新型壳聚糖基温敏凝胶膜引导骨再生性能的体外实验研究

上海口腔医学 ›› 2015, Vol. 24 ›› Issue (4): 442-446.

新型壳聚糖基温敏凝胶膜引导骨再生性能的体外实验研究

梁杰¹, 崔军², 许胜¹, 周倜¹, 徐伟成¹, 徐欣^{3, 4}

1.烟台市口腔医院,山东烟台 264001; 2.济南市口腔医院,山东济南 250001; 3.山东大学口腔医学院口腔种植中心,山东济南 250012; 4.山东省口腔生物医学重点实验室,山东济南 250012

收稿日期:2014-07-24 出版日期:2015-08-20 发布日期:2015-09-10
通讯作者: 徐欣,Tel:0531-88382636,E-mail: xinxu@sdu.edu.cn E-mail:xinxu@sdu.edu.cn
作者简介:梁杰(1986-),女,硕士研究生,医师,E-mail：kathyliangjie@163.com
基金资助:
烟台市科技发展计划项目（2013ws248）; 山东省中青年科学家科研奖励基金计划项目（BS2013YY056）

In vitro study on the properties of guided bone regeneration of new type chitosan-based thermosensitive hydrogel membranes

LIANG Jie¹, CUI Jun², XU Sheng¹, ZHOU Ti¹, XU Wei-cheng¹, XU Xin^{3, 4}

(1.Yantai Stomatological Hospital. Yantai 264001; 2. Jinan Stomatological Hospital. Jinan 250001; 3. Department of Implantology, School of Stomatology Shandong University. Jinan 250012; 4. Key Lab of Oral Biomedicine of Shandong Province. Jinan 250012, Shandong Province, China

Received:2014-07-24 Online:2015-08-20 Published:2015-09-10
Supported by:
Supported by Science and Technology Development Planning Project of Yantai City(2013WS248) and Science and Technology Award Program for Young and Middle-aged Scientists of Shandong Province(BS2013YY056)

摘要/Abstract

摘要： 目的制备负载釉基质蛋白（enamel matrix proteins,EMPs）的壳聚糖/β甘油磷酸钠(chitosan/β-glycerophosphate salt,CS/β-GP)温敏凝胶膜,探讨其体外引导骨再生的能力。方法利用CS/β-GP体系的温敏相转变特性,制备新型生物膜并添加牛血清白蛋白（bovine serum albumin,BSA）,利用蛋白浓度测定试剂盒（加强型）检测不同时点的蛋白释放浓度,绘制蛋白缓释曲线。添加EMPs的CS/β-GP（1.0 g）复合膜作为A组; 单纯CS/β-GP（1.0 g）复合膜作为B组;同时,设空白对照组即C组,分别与ST2细胞共培养。检测膜的理化性能, MTT法检测复合膜的生物相容性, PNPP法检测碱性磷酸酶（alkaline phosphatase, ALP）的活性。采用SPSS16.0软件包对数据进行统计学分析。结果不同浓度的复合膜可缓慢释放蛋白12 d以上,并且随着β-GP浓度的升高,蛋白缓释总量增大。负载蛋白后,复合膜的理化性能均未发生显著变化（P>0.05）。MTT检测A、B、C 3组的吸光度（OD）值,组间差异具有显著性,A、B组OD值均高于C组,A组OD值高于B组（P<0.05）。A、B组ALP表达高于空白对照组（P<0.05）,A、B 2组之间ALP活性表达亦具有显著差异,A组高于B组（P<0.05）。结论负载EMPs的新型壳聚糖基温敏凝胶膜在体外实验中表现出一定的引导骨再生活性,是一种具有应用潜力的新型生物膜材料。

