中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (14): 2177-2182.doi: 10.3969/j.issn.2095-4344.1656

• 组织工程口腔材料 tissue-engineered oral materials • 上一篇    下一篇

血小板胶-胶原生物活性复合膜修复大鼠牙周骨组织缺损

张旭凤1，符起亚1，郑根建1，郭玉苏1，陈丹宇1，符方满1，吴 慧1，王 琳2   

  1. 1海南医学院第一附属医院口腔内科， 海南省海口市 570102；2海南省人民医院口腔科， 海南省海口市 570311
  • 收稿日期:2018-11-12
  • 通讯作者: 王琳，博士，主任医师，海南省人民医院口腔科， 海南省海口市 570311
  • 作者简介:张旭凤，女，1976年生，河北省深州市人，汉族，2005年青海医学院毕业，硕士，副主任医师，主要从事口腔医学研究。
  • 基金资助:

    2014年度海南省自然科学基金项目(814315)，项目负责人：王琳

Autologous platelet gel-collagen biologically active composite membrane for repair of periodontal bone defect in rats

Zhang Xufeng1, Fu Qiya1, Zheng Genjian1, Guo Yusu1, Chen Danyu1, Fu Fangman1, Wu Hui1, Wang Lin2   

  1. 1Department of Oral Medicine, the First Affiliated Hospital of Hainan Medical University, Haikou 570102, Hainan Province, China; 2Department of Stomatology, Hainan Provincial People’s Hospital, Haikou 570311, Hainan Province, China
  • Received:2018-11-12
  • Contact: Wang Lin, MD, Chief physician, Department of Stomatology, Hainan Provincial People's Hospital, Haikou 570311, Hainan Province, China
  • About author:Zhang Xufeng, Master, Associate chief physician, Department of Oral Medicine, the First Affiliated Hospital of Hainan Medical University, Haikou 570102, Hainan Province, China
  • Supported by:

    the Natural Science Foundation of Hainan Province in 2014, No. 814315 (to WL)

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摘要：

文章快速阅读：

 

文题释义：
富血小板血浆：将采集的抗凝血通过梯度离心后，与牛凝血酶、钙离子混合后可激活血小板起止血作用，并刺激颗粒释放不同类型的生长因子的物质。
骨形态发生蛋白2：是诱导新骨形成中最强的一类活性生长因子，能够不断诱导未分化的间充质细胞的定向迁徙、增殖和继续分化，从而发挥骨诱导作用。
 
 
背景：近些年，组织工程学的发展为牙周组织缺损的治疗与组织再生提供了新途径。组织工程学治疗手段包括种子细胞、支架和生长因子。血小板胶因含大量血小板生长因子，胶原常被用于支架材料的制备，故血小板胶与胶原生物活性复合膜可为缺损组织提供支架及生长因子。
目的：观察血小板胶-胶原生物活性复合膜在修复大鼠牙周骨组织缺损中的作用。
方法：实验选取Wistar大鼠42只(由上海西普尔-必凯实验动物有限公司提供)。①将胶原裁成5 mm×2 mm大小，抽取6只大鼠10 mL全血后，从大鼠新鲜全血中获取富血小板血浆，按一定比例加入牛凝血酶、氯化钙和胶原制成血小板胶-胶原复合膜。测定全血及富血小板血浆中血小板计数值，ELISA法测定全血及富血小板血浆中血小板衍生生长因子AB、转化生长因子β、碱性成纤维细胞生长因子和血管内皮生长因子的水平。②将剩余36只大鼠建立下颌牙周骨缺损模型(骨缺损大小为5 mm×2 mm，磨除牙根面牙骨质)后，随机分成2组。血小板胶-胶原组将血小板胶-胶原复合膜置于骨缺损处，而对照组不放置任何材料。2，4和8周后获取2组大鼠牙周组织标本进行苏木精-伊红染色，测定新生牙周骨率、新生牙骨质、新生牙槽骨、新生牙周膜组织高度。免疫组织化学染色观察骨形成蛋白2的表达。

