本实验分为3组：空白组(A组)、骨粉组(B组)以及骨化三醇复合骨粉组(C组)。以2、4、6周为研究时点,每一时点6只兔子。每只兔子颅顶制各4个8 mm直径的圆形骨缺损,每一研究时点有24个圆形骨缺损,随机分配给A、B、C三组。2.材料制备及处理用无水乙醇溶解骨化三醇(Sigma Chemical Co. 1a 25-Dihydroxyvitamin D3 D1530)后,将含有6μg 1,25(OH)2D3的无水乙醇溶液与5 mg Bio-Oss骨粉(瑞士盖氏制药有限公司,注册证号：国食药监械(进)字2014第3460922号,进口产品标准编号：YZB/SWI8428-2013,0.25-1 mm)混合,真空低温冻干,钴60辐照消毒后备用。3.手术过程术前30分钟肌注庆大霉素(1 mg/kg),预防术中及术后感染,术区备皮,以速眠新0.2 mL/kg后腿肌肉注射诱导麻醉,待诱导麻醉起效后,3%戊巴比妥钠注射液30 mg/kg耳缘静脉注射麻醉,全麻后取俯卧位,术区以碘伏消毒,铺一次性孔巾,2%盐酸利多卡因术区皮下局部浸润麻醉。扪及颅骨中缝骨性突起标志,沿中线作垂直切口,直达骨面,沿中缝翻起全厚瓣,前界达额骨的中部,后界至项骨的底部,两侧完全暴露顶骨的侧边缘,充分暴露术区,以外径为8 mm的环形骨钻于颅顶制各4个环形骨缺损,缺损之间相距大于2 mm,期间以4℃生理盐水充分冷却,注意勿伤及硬脑膜。按分组置入相应单纯骨粉或骨化三醇复合骨粉,分层严密缝合。术后肌肉注射庆大霉素(20万/d×5d)预防感染,必要时增加肌注青霉素类抗生素如氨苄西林钠或头孢拉定0.1 g/kg/d×5d。在动物处死之前的第3天和第4天颈部皮下注射钙黄绿素6 mg/kg,处死之前的第13天和第14天,在兔子颈部皮下注射四环素30 mg/kg。所有动物按照观察时间点2周,4周,6周分批以耳缘静脉空气栓塞法处死,取材,10%甲醛溶液固定48 h后,流水冲洗24小时,一半直接制作硬组织切片,一半置于10%EDTA溶液中脱钙约3个月,制作石蜡切片。4.主要检测指标大体观察、组织学观察、荧光染色钙沉积观察、骨计量学检测。结果1.术中及术后大体观察手术共制备72个圆形骨缺损,均未损及硬脑膜。术后所有动物精神状况良好,无死亡,创缘可见略有红肿,饮食及排泄均正常。术后1周术创区肿胀基本消退,缝线无松动、脱落,创区无感染现象,一期愈合。术后2周,术区完全愈合,缝线自然脱落,A组骨缺损区为纤维组织覆盖,边界清晰可辨,手指轻触柔软,B组及C组手指轻触稍软,较完全空白组硬实,骨粉颗粒间见纤维组织长入,缺损边缘较清晰。术后4周,A组骨缺损区见骨缺损边缘有少量新骨生成,缺损边缘分界模糊,缺损中心仍为软组织,手指轻触柔软,B组及C组手指轻触较硬,骨粉颗粒边缘较2周时模糊,缺损边缘模糊。术后6周,A组骨缺损边缘新生骨组织继续向中心长入,仍未完全长满,B组及C组骨粉间可见明显骨组织存在,手指触感极硬,缺损边界模糊不清,难以辨别。2.组织学观察术后2周,A组见缺损边缘有少量类骨质形成,中央为纤维组织,大量炎性细胞广泛浸润,结缔组织内见少量的骨胶原团块；B组见缺损中央及边缘处均可见骨粉颗粒周围有少量新骨及类骨质形成；C组所见与B组类似,但新骨及类骨质面积更大。术后4周,A组见缺损边缘新生骨向中央延伸,面积增大,中央仍为纤维组织充填,可见粗大的胶原纤维,散在少量炎性细胞；B组骨粉周围新生骨质染色加深,更趋成熟,面积增大：C组新生骨质面积较B组大,骨小梁稀疏交织成网状,周围环绕着扁平的呈单层排列的成骨细胞,可见大量尚未发育成熟的、由成骨细胞转化而来的骨细胞,包绕在这些骨细胞周围的骨陷窝间隙较为狭小,成骨细胞呈栅栏状排列在新生骨周围。术后6周,A组缺损边缘新生骨质继续向中央延伸,但仍未对接；B组缺损内广泛可见新生骨质围绕于骨粉颗粒周围,骨质成熟；C组较B组新生骨质面积增大,骨质更成熟,见大量的骨小梁融合成片形成板层骨,其间散在着少量形态不规则的间隙,间隙周围排列着均匀且连续的成骨细胞。3.荧光染色钙沉积观察术后2周,A组钙沉积较少,仅在缺损边缘有部分亮绿色的钙沉积；B组较完全空白组钙沉积范围扩大,且在骨缺损中央也出现了部分钙沉积,C组钙沉积范围较前两组又有增加,且亮度更高,表明钙沉积量较A、B两组也有一定程度增加。术后4周,三组钙沉积范围均较前扩大,钙沉积量依然显示出C组B组A组。术后6周,A组骨缺损中央形成骨岛,边缘可见钙沉积,但缺损中央仍有部分未见钙沉积,B组及C组缺损范围内均可见广泛钙沉积,C组较B组钙沉积所占面积更大,亮度更高。4.骨计量学观察术后2周,A组新生骨面积比为9.58±1.21%,成骨细胞数为15.2±1.68；B组新生骨面积比为12.52±1.09%,成骨细胞数为23.8±1.25；C组新生骨面积比为15.12±0.80%,成骨细胞数为24.8±2.12。术后4周,A组新生骨面积比为12.75±1.13%,成骨细胞数为20.3±2.15；B组新生骨面积比为15.88±1.04%,成骨细胞数为24.1±2.23;C组新生骨面积比为20.95±1.34%,成骨细胞数为23.8±1.79。术后6周,A组新生骨面积比为18.94±1.20%,成骨细胞数为25.0±2.26；B新生骨面积比为21.37±1.32%,成骨细胞数为25.8±1.69；C组新生骨面积比为25.10±1.27%,成骨细胞数为26.6±3.50。结果进行单向方差分析,3个时间点均显示出新生骨面积比三组组间有显著差异(P0.05),且C组B组A组,但随着时间的延长,3组差距呈逐渐缩小的趋势,而成骨细胞数量3组并无显著差异。结论1.15-16周龄,体重2.9-3.2 kg的雄性新西兰大白兔颅顶骨可制备4个直径8mm的圆形骨缺损。2.兔颅骨8mm直径的骨缺损在6周的观察时间内,可作为标准骨缺损对药物或骨代用品成骨效能进行相关研究。3.骨化三醇的局部应用,并未使骨缺损内的成骨细胞数量产生差异。4.骨化三醇的局部应用在骨缺损修复中可以促进早中期成骨,促进钙盐的沉积。
[Abstract]:The repair of bone defects has always been a focus of attention in clinical work. In oral implant repair, it often faces the problem of insufficient bone mass, and the most important prerequisite for the long-term maintenance of implant bone union is that there is sufficient healthy bone mass at the implant site, allowing the implantation of appropriate length and thickness with bone height and thickness. The diameter of the implant. However, more than half of the implant sites are deficient in bone mass. Bone mass amplification must be carried out at the same time or in stages of implant implantation. Autogenous bone is considered as a "gold standard" for bone repair and reconstruction, but due to its source limitations, complications such as blood loss, pain and infection after the operation of the bone region are used with bone citation. Artificial bone graft material is the main way to repair bone defect in clinical. Artificial bone grafting material mainly maintains bone space in the process of bone defect repair, stabilizing blood clot, promoting healing, its effect on promoting new bone growth is limited, bone formation is slow, bone formation efficiency is poor, and it can not meet the demands of early load in clinical. In order to obtain better osteogenic effects, it is a hot spot to try to combine different bone growth factors to improve the quality of osteogenesis and accelerate the formation of new bone. The bone growth factors are often used as protein or polypeptide molecules, such as bone morphogenetic protein (BMP), transforming growth factor (TGF- beta), and so on. They will be protein in the body. The enzyme degradation, or the rapid