摘要
牙周病是发生在牙周支持组织的细菌感染性疾病。目前公认细菌菌斑及其产物是引发牙周病必需的始动因子。因此合理正确的使用抗生素治疗牙周病是必要的辅助手段。具有可生物降解和原位注射温度敏感水凝胶被认为是最方便、最可靠且最能够满足口腔科医师需要的牙周病局部给药剂型。
壳聚糖(Chitosan,CS)是天然生物多糖,因其良好生物医学特性,在生物医药领域被广泛用作药物释放载体、组织工程支架及抑菌剂等。壳聚糖季铵盐(Quaternized chitosan,HTCC)是将CS的氨基引入季铵基团引入CS分子链中而得到的一类壳聚糖衍生物,以改善CS水溶性差的缺点。
本试验中,利用CS与醚化剂2,3-环氧丙基三甲基氯化铵(GTMAC)反应,制备HTCC。以CS及HTCC配合α,β-甘油磷酸(α,β-GP)制备原位可注射物理交联温敏水凝胶(CS/HTCC-GP水凝胶)。其溶胶-凝胶的转变时间可被控制在25°C以上。CS与HTCC的比例、α,β-GP的浓度均可控制转变温度和时间。CS与HTCC的重量比为5/1、α,β-GP的终浓度为8.33%,37°C时的溶胶-凝胶转变时间为3 min。CS/HTCC-GP温敏水凝胶超微结构疏松、多孔,以利于水分子的自由通过。CS/HTCC-GP温敏水温敏水凝胶能够有效的延缓奥硝唑和氯已啶的体外释放率。
选用牙龈卟啉单胞菌、中间普氏菌和伴防线放线杆菌3种牙周常见G-厌氧致病菌株为实验菌种,采用美国国立标准化委员会推荐厌氧菌药敏培养方法琼脂稀释法测定CS、HTCC及CS/HTCC-GP温敏水凝胶对三种牙周常见菌的最低抑菌浓度和抑菌环直径。结果表明,CS及HTCC对三种致病菌有较强的抑制活性,HTCC的抑菌活性较CS略强。WCS/WHTCC=5/1时具有最强的抑菌活性。载药温敏水凝胶较水凝胶基质或抗生素单纯对牙周常见致病菌的MIC显著减少,抑制活性显著增强。CS-HTCC/GP温敏水凝胶的抑菌环直径较CS-GP的大。
原代培养牙周膜成纤维细胞(HPDLCs),MTT比色法结果显示CS在浓度0.2-2000μg/mL均对HPDLCs的增殖有促进作用。HTCC在浓度为0.2-10μg/mL能够促进HPDLCs的增殖,而浓度为50-2000μg/mL时,则有抑制细胞增殖的作用。含有不同浓度CS/HTCC-GP温敏水凝胶浸提液的培养基均有不同程度促进HPDLCs增殖的作用。
细胞透射电镜显示:浓度为1000μg/mL和3μg/mL的CS以及3μg/mL的HTCC作用下的HPDLCs的超微结构与正常对照组无显著差别。而浓度为1000μg/mL的HTCC表现出细胞溶解等严重的细胞破坏现象。含50%CS/HTCC-GP温敏水凝胶的浸提液的DMEM培养的HPDLCs的超微结构正常,未见细胞破坏现象。应用流式细胞仪(FCM)对HPDLCs的细胞周期影响结果表明:浓度为3μg/mL和3 mg/mL的CS能使细胞的G1%增多,S%减少。浓度为3μg/mL的HTCC,G1%细胞较对照组显著减少,S%显著增多;而浓度为1 mg/mL和3 mg/mL时均发生细胞凋亡率,凋亡率分别为10.6%和51.8%。CS/HTCC-GP温敏水凝胶可以加速HPLDCs的分化与增殖。
通过ELISA法测定对LPS介导HPDLCs分泌IL-1β和TNF-α的影响。CS在最初的48 h能够抑制LPS介导的HPDLCs分泌IL-1β和TNF-α。而HTCC在最初24 h就促进HPDLCs分泌IL-1β和TNF-α。bFGF对LPS介导的HPDLCs分泌IL-1β和TNF-α有显著的抑制作用。CS/HTCC-GP温敏水凝胶的浸提液能够不同程度的减少LPS介导的HPDLCs对IL-1β和TNF-α的分泌。
CS/HTCC-GP温敏水凝胶大鼠股部肌肉内原位注射结果表明,此水凝胶具有良好的组织相容性。毒理学实验结果表明:CS、HTCC、CS/HTCC-GP、CS/HTCC-GP-1%Onz和CS/HTCC-GP-0.1%Chx温敏水凝胶临床使用安全。
通过建立犬牙周组织缺损病理模型,检测CS/HTCC-GP温敏水凝胶对牙周组织再生的作用。结果表明CS/HTCC-GP温敏水凝胶是良好的生长因子载体,具有较强的促进组织再生的作用。
以上实验结果提示,CS/HTCC-GP温度敏感水凝胶表现出良好的生物相容性和生物功能性,是牙周局部缓释药物载体和组织工程支架的理想选择。
Periodontal disease is a world-wide prevalent chronic infection which is caused by acummulation of bacterial in dental plaque. Biodegradable and injectable thermosensitive hydrogel was called one of the most convenient, reliable methods to prevent periodontal disease.