关键词: 壳聚糖, β, 甘油磷酸钠, 温敏凝胶, 引导骨再生, 牛血清白蛋白, 釉基质蛋白

Abstract: PURPOSE：To prepare chitosan/β-glycerophosphate salt (CS/β-GP) thermosensitive hydrogel membranes loaded enamel matrix proteins and detect the guided bone regeneration properties. METHODS：A newly membrane was synthesized using thermal phase inversion property of the CS/β-GP system. The membrane was synthesized and added with protein BSA. The concentration of protein was detected at different time points by enhanced protein assay kit, and the protein release curve was drawn. CS/β -GP membrane added EMPs (1.0 g) as group A, CS/β -GP (1.0 g) membrane as group B and nothing as blank control group (group C). They were co-cultured with ST2 cells. The mechanical properties of the membranes were tested in vitro, and the compatibility properties were detected by MTT method. The activity of ALP was assayed by PNPP method. The data was analyzed by SPSS 16.0 software package. RESULTS：Membranes with different concentration of CS/β -GP could release protein slowly more than 12 days, and the total quantity of the released protein increased with the concentration of the β-GP. The changes of mechanical properties of the membranes were not significant（P>0.05）. The OD value of group A, B and C had statistically significant difference in the MTT test. The values of group A and B were higher than that of group C, while the value of group A was higher than that of group B (P<0.05). The activities of ALP were different in the three groups .The activities of group A and B were higher than that of the blank control group(P<0.05）.The difference in expression of ALP between group A and B was also statistically significant. The expression in group A was higher than that in group B (P<0.05). CONCLUSIONS: The new type CS/β-GP membrane shows property of guided bone regeneration in vitro, which have the potentials for clinical use.

Key words: Chitosan, β-Glycerophosphate salt, Thermosensitive hydrogel, Guided bone regeneration, Bovine serum albumin, Enamel matrix proteins Shanghai J Stomatol, 2015, 24(4):442-446.

中图分类号:

R781.34

梁杰, 崔军, 许胜, 周倜, 徐伟成, 徐欣. 新型壳聚糖基温敏凝胶膜引导骨再生性能的体外实验研究[J]. 上海口腔医学, 2015, 24(4): 442-446.

LIANG Jie, CUI Jun, XU Sheng, ZHOU Ti, XU Wei-cheng, XU Xin. In vitro study on the properties of guided bone regeneration of new type chitosan-based thermosensitive hydrogel membranes[J]. Shanghai Journal of Stomatology, 2015, 24(4): 442-446.

参考文献

[1] Yan J, Yang L, Wang G, et al. Biocompatibility evaluation of chitosan-based injectable hydrogels for the culturing mice mesenchymal stem cells in vitro [J]. J Biomater Appl, 2010, 24(7): 625-637.
[2] 吴广升, 张艺文, 王新文, 等. 壳聚糖温敏凝胶负载釉基质蛋白对骨髓基质细胞的作用[J]. 上海口腔医学,2009,18(2):178-182.
[3] Shi W, Ji Y, Zhang X, et al. Characterization of ph- and thermosensitive hydrogel as a vehicle for controlled protein delivery [J]. J Pharm Sci, 2011,100(3): 886-895.
[4] CUI Jun, JIANG Bao-Qi, LIANG Jie, et al. Preparation and characterization of chitosan/β-GP membrane for guided bone regeneration[J]. J Wuhan Univ Tech (Mater Sci), 2011, 26(2): 242-246.
[5] 宋爱梅, 束蓉, 谢玉峰, 等. 釉基质蛋白对猪骨髓基质细胞增殖和根面附着生长的影响[J]. 实用口腔医学杂志, 2006, 22(5): 679-683.
[6] 梁杰, 崔军, 孙康, 等. 壳聚糖温敏凝胶引导组织再生膜的制备及其细胞生物相容性[J].山东大学学报(医学版), 2011, 49(6): 80-83.
[7] Schwartz Z, Carnes DL Jr, Pulliam R, et al. Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells [J]. J Periodontol, 2000, 71(8): 1287-1296.
[8] 束蓉, 张濒, 刘正, 等.釉基质蛋白诱导牙周组织再生的量效实验研究 [J].牙体牙髓牙周病学杂志, 2000, 10(1): 1-3.
[9] 宋爱梅, 束蓉, 蒋欣泉, 等. 釉基质蛋白对猪骨髓基质细胞黏附、伸展及增殖的影响[J]. 上海口腔医学,2005,14(6): 624-628.
[10] 杜岩,吴织芬,董广英, 等.釉基质蛋白对体外培养的兔骨髓基质细胞生物学功能的影响[J].口腔医学研究,2004,20(5):457-461.
[11] Jin R, Moreira Teixeira LS, Dijkstra PJ, et al. Injectable chitosan-based hydrogels for cartilage tissue engineering [J]. Biomaterials, 2009, 30(13): 2544-2551.
[12] Wang L, Stegemann JP. Thermogelling chitosan and collagen composite hydrogels initiated with beta-glycerophosphate for bone tissue engineering[J]. Biomaterials, 2010, 31(14): 3976-3985.
[13] Cui J, Liang J, Wen Y, et al. In vitro and in vivo evaluation of chitosan/β-glycerol phosphate composite membrane for guided bone regeneration[J]. J Biomed Mater Res A, 2014, 102(9): 2911-2917.