dilution of the body fluid, the half-life is very short, it is difficult to maintain the effective treatment concentration, and it has a certain immunogenicity, which may cause the immune rejection. In addition, this kind of large molecular drug has a high price problem in any way, which makes the clinical application of bone growth factor greatly limited. The use of non protein small molecules to activate the intracellular signal transduction pathway to promote osteogenesis is a new direction in the study of bone defect repair. In recent years, some scholars have noticed that a previously unlocalized small molecule drug, three alcohol [1,25 (OH) 2D3]., has been found in many in vitro experiments, which can promote the maturation of osteoblasts and bone cells. In addition, it has synergistic effect with B VIII -2 to promote mineralization and promote osteogenesis. At the same time, it can inhibit the formation of osteoclasts by downregulating the expression of c-Fos and RANK. At present, the synthesis of ossified three alcohol and its analogues is very mature, and the ossification of three alcohol has no immunogenicity and does not lead to the rejection of the body. It is an ideal bone growth factor. Three alcohol is the highest bioactivity of vitamin D, the active component produced by vitamin D after a series of metabolism through the liver and kidney, and eventually into the blood circulation and plays an important role in regulating bone formation and bone development. At present, many animal models (chicken, mice, rabbits) support systemic vitamin D and vitamin C The positive correlation between bone healing, in these experiments, vitamin D metabolites promote the healing of the fracture and increase the strength of the callus. However, a few of the ossification three alcohol through the systemic circulation to the target organ can only maintain the basic metabolic balance of the body, especially in menopause women, abnormality of bone metabolism, or bone thinning. In pine patients, the content of vitamin D in the body will be greatly reduced, and the efficacy of promoting the repair of local bone defect is limited, and a large number of oral ossification three alcohol will produce drug toxicity, causing hypercalcemia, anorexia, vomiting, diarrhea, and even ectopic ossification of soft tissue. Therefore, the researchers began to study the effect of local application of three alcohols. Salomo and other local application of vitamin D2 to the implant of the mandible of the Beagle dog. It is found that it can reduce the loss of bone and increase the area of bone binding. But the drug used as the precursor of the ossification three alcohol, and the local application of this kind of medicine directly, is easy to be degraded quickly by the humoral dilution, and is difficult to be localized. At the same time, the problem of reaching the effective concentration will also become disorderly and disorderly due to lack of scaffold, which can not effectively play an effective role in promoting osteoblast maturation and promoting bone matrix mineralization. In the study of the animal model of the Beagle dog, Hsiang-Hsi Hong and other scholars have overcome the problem of rapid dilution of the direct local application of ossification of three alcohol by the body fluid. This method was used to study the osteogenesis efficiency of the local application of ossification of three alcohol by injection of a finished product with low three alcohol content around the circumferential bone defect of the mandible every week. In order to overcome the above problems, we need to find another way to overcome the above problems, and to prepare the standard bone defect for the preparation of the standard bone defect for examination. It is reported that the hydroxyapatite group, such as -OH-NH2, -COOH-, has the function of non covalent growth factor, and it is a good growth factor sustained-release carrier. The open connected irregular porous structure can increase the acreage of the growth factor of the scaffold, be beneficial to the adsorption and release of growth factors. Therefore, the hydroxy phosphorus The limestone is often used as a scaffold material to carry