Chitosan (CS), a nature well-known parvus polysaccharide, got much application in pharmaceutical and medical fields as a promising biomaterial due to many unique advantages. Quaternized chitosan - N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC) introduced quaternary amino groups into CS chain to render it soluble in water.
An in situ injectable thermosensitive hydrogel constitutes of CS, HTCC withα,β-glycerophosphate (α,β-GP) was synthesized. The gelling point can be set at a temperature above 25°C. The transition process can be controlled by adjusting the weight ratio of CS to HTCC, or different final concentration ofα,β-GP and it effectively retarded the release of ornidazole (Onz) and chlorhexidine (Chx). The antibacterial activity to Porphyromonas gingivalis,Prevotella intermedia and Actinobacillus actinomycetemcomitans was determined through minimum inhibitory concentrations (MIC) and inhibitory zone measurement which are recommended by CLSI/NCCLS. Three periodontal pathogens strains were susceptible to CS and HTCC and a combination of CS and HTCC at weight ratio of 5 to 1 was the most potent inhibitor mixture. In addition, CS-HTCC/α,β-GP thermosensitive hydrogel exhibited stronger antibacterial activity with the larger inhibitory zone than CS/GP thermosensitive hydrogel.
The MTT assay revealed that CS at 0.2 g/mL to 2000 g/mL exhibit excellent cytocompatibility to human periodontal liganment cells(HPDLCs), While HTCC showed strong cytotoxicity to HPDLCs at the concentration of 2000, 1000, 100 and 50 g/mL. Transmission electron microscope (TEM) scan indicated that the architecture of HPDLCs at a concentration of 1000 g/mL was seriously destroyed. CS/HTCC-GP thermosensitive hydrogel was found to increase the proliferation of HPDLCs and promote the ALP activity in vitro.
Flow cytometry (FCM) revealed that CS/HTCC-GP thermosensitive hydrogel can increase the proliferation activity of HPDLCs. Whereas, HTCC at 3 mg/mL we observed apoptosis, and there was not observed in CS at the same concentration. HTCC can increase the secret of the volume of IL-1βand TNF-αcompared with CS. And basic fibroblast growth factor (bFGF) can inhibit the secret of IL-1βand TNF-α. The extract of CS/HTCC-GP thermosensitive hydrogel can decrease the secret of IL-1βand TNF-α.
The CS/HTCC-GP solution was injected intramuscularly into the rumps of rats and resulted in non-specific inflammation reaction. CS/HTCC-GP thermosensitive hydrogel loading with or without bFGF is promising to enhance new bone regeneration, periodontal ligament formation and new cement of dogs. All results indicated that CS/HTCC-GP thermosensitive hydrogel exhibit excellent biocompatibility and biological function and has potential to utilize as an injectable local drug delivery vehicle and as a tissue-engineering scaffold for periodontal disease therapy.
引文
1.蒋挺大.甲壳素.北京:化学工业出版社, 2003.
2. Rao SB, Sharma CP. Use of chitosan as a biomaterial: studies on its safety and hemostatic potential. Journal of biomedical materials research, 1997, 34: 21-28.
3. Molinaro G, Leroux JC, Damas J, et al. Biocompatibility of thermosensitive chitosan-based hydrogels: an in vivo experimental approach to injectable biomaterials. Biomaterials, 2002, 23: 2717-2722.
4. Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnology advances, 2008, 26: 1-21.
5. Schmitz T, Grabovac V, Palmberger TF, et al. Synthesis and characterization of a chitosan-N-acetyl cysteine conjugate. International journal of pharmaceutics, 2008, 347: 79-85.
