壳聚糖-PVA-羧甲基壳聚糖屏障膜基本生物学性能的初步实验研究
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摘要
目的：探讨几丁质及其衍生物几丁聚糖制成的壳聚糖-PVA-羧甲基壳聚糖膜(C-CM膜)的组织相容性，可降解性，促进创面愈合，止血及抑菌作用，以便对C-CM膜用于牙周引导组织再生的可行性进行初步的生物学评价。
方法：64只大鼠分成3组。第一组42只，3种不同厚度的膜植入大鼠腿部肌肉与植入外科缝合丝线组作对照，按照1周和2，3，4，5，6周时间点取标本做组织病理学检查，考察其体内降解性及其组织相容性；第二组12只大鼠，在每只大鼠背部两侧制备全厚皮片缺损促进愈合的动物模型，4个1.0×1.0cm创面，一侧敷以C-CM膜，另一侧裸露作空白对照，测量伤口愈合面积；第三组10只大鼠，制备大鼠全厚皮片缺损止血的动物模型，在每只大鼠背部两侧做4个1.0×1.0cm创面，随机分别用C-CM膜和纱布止血，观察与创面的黏附情况，记录出血时间，45min后，取下C-CM膜和纱布，并用紫外分光光度仪测膜或纱布中血红蛋白光度吸收值表示出血量，测量其止血效果；采用固体培养基体外抑菌法，观察C-CM膜对细菌(金黄色葡萄球菌ATCC 27583、大肠杆菌ATCC25922、白色念珠菌ATCC 14053)生长的影响。
结果：C-CM膜植入动物肌肉中，周围组织无明显炎症，未发现组织变性和坏死，可在组织内缓慢降解，具有良好的组织相容性，6周后反应接近外科缝合丝线组；止血实验结果发现：C-CM膜组和纱布组的出血时间分别为83.5±9.333s和117±15.9275s，血红蛋白光度吸收值为0.9204±0.7559和1.9764±0.7205，二组比较差异显著(P＜0.05)，说明C-CM膜有一定的止血作用；促进创面愈合实验结果显示：不同时间段C-CM膜组肉芽组织较对照组厚，其中胶原纤维含量亦较多，C-CM膜组的创面愈合时间早于空白对照组，伤口愈合较快；抑菌实验显示C-CM膜对三种菌产生接触性抑制。
结论：C-CM膜有良好的生物学性能，能满足作为牙周引导组织再生膜的基本要求，有可能成为一种较理想的牙周引导组织再生的生物膜材料。
Objective: The objective of the study was to explore the biocompatibility,
biodegradability ,hemostatic and biological effects of chitosan-PVA-cm-chitosan membranes(C-CM), for the possibility of using C-CM membrane in guided tissue regeneration.
Methods: 64 Wistar rats were divided into 3 groups. In the first group, the 3
different thick C-CM membranes were implanted in 42 rats leg muscular porch .The specimen were examined histologically 1 week and 2. 3. 4. 5. 6 weeks respectively ;In the second group , full thicle flap wounds were made in 12 Wistar rats bilaterally. One wound in each animal was redressed by C-CM fiim ,the other was exposed as the control. The healing process was studied by the changes of depth and width of wounds. The third group included 10 Wistar rats . Four full thick flap wounds were made on the back of the rats. Using a randomized design, two wounds in each animal was treated with C-CM film, the others were treated with gauze. Bleeding times were measured for the bilateral back wounds. After bleeding sloped, the hemoglobin amount in films or gauzes were measured to determine the hemostatic effect Using a randomized design, one incision in each animal was treated with C-CM membrane, the influence of C-CM film on the growth of bacteria (Staphylococcus aureus ATCC 27853. Escherichia coli ATCC 25922. Monil
lia albicans ATCC 14053)were observed in vitro with solid medium.
Rusults: It was observed that C-CM membrane had no toxic effects on
surrounding tissue and was slowly degraded after implanted into rat leg muscular porch ; The results of hemostatic experiment showed that C-CM film adhered well to incisions. Bleeding time and hemoglobin amouts in C-CM film group were less than those in gauze group (P<0.05), the content of granulation tissue and collagen formation in the C-CM films redressed wounds was more
short than the control; The results showed the C-CM membrane touched inhibition on the three kinds of tested bacteria .
Conclusion: The C-CM membrane possesses satisfactory properties in
vivo,and the C-CM membrane could be therefore applied as a biological material for GTR.