PDGF-BB, VEGF, BMP-2 and other growth factors to repair all kinds of bone defects. The main ingredient of the mineral demineralized calf bone is hydroxyapatite. It is also the most widely used and most widely studied bone material. Therefore, this study is to be used as the carrier of the mineral calf bone powder and after the complex ossification of three alcohol. The effect of local application of ossification three alcohol on bone formation in the early and middle period of bone defect repair in rabbit skull defect. Objective to study the effect of local application of ossification of three alcohol on early and mid-term osteogenesis. Method 1. animal model and group 18 male New Zealand white rabbits, 15-16 weeks old, body weight 2.9-3.2kg, from Guangdong Medical Laboratory Center The experimental process and postoperative treatment were all in accordance with the standard of animal ethics. After a week of adaptive feeding, the experiment was carried out in Guangdong animal laboratory. The experiment was divided into 3 groups: blank group (group A), bone powder group (group B) and ossified three alcohol compound bone powder group (group C). At the time point of study, 6 rabbits and each rabbit cranium at every point. 4 round bone defects of each 8 mm diameter were made with 24 circular bone defects at each time point, and randomly assigned to A, B, and C three groups of.2. materials for the preparation and treatment of three alcohol (Sigma Chemical Co. 1a 25-Dihydroxyvitamin D3 D1530) and 5 alcohol solution and 5 bone powder (Switzerland). Gai Pharmaceutical Co., Ltd., registration number: National Food Drug Supervision (Jin) 2014 3460922nd, imported product standard number: YZB/SWI8428-2013,0.25-1 mm) mixed, vacuum cryo freeze dry, cobalt 60 radiation disinfection after.3. operation 30 minutes before the operation of gentamicin (1 mg /kg), prevention of intraoperative and postoperative infection, surgical area preparation, to sleep a new 0. to sleep a new 0. 2 mL/kg hind leg muscle injection induction anesthesia, after the induction of anesthesia, 3% pentobarbital sodium injection 30 mg/kg ear vein injection anesthesia, after general anesthesia, take the prone position, the operation area with Iodophor disinfection, a disposable hole towel, 2% hydrochloric lidocaine area subcutaneous local infiltration anesthesia. Directly to the bone surface, the full thick flap was turned over the middle of the middle of the frontal bone, the posterior border to the bottom of the bone, the side of the parietal bone exposed on both sides, full exposure of the surgical area. The circumferential bone of 8 mm of the outer diameter was drilled on the cranial top to make 4 ring bone defects, the distance between the defect was more than 2 mm, and the physiological saline was cooled fully during the period, and the hard brain was not injured. Membrane. The corresponding bone powder or ossified three alcohol compound bone powder was inserted into the group. The intramuscular injection of gentamicin (200 thousand /d x 5d) to prevent infection after operation, and the increase of the penicillin antibiotics such as ampicillin sodium or Cefradine 0.1 g/kg/d x 5d. for third and fourth day neck subcutaneous injection of calcium yellow green before the death of the animals. 6 mg/kg, thirteenth days and fourteenth days before death, all animals were subcutaneously injected with tetracycline 30 mg/kg. in the neck of the rabbit, and all animals were killed in accordance with the observation time for 2 weeks, 4 weeks, and 6 weeks by ear vein air embolism. After 10% Formaldehyde Solution fixed 48 h, water was washed for 24 hours, half of the hard tissue sections were made directly, half placed in 10%EDTA solution. About 3 months of decalcification, the main detection indexes of paraffin section.4. were made by gross observation, histological observation, fluorescence staining calcium deposition and bone metrology. Results 72 circular bone defects were prepared in 1. operations and after operation. All of them were not damaged by the dura mater. 