6. Jumaa M, Furkert FH, Muller BW. A new lipid emulsion formulation with high antimicrobial efficacy using chitosan. Eur J Pharm Biopharm, 2002, 53: 115-123.
7. Kim KW, Thomas RL, Lee C, et al. Antimicrobial activity of native chitosan, degraded chitosan, and O-carboxymethylated chitosan. Journal of food protection, 2003, 66: 1495-1498.
8. Lehr C, Bouwstra J, Schacht E, et al. In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers. International journal of pharmaceutics, 1992, 78: 43-48.
9. Ikinci G, Senel S, Akincibay H, et al. Effect of chitosan on a periodontal pathogen Porphyromonas gingivalis. International journal of pharmaceutics, 2002, 235: 121-127.
10. Chung YC, Wang HL, Chen YM, et al. Effect of abiotic factors on the antibacterial activity of chitosan against waterborne pathogens. Bioresource technology, 2003, 88: 179-184.
11. Choi BK, Kim KY, Yoo YJ, et al. In vitro antimicrobial activity of a chitooligosaccharide mixture against Actinobacillus actinomycetemcomitans and Streptococcus mutans. International journal of antimicrobial agents, 2001, 18:553-557.
12. Park YJ, Lee YM, Park SN, et al. Enhanced guided bone regeneration by controlled tetracycline release from poly(L-lactide) barrier membranes. Journal of biomedical materials research, 2000, 51: 391-397.
13. Bae K, Jun EJ, Lee SM, et al. Effect of water-soluble reduced chitosan on Streptococcus mutans, plaque regrowth and biofilm vitality. Clinical oral investigations, 2006, 10: 102-107.
14. Tarsi R, Muzzarelli RA, Guzman CA, et al. Inhibition of Streptococcus mutans adsorption to hydroxyapatite by low-molecular-weight chitosans. Journal of dental research, 1997, 76: 665-672.
15.张澄波,梅学文.壳多糖对巨噬细胞精氨酸酶活性的影响.首都医科大学学报, 1992, 13: 63-64.
16. Muzzarelli R, Baldassarre V, Conti F, et al. Biological activity of chitosan: ultrastructural study. Biomaterials, 1988, 9: 247-252.
17. Ruel-Gariepy E, Chenite A, Chaput C, et al. Characterization of thermosensitive chitosan gels for the sustained delivery of drugs. International journal of pharmaceutics, 2000, 203: 89-98.
18. Wu J, Su ZG, Ma GH. A thermo- and pH-sensitive hydrogel composed of quaternized chitosan/glycerophosphate. International journal of pharmaceutics, 2006, 315: 1-11.
19. Zhang WF, Chen XG, Li PW, et al. Preparation and characterization of theophylline loaded chitosan/beta-cyclodextrin microspheres. Journal of materials science, 2008, 19: 305-310.
20. Borges O, Cordeiro-da-Silva A, Romeijn SG, et al. Uptake studies in rat Peyer's patches, cytotoxicity and release studies of alginate coated chitosan nanoparticles for mucosal vaccination. J Control Release, 2006, 114: 348-358.
21. Wang LC, Chen XG, Zhong DY, et al. Study on poly(vinyl alcohol)/carboxymethyl-chitosan blend film as local drug delivery system. Journal of materials science, 2007, 18: 1125-1133.
22. Needleman IG, Smales FC. In vitro assessment of bioadhesion for periodontal and buccal drug delivery. Biomaterials, 1995, 16: 617-624.
23. Barat R, Srinatha A, Pandit JK, et al. Chitosan inserts for periodontitis: influence of drug loading, plasticizer and crosslinking on in vitro metronidazole release. Acta pharmaceutica (Zagreb, Croatia), 2007, 57: 469-477.
24. El-Kamel AH, Ashri LY, Alsarra IA. Micromatricial metronidazole benzoate film as a local mucoadhesive delivery system for treatment of periodontal diseases. AAPS PharmSciTech, 2007, 8: E75.
25. Perugini P, Genta I, Conti B, et al. Periodontal delivery of ipriflavone: new chitosan/PLGA film delivery system for a lipophilic drug. International journal of pharmaceutics, 2003, 252: 1-9.
26. Govender S, Lutchman D, Pillay V, et al. Enhancing drug incorporation into tetracycline-loaded chitosan microspheres for periodontal therapy. Journal of microencapsulation, 2006, 23: 750-761.