方法：64只大鼠分成3组。第一组42只，3种不同厚度的膜植入大鼠腿部肌肉与植入外科缝合丝线组作对照，按照1周和2，3，4，5，6周时间点取标本做组织病理学检查，考察其体内降解性及其组织相容性；第二组12只大鼠，在每只大鼠背部两侧制备全厚皮片缺损促进愈合的动物模型，4个1.0×1.0cm创面，一侧敷以C-CM膜，另一侧裸露作空白对照，测量伤口愈合面积；第三组10只大鼠，制备大鼠全厚皮片缺损止血的动物模型，在每只大鼠背部两侧做4个1.0×1.0cm创面，随机分别用C-CM膜和纱布止血，观察与创面的黏附情况，记录出血时间，45min后，取下C-CM膜和纱布，并用紫外分光光度仪测膜或纱布中血红蛋白光度吸收值表示出血量，测量其止血效果；采用固体培养基体外抑菌法，观察C-CM膜对细菌(金黄色葡萄球菌ATCC 27583、大肠杆菌ATCC25922、白色念珠菌ATCC 14053)生长的影响。
结果：C-CM膜植入动物肌肉中，周围组织无明显炎症，未发现组织变性和坏死，可在组织内缓慢降解，具有良好的组织相容性，6周后反应接近外科缝合丝线组；止血实验结果发现：C-CM膜组和纱布组的出血时间分别为83.5±9.333s和117±15.9275s，血红蛋白光度吸收值为0.9204±0.7559和1.9764±0.7205，二组比较差异显著(P＜0.05)，说明C-CM膜有一定的止血作用；促进创面愈合实验结果显示：不同时间段C-CM膜组肉芽组织较对照组厚，其中胶原纤维含量亦较多，C-CM膜组的创面愈合时间早于空白对照组，伤口愈合较快；抑菌实验显示C-CM膜对三种菌产生接触性抑制。
结论：C-CM膜有良好的生物学性能，能满足作为牙周引导组织再生膜的基本要求，有可能成为一种较理想的牙周引导组织再生的生物膜材料。
Objective: The objective of the study was to explore the biocompatibility,
biodegradability ,hemostatic and biological effects of chitosan-PVA-cm-chitosan membranes(C-CM), for the possibility of using C-CM membrane in guided tissue regeneration.
Methods: 64 Wistar rats were divided into 3 groups. In the first group, the 3
different thick C-CM membranes were implanted in 42 rats leg muscular porch .The specimen were examined histologically 1 week and 2. 3. 4. 5. 6 weeks respectively ;In the second group , full thicle flap wounds were made in 12 Wistar rats bilaterally. One wound in each animal was redressed by C-CM fiim ,the other was exposed as the control. The healing process was studied by the changes of depth and width of wounds. The third group included 10 Wistar rats . Four full thick flap wounds were made on the back of the rats. Using a randomized design, two wounds in each animal was treated with C-CM film, the others were treated with gauze. Bleeding times were measured for the bilateral back wounds. After bleeding sloped, the hemoglobin amount in films or gauzes were measured to determine the hemostatic effect Using a randomized design, one incision in each animal was treated with C-CM membrane, the influence of C-CM film on the growth of bacteria (Staphylococcus aureus ATCC 27853. Escherichia coli ATCC 25922. Monil
lia albicans ATCC 14053)were observed in vitro with solid medium.
Rusults: It was observed that C-CM membrane had no toxic effects on
surrounding tissue and was slowly degraded after implanted into rat leg muscular porch ; The results of hemostatic experiment showed that C-CM film adhered well to incisions. Bleeding time and hemoglobin amouts in C-CM film group were less than those in gauze group (P<0.05), the content of granulation tissue and collagen formation in the C-CM films redressed wounds was more
short than the control; The results showed the C-CM membrane touched inhibition on the three kinds of tested bacteria .
Conclusion: The C-CM membrane possesses satisfactory properties in
vivo,and the C-CM membrane could be therefore applied as a biological material for GTR.