1 weeks after the operation, the swelling of the wound area basically subsided, the sutures were not loosened, the sutures were not loose, and the wound area had no infection, the first period of healing. The operation area was completely healed and the suture naturally fell off, the bone defect area of group A was covered with fibrous tissue, the boundary was clearly distinguishable, the fingers touch softly, and the fingers in group B and C were slightly soft, and the complete blank group hard. 4 weeks after the operation, a small amount of new bone formation was found on the edge of bone defect in group A, and the edge of the defect was blurred, the defect center was soft tissue, the finger touch was soft, the fingers of B and C were hard, the edge of bone powder particles was blurred at 2 weeks, and the edge of the defect was blurred. 6 weeks after operation, A In group B and group C, the bone tissues of group B and group C were still not fully full. There was obvious bone tissue in the bone powder between group and group. The finger felt very hard and the defect boundary was blurred. It was difficult to distinguish the 2 weeks after the histological observation. In group A, there was a small amount of osteoid formation on the edge of the defect, the center was fibrous tissue, and a large number of inflammatory cells were widely used. In group B, a small amount of bone gelatin was found in the connective tissue. In group B, a small amount of new bone and osteoid formed around bone powder particles were found at the center and edge of the defect. Group C was similar to that in group B, but the area of new bone and osteoid was larger. In group A, the new bone on the edge of the defect was extended to the Central Center, the area was enlarged, and the central area was still filled with fibrous tissue. There was a large amount of collagen fiber and scattered in a small amount of inflammatory cells. In group B, the bone powder surrounding the bone powder was deepened, more mature, and the area increased: the area of the new bone in group C was larger than that of the B group, the trabecular bone was sparsely interwoven into the reticulation, surrounded by a flat single layer of osteoblasts, which showed a large number of undeveloped mature cells, from osteoblasts. The bone cells surrounded by these cells were narrower and narrower around the bone lacunae around these bone cells. Osteoblasts were arranged around the new bone in a palisade. 6 weeks after the operation, the defect margin of the A Group continued to extend to the center, but still did not dock. In group B defects, the new bone mass was widely surrounded by bone powder particles and bone matured; C group was more than B The area of the new bone in the group was increased and the bone was more mature. A large number of small bone Liang Rong synthetic tablets were found to form the lamellar bone, which scattered in a small amount of irregular space. The.3. fluorescent staining calcium deposition was arranged around the space, and the calcium deposition in the A group was less, and the calcium deposition was only partly bright green on the edge of the defect. The extent of calcium deposition in B group was enlarged, and some calcium deposits appeared in the center of bone defect. The range of calcium deposition in group C was higher than that in the first two groups, and the luminance was higher, indicating that calcium deposition was more than A, and B two groups also increased to a certain extent. The three groups of calcium deposits were expanded at 4 weeks after the operation, and the calcium deposition still showed A group in group B group C. 6 weeks after the operation, the bone island in the A group formed bone island with calcium deposition on the edge, but there was still no calcium deposition in the center of the defect. There was extensive calcium deposition in the B and C group. The area C was larger than the B group, the brightness was higher than the.4. bone metrology, the ratio of the bone area of the A group was 9.58 + 1.21% and the number of osteoblasts was 15. .2 + 1.68; B group new bone area ratio was 12.52 + 1.09%, osteoblast number was 23.8 + 1.25; C group new bone area ratio was 15.12 + 0.80%.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R683

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