[1]	谢雪梅, 张佳园, 张野, 俞晓佳, 刘李明, 王健, 于德栋. 龈沟液中sICAM-1、IL-1β和HIF-1α水平与义齿种植修复并发种植体周围炎的关系[J]. 上海口腔医学, 2023, 32(1): 47-51.
[2]	吴靖, 邹多宏, 张志愿. 壳聚糖-藻酸钙-锂皂石纳米片止血复合膜的制备及其对口腔内创面的治疗作用[J]. 上海口腔医学, 2022, 31(6): 569-575.
[3]	毛新霞, 林晓炜, 魏轩, 李琴. 自体牙本质颗粒与Bio-Oss骨粉植入引导骨再生治疗牙周骨缺损效果比较[J]. 上海口腔医学, 2022, 31(5): 530-534.
[4]	涂慧娟, 李凌宇, 李医丹, 罗真兰, 俞明. Er:YAG激光联合引导骨再生治疗种植体周围炎伴骨缺损效果评价[J]. 上海口腔医学, 2022, 31(4): 418-422.
[5]	白月辉, 刘阳, 崔玉兰, 姜杉杉, 商庆龙, 赵琛. 慢性睡眠剥夺对大鼠髁突软骨的影响[J]. 上海口腔医学, 2022, 31(2): 148-155.
[6]	徐方芳, 蒋备战. DKK1对脂多糖感染人牙髓细胞生物学行为的影响[J]. 上海口腔医学, 2021, 30(4): 355-359.
[7]	马春燕, 潘清, 何磊, 杨海兵. 细胞外基质PDMS硬度对牙髓干细胞增殖和成骨分化的影响[J]. 上海口腔医学, 2021, 30(3): 253-257.
[8]	孙小娟, 刘萍萍, 周忠伟, 秦立业, 付浩, 郑蓝天, 曹昆. TC-PHBHHx/β-TCP复合物治疗SD大鼠干槽症的实验研究[J]. 上海口腔医学, 2019, 28(5): 460-466.
[9]	梅梅, 易杰, 张疆弢, 黄瑾, 江策, 丰雷. 外源性TGF-β1与PDGF-BB联合应用对正畸牙压力侧Pyk2蛋白和基因表达的影响[J]. 上海口腔医学, 2019, 28(5): 472-477.
[10]	李宇崚, 王卫忠, 龚忠诚. 二甲胺四环素联合茶多酚对早期种植体周围软组织炎患者IL-1β、IL-17F水平的影响[J]. 上海口腔医学, 2019, 28(5): 494-498.
[11]	周琳怡, 张楚南, 顾迎新, 赖红昌. 光动力疗法对种植体周炎症因子含量的影响[J]. 上海口腔医学, 2019, 28(1): 63-66.
[12]	陈婉红, 蔡世雄, 孙玉环. 视觉模拟评分法联合疼痛相关因子评估不同正畸力对人活体牙髓的影响[J]. 上海口腔医学, 2017, 26(5): 561-564.
[13]	翟越, 黄枧英, 朴玄, 纪芳, 费照亮, 陶疆. 人β防御素在牙髓组织中的表达及与炎症因子的关系[J]. 上海口腔医学, 2017, 26(4): 368-373.
[14]	胡琴, 徐小淋, 周建萍, 戴红卫. 釉基质蛋白衍生物对大鼠牙移动后复发和牙根修复的影响[J]. 上海口腔医学, 2017, 26(2): 156-161.
[15]	郭珲, 潘克清, 邓婧, 刘学玉, 宋佳. 羧甲基壳聚糖锌多肽复合材料对人牙周膜成纤维细胞的毒性[J]. 上海口腔医学, 2017, 26(2): 162-166.