引文
1 Isidor F, Karring T, Nyman S, et al. The significance of coronal growth of periodontal ligament tissue for new attachment formation. J Clin Periodontol, 1986, 13(2): 145-150
2 Simao Kon, et al. Regeneration of periodontal ligament using resorbable and nonresorbable membranes: Clinical, histologic, and histometric study in dogs. Int J Periodont Rest Dent, 1991;11:59
3 one Mun Chung, Salkin LM, Stein MD, et al. Clinical evaluation of a biodegradable collagen membrane in guided tissue regeneration. J Periodontol 1990;61:732
4 Gager AH, Schultz AJ. Treatment of periodontal defects with an absorbable membrane (polyglactin 910)with and without osseous grafting; Case reports. J Periodontol 1991;18:276
5 侯春林，顾其胜主编．几丁质与医学，上海．上海科学技术出版社，2001．2
6 侯春林，卢建熙，曹梅讯等．几丁质生物学特性的研究．中国修复与重建外科杂志，1993，2：215
7 陈富林，毛天球，曹梅讯等．几丁质作为植骨材料生物相容性的实验研究．使用口腔医学杂志，1999；15(4)：246-248
8 庄昭霞，林志勇，卢凤琦等．壳聚糖生物降解与组织反应的实验研究．口腔材料器械杂志，1997；6 (4) 178-180
9 顾汉卿，徐国凤．生物医学材料[M]．天津：天津科技翻译出版公司，1993．
10 孟舒献，温晓娜，冯亚青等．聚乙烯醇生物相容性的研究．天津大学学报，2003，36(4)：473-477
11 Galgut P, Pitrola R, Waite P, et al. Histological evaluation of biodegradable and non-degradable membrance placed transcutaneously in rats. J Clin Periodontol, 1991, 18(6):581-586
12 Piattelli A, Scarano A, Coraggio F et al. Early tissue reactions to polylactie acid resorbable membranes: A histological and histochemical study in rabbit. Biomater, 1998;19(10):889-896
13 Riccado Muzzarelli. Chitin in Nature and Technology. [M]New York:Plerum press, 1986.435-442
14 Takashi M, et al. Effect of chitin and its derivatives on the
proliferation and cytokine production of fibroblasts in vitro. [J]Biomaterials, 1997,18:947-951
15 陈西广，王真，刘万顺等．6-0-羧甲基壳聚糖的细胞毒性效应研究．青岛海洋大学学报，2000，30(2)：221-224
16 Xi-GuangChen, Zhen Wang, Wan-Shun Liu, et al. The effect of carboxymethyl-chitosan on prolifertion and collagen secretion of normal and keloid skin fibroblasts. Biomaterials 23(2002)4609-4614
17 S. Hirano, et al. Effect of sulfated derivatives of chitosan on some blood coagulant factors. Carbohydr. Res;1985;137:205-215
18 S.I. Nishimura, et al. Inhibition of the hydrolytic activity of thrombin by chitin heparinoids; Carbohydr. Res;1986;156:286-292
19 R.A. Muzzarelli, et al. Sulfated, N-(arboxymethyl)chitosans:Novel. Blood Anticoagulants; Res;1984;126:225-231
20 斯洁庆，侯春林，蒋丽霞．几丁糖膜的制备及其止血性能的研究．解放军医学杂志，2002；27(12)：1055-1056
21 朱爱民，李振玲，杜平华．壳聚糖体外抗菌活性的研究．海峡药学，2001；13(4)：122-123
22 刘艳如，余萍，郑怡．水溶性壳聚糖的抑菌作用研究．中国海洋药物，2001；2：42-43
23 郑磊，王前，裴国献．可降解聚合物在骨组织工程中的应用进展．中华修复重建外科杂志，2000，14(3)：175-180
24 Pritlove CS, Immunohistochemical analysis of tissues regenerated from within periodontal defects treated with expanded polyterafluoroethylene membranes. J Periodontol, 1994, 65(2): 134
25 Ross Hardwick, et al. Devices for dentoalvedar regeneration; an up-to-date lierature review. J Periodontol, 1995;66:495
26 IgIhaut J. AuRhil I. Simpson DM, et al. Progenitor cell kinotics during guided tissue regeneration inexperimental periodontal wound. J Periondontal Res, 1988, 23:107
27 Vergara JA, Nasjleti CE, Caffesse RG, et al. Vascular response to guided tissue regeneration procedures using nonresorbable and bioabsorbable membranes in dogs[J]. J Periodontol, 1997, 68(3):217-224
28 Reiss RF, Oz MC. Autologous fibrin glue:production and clinical use
[J]. Transfus Med Rev, 1996,10(2):85
29 Radosevich M, Goubran HI, Burnouf T. Fibrin sealant: scientific rationale, production methods, properties, and current clinical use[J]. Vox Sang, 1997, 7(23):133
