富血小板血浆支架体内诱导牙髓再生的初步研究

发布时间：2018-07-03 01:56

本文选题：富血小板血浆 + 白细胞　；参考：《广西医科大学》2014年硕士论文

【摘要】：目的：分别探讨不同类型的富血小板血浆(platelet-rich plasma, PRP）支架和复合牙髓细胞的PRP支架体内诱导牙髓再生的可行性；评价不同类型的PRP支架募集内源性细胞归巢的能力，为PRP支架应用于牙髓再生的临床治疗提供实验基础和理论依据。方法： 1.实验一：改良的Curasn’s方法制备富白细胞富血小板血浆（leucocyte-rich plasma,L-PRP）支架，将小型猪乳牙牙根段进行化学预备，随机分为4组，每组6个样本，分别为①全血组；②100%L-PRP组；③50%L-PRP组：将100%L-PRP用DMEM培养基稀释成50%L-PRP（v/v）④空白组，空的牙根段。分组植入裸鼠背部皮下，5周后取出样本进行组织学观察。 2.实验二：二次离心法制备贫白细胞富血小板血浆（leucocyte-poorplatelet-rich plasma，P-PRP）支架，组织块法分离培养人牙髓细胞（humandental plus cells, hDPCs）并进行鉴定。将人牙根管段进行机械化学预备，随机分为3组，，每组8个样本,分别为①阴性对照组：P-PRP支架；②实验组：P-PRP支架复合hDPCs；③空白组，空的牙根段。分组植入裸鼠背部皮下，8周后取出样本进行组织学观察分析。结果： 1.小型猪全血制备的100%L-PRP中血小板和白细胞的含量分别是全血的6倍和4倍。移植了100%L-PRP的两只裸鼠出现皮肤损害和炎症反应。 2.50%L-PRP组根管内有新的结缔组织形成，没有血管生成；100%L-PRP组根管内充满了大量的炎性细胞，可见少量散在的梭形细胞。 3.培养的第4代hDPCs矿化诱导后碱性磷酸酶染色呈阳性，茜素红，Von Kossa染色，可观察到少量钙结节生成。人全血制备的P-PRP中血小板的含量是全血的1.55倍，白细胞含量远远低于全血中的含量。单独移植P-PRP支架的根管内观察到不规则的组织形成。 4．P-PRP支架复合hDPCs的根管内观察到有血管化的牙髓样组织形成，但是没有牙本质样组织生成。根管内大部分细胞抗人线粒体免疫组织化学染色阳性，表明新生组织中细胞主要是人来源的细胞，但在靠近根管开口附近有较集中的抗人线粒体免疫组织化学染色阴性的梭形细胞，表明有内源性宿主细胞参与组织再生。结论： 1.复合hDPCs的P-PRP支架在体内再生牙髓样组织是可行的。 2.单独移植L-PRP支架或者P-PRP支架可以募集内源性细胞参与组织再生，但是募集的细胞类型和数量有限。
[Abstract]:Objective: to investigate the feasibility of inducing pulp regeneration in different types of platelet-rich plasma (PRP-rich) stents and composite dental pulp cells, and to evaluate the ability of different types of platelet-rich stents to recruit endogenous cells to homing. To provide experimental and theoretical basis for the application of PRP stent in the clinical treatment of dental pulp regeneration. Methods: 1. Experiment 1: modified Curasnines method was used to prepare leucocyte-rich plasma-rich plasma (L-PRP) stents. The root segments of primary teeth of miniature pigs were chemically prepared and randomly divided into 4 groups, 6 samples in each group: 1 whole blood group (2100g / L PRP group); 350L-PRP group: 100% L-PRP was diluted into 50 L-PRP (v / v) 4 blank group with DMEM medium, empty root segment. After 5 weeks of subcutaneous implantation in nude mice, the samples were taken out for histological observation. 2. Experiment 2: leucocyte-poorplatelet-rich plasma-rich plasma (leucocyte-poorplatelet-rich PPRP) scaffolds were prepared by secondary centrifugation, and (humandental plus cells, hDPCs were isolated and identified by tissue block method. The human root canal segment was prepared by mechanochemistry and was randomly divided into 3 groups with 8 samples in each group: 1 negative control group: 1: P-PRP stent and 2 experimental group: 1 P-PRP stent combined with hDPCs3 blank group, empty root segment. After 8 weeks of subcutaneous implantation of nude mice, the samples were taken out for histological observation and analysis. Results: 1. The contents of platelet and leukocyte in 100% of L-PRP prepared from whole blood of miniature pig were 6 times and 4 times of that of whole blood, respectively. Two nude mice transplanted with 100% L-PRP had skin damage and inflammatory reaction. 2.50 L-PRP group had new connective tissue formation in the root canal, and the root canal in the 100L-PRP group was filled with a large number of inflammatory cells without angiogenesis. A few scattered fusiform cells can be seen. 3. After mineralization induction of the fourth generation of hDPCs, alkaline phosphatase staining was positive, alizarin red Von Kossa staining, a small amount of calcium nodule formation could be observed. The platelet content of P-PRP prepared by human whole blood was 1.55 times of that of the whole blood, and the white blood cell content was much lower than that of the whole blood. Irregular tissue formation was observed in the root canal of P-PRP scaffold alone. 4. The formation of vascularized dental pulp tissue was observed in the root canal of P-PRP scaffold combined with hDPCs, but no dentin tissue formation was observed. Most of the cells in the root canal were positive for anti-human mitochondria immunohistochemical staining, indicating that the cells in the newborn tissue were mainly human derived cells. However, near the root canal opening, there were concentrated spindle cells with negative anti-human mitochondria immunohistochemical staining, indicating that endogenous host cells were involved in tissue regeneration. Conclusion: 1. P-PRP scaffold combined with hDPCs is feasible to regenerate pulp tissue in vivo. 2. 2. L-PRP stents or P-PRP stents alone could recruit endogenous cells to participate in tissue regeneration, but the type and number of cells recruited were limited.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.3

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