结果与结论：①富血小板血浆中血小板计数均值为全血的4.78倍，即全血制备成富血小板血浆后血小板数量显著上升(P < 0.05)。富血小板血浆中血小板衍生生长因子AB、转化生长因子β、碱性成纤维细胞生长因子和血管内皮生长因子的水平分别是全血中的3.10倍、3.45倍、7.17倍、5.45倍(P < 0.05)；②苏木精-伊红染色观察结果：两组新生骨率、新生牙骨质、新生牙槽骨、新生牙周膜组织高度在2周时差异无显著性意义(P > 0.05)，第4和第8周时，血小板胶-胶原组上述指标均显著高于对照组(P < 0.05)；③免疫组化结果显示，在2周时，血小板胶-胶原组骨形态发生蛋白2开始表达，4周时骨形态发生蛋白2表达最强(P < 0.05)，而8周时阳性表达减弱(P > 0.05)；④结果提示，血小板胶-胶原生物活性复合膜可明显促进新生牙组织再生，并伴随着骨形态发生蛋白2的表达，减小牙周组织缺损后的修复时间。

ORCID: 0000-0003-2308-8183(张旭凤)

关键词: 血小板胶, 胶原, 生物复合膜, 下颌牙周骨缺损, 牙组织再生, 富血小板血浆, 骨形态发生蛋白2, 组织构建

Abstract:

BACKGROUND: In recent years, the development of tissue engineering has provided a new approach for the treatment of periodontal bone defect. Tissue engineering therapy includes seed cells, scaffolds and growth factors. Platelet gel contains a large number of platelet growth factors, and collagen is often used for the preparation of scaffold materials. Therefore, the platelet gel and collagen biologically active composite membrane can provide scaffolds and growth factors for the defect bone.

OBJECTIVE: To investigate the effect of autologous platelet gel-collagen biologically active composite membrane on the repair of periodontal bone defect in rats.
METHODS: Forty-two Wistar rats (Shanghai Xipuer-Bikai Experimental Animal Co., Ltd., China) were selected. (1) Collagen was cut into 5 mm×2 mm size, and 10 mL of whole blood was extracted from 6 rats to obtain platelet-rich plasma. Autologous platelet gel-collagen composite membrane was prepared by adding bovine thrombin, calcium chloride and collagen in a certain proportion. Platelets in whole blood and in platelet-rich plasma were detected. The levels of platelet derived growth factor AB, transforming growth factor-β, basic fibroblast growth factor and vascular endothelial growth factor in whole blood and platelet-rich plasma were detected by ELISA. (2) The models of mandibular periosteal defect were established in 36 rats (the size of the bone defect was 5 mm×2 mm, and the root surface cementum was removed), and randomly divided into two groups. Autologous platelet gel-collagen group placed the autologous platelet gel-collagen composite membrane in the bone defect, and the control group did not place any materials. The hematoxylin-eosin staining of periodontal tissues of rats in each group was analyzed at 2, 4 and 8 weeks after surgery. Rate of new born, new centumum formation, new alveolar bone formation, and new periodontal ligament tissue formation height were measured. The expression of bone morphogenetic protein-2 was detected by immunohistochemical staining.
RESULTS AND CONCLUSION: (1) The mean platelet count in platelet-rich plasma was 4.78 times as high as the whole blood, indicating that the number of platelets increased significantly after prepared into platelet-rich plasma (P < 0.05). The levels of platelet derived growth factor AB, transforming growth factor-β, basic fibroblast growth factor and vascular endothelial growth factor in platelet-rich plasma were 3.10, 3.45, 7.17 and 5.45 times of the whole blood, respectively (P < 0.05). (2) The results of hematoxylin-eosin staining observed that the rate of new born, new centumum formation, new alveolar bone formation, and new periodontal ligament tissue formation height at 2 weeks in the autologous platelet gel-collagen group showed no significant difference from the control group (P > 0.05). At 4 and 8 weeks, all above indexes in the autologous platelet gel-collagen group were significantly higher than those in the control group (P < 0.05). (3) Results of immunohistochemical staining revealed that at 2 weeks, bone morphogenetic protein-2 in the autologous platelet gel-collagen group began to express, and the expression of bone morphogenetic protein-2 was highest at 4 weeks (P < 0.05), and the positive expression was weakened at 8 weeks (P > 0.05). (4) Our results clarify that autologous platelet gel-collagen bioactive composite membrane can significantly promote the regeneration of new tooth, which is associated with the expression of bone morphogenetic protein-2, and reduce the repair time after periodontal tissue defect.

Key words: Platelet-Rich Plasma, Collagen, Bone Morphogenetic Proteins, Tissue Engineering

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