30 储庆，吴织芬，万灵等．牙周组织再生术后的聚四氟乙烯膜扫描电镜观察[J]．牙体牙髓牙周病学杂志，2003，13(8)：436
2 Simao Kon, et al. Regeneration of periodontal ligament using resorbable and nonresorbable membranes: Clinical, histologic, and histometric study in dogs. Int J Periodont Rest Dent, 1991;11:59
3 one Mun Chung, Salkin LM, Stein MD, et al. Clinical evaluation of a biodegradable collagen membrane in guided tissue regeneration. J Periodontol 1990;61:732
4 Gager AH, Schultz AJ. Treatment of periodontal defects with an absorbable membrane (polyglactin 910)with and without osseous grafting; Case reports. J Periodontol 1991;18:276
5 侯春林，顾其胜主编．几丁质与医学，上海．上海科学技术出版社，2001．2
6 侯春林，卢建熙，曹梅讯等．几丁质生物学特性的研究．中国修复与重建外科杂志，1993，2：215
7 陈富林，毛天球，曹梅讯等．几丁质作为植骨材料生物相容性的实验研究．使用口腔医学杂志，1999；15(4)：246-248
8 庄昭霞，林志勇，卢凤琦等．壳聚糖生物降解与组织反应的实验研究．口腔材料器械杂志，1997；6 (4) 178-180
9 顾汉卿，徐国凤．生物医学材料[M]．天津：天津科技翻译出版公司，1993．
10 孟舒献，温晓娜，冯亚青等．聚乙烯醇生物相容性的研究．天津大学学报，2003，36(4)：473-477
11 Galgut P, Pitrola R, Waite P, et al. Histological evaluation of biodegradable and non-degradable membrance placed transcutaneously in rats. J Clin Periodontol, 1991, 18(6):581-586
12 Piattelli A, Scarano A, Coraggio F et al. Early tissue reactions to polylactie acid resorbable membranes: A histological and histochemical study in rabbit. Biomater, 1998;19(10):889-896
13 Riccado Muzzarelli. Chitin in Nature and Technology. [M]New York:Plerum press, 1986.435-442
14 Takashi M, et al. Effect of chitin and its derivatives on the
proliferation and cytokine production of fibroblasts in vitro. [J]Biomaterials, 1997,18:947-951
15 陈西广，王真，刘万顺等．6-0-羧甲基壳聚糖的细胞毒性效应研究．青岛海洋大学学报，2000，30(2)：221-224
16 Xi-GuangChen, Zhen Wang, Wan-Shun Liu, et al. The effect of carboxymethyl-chitosan on prolifertion and collagen secretion of normal and keloid skin fibroblasts. Biomaterials 23(2002)4609-4614
17 S. Hirano, et al. Effect of sulfated derivatives of chitosan on some blood coagulant factors. Carbohydr. Res;1985;137:205-215
18 S.I. Nishimura, et al. Inhibition of the hydrolytic activity of thrombin by chitin heparinoids; Carbohydr. Res;1986;156:286-292
19 R.A. Muzzarelli, et al. Sulfated, N-(arboxymethyl)chitosans:Novel. Blood Anticoagulants; Res;1984;126:225-231
20 斯洁庆，侯春林，蒋丽霞．几丁糖膜的制备及其止血性能的研究．解放军医学杂志，2002；27(12)：1055-1056
21 朱爱民，李振玲，杜平华．壳聚糖体外抗菌活性的研究．海峡药学，2001；13(4)：122-123
22 刘艳如，余萍，郑怡．水溶性壳聚糖的抑菌作用研究．中国海洋药物，2001；2：42-43
23 郑磊，王前，裴国献．可降解聚合物在骨组织工程中的应用进展．中华修复重建外科杂志，2000，14(3)：175-180
24 Pritlove CS, Immunohistochemical analysis of tissues regenerated from within periodontal defects treated with expanded polyterafluoroethylene membranes. J Periodontol, 1994, 65(2): 134
25 Ross Hardwick, et al. Devices for dentoalvedar regeneration; an up-to-date lierature review. J Periodontol, 1995;66:495
26 IgIhaut J. AuRhil I. Simpson DM, et al. Progenitor cell kinotics during guided tissue regeneration inexperimental periodontal wound. J Periondontal Res, 1988, 23:107
27 Vergara JA, Nasjleti CE, Caffesse RG, et al. Vascular response to guided tissue regeneration procedures using nonresorbable and bioabsorbable membranes in dogs[J]. J Periodontol, 1997, 68(3):217-224
28 Reiss RF, Oz MC. Autologous fibrin glue:production and clinical use
[J]. Transfus Med Rev, 1996,10(2):85
29 Radosevich M, Goubran HI, Burnouf T. Fibrin sealant: scientific rationale, production methods, properties, and current clinical use[J]. Vox Sang, 1997, 7(23):133
30 储庆，吴织芬，万灵等．牙周组织再生术后的聚四氟乙烯膜扫描电镜观察[J]．牙体牙髓牙周病学杂志，2003，13(8)：436