27. Needleman I, Mather SJ, Martin GP, et al. Periodontal pocket clearance by gamma scintigraphy in human volunteers. Journal of clinical periodontology, 2000, 27: 904-909.
28. Tarsi R, Corbin B, Pruzzo C, et al. Effect of low-molecular-weight chitosans on the adhesive properties of oral streptococci. Oral microbiology and immunology, 1998, 13: 217-224.
29. Park YJ, Lee JY, Yeom HR, et al. Injectable polysaccharide microcapsules for prolonged release of minocycline for the treatment of periodontitis. Biotechnology letters, 2005, 27: 1761-1766.
30. Akncbay H, Senel S, Ay ZY. Application of chitosan gel in the treatment of chronic periodontitis. Journal of biomedical materials research, 2007, 80: 290-296.
31. Chenite A, Buschmann M, Wang D, et al. Rheological characterisation of thermogelling chitosan/glycerol-phosphate solutions. Carbohydrate Polymers, 2001, 46: 39-47.
32. Chenite A, Chaput C, Wang D, et al. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials, 2000, 21: 2155-2161.
33. Dung TH. Chitosan-TPP nanoparticle as a release system of antisense oligonucleotide in the oral environment. J Nanosci Nanotechnol, 2007, 7: 3695–3699.
34. Ma ZW, Zhang YJ, Wang R, et al. An animal experiment for the regeneration of periodontal defect by application of the dual-release chitosan thermosensitive hydrogel system. Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology, 2008, 43: 273-277.
35. Park JS, Choi SH, Moon IS, et al. Eight-week histological analysis on the effect of chitosan on surgically created one-wall intrabony defects in beagle dogs. Journal of clinical periodontology, 2003, 30: 443-453.
36. Lee YM, Park YJ, Lee SJ, et al. Tissue engineered bone formation using chitosan/tricalcium phosphate sponges. Journal of periodontology, 2000, 71: 410-417.
37. Park YJ, Lee YM, Park SN, et al. Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration. Biomaterials, 2000, 21: 153-159.
38. Hollinger JO, Battistone GC. Biodegradable bone repair materials. Synthetic polymers and ceramics. Clinical orthopaedics and related research, 1986: 290-305.
39.许春姣,郭峰,高清平, et al.骨髓基质干细胞与黄芪-壳聚糖/聚乳酸支架对犬牙周骨缺损再生的影响中南大学学报(医学版) 2006, 4: 512-517.
40. Scantlebury TV. 1982-1992: a decade of technology development for guided tissue regeneration. Journal of periodontology, 1993, 64: 1129-1137.
41. Jovicic B, Lazic Z, Nedidc M. Therapeutic efficacy of guided tissue regeneration and connective tissue autotransplants with periosteum in the management of gingival recession. Vojnosanitetski pregled, 2008, 65: 758-762.
42. Zucchelli G, Brini C, De Sanctis M. GTR treatment of intrabony defects in patients with early-onset and chronic adult periodontitis. The International journal of periodontics & restorative dentistry, 2002, 22: 323-333.
43. Hamano T, Teramoto A, Iizuka E, et al. Effects of polyelectrolyte complex (PEC) on human periodontal ligament fibroblast (HPLF) function. I. Three-dimensional structure of HPLF cultured on PEC. Journal of biomedical materials research, 1998, 41: 257-269.
44.张毅,庄昭霞,卢凤琦, et al.牙周引导组织再生壳聚糖膜的生物相容性研究.山东大学学报(医学版), 2002, 40: 115-117.
45. Illum L. Chitosan and its use as a pharmaceutical excipient. Pharmaceutical research, 1998, 15: 1326-1331.
46. Artursson P, Lindmark T, Davis SS, et al. Effect of chitosan on the permeability of monolayers of intestinal epithelial cells (Caco-2). Pharmaceutical research, 1994, 11: 1358-1361.
47. Stoltenberg JL, Osborn JB, Pihlstrom BL, et al. Prevalence of periodontal disease in a health maintenance organization and comparisons to the national survey of oral health. Journal of periodontology, 1993, 64: 853-858.
48. Southard GL, Godowski KC. Subgingival controlled release of antimicrobial agents in the treatment of periodontal disease. International journal of antimicrobial agents, 1998, 9: 239-253.
49. Haffajee AD, Socransky SS. Attachment level changes in destructive periodontal diseases. Journal of clinical periodontology, 1986, 13: 461-475.
50. Vyas SP, Sihorkar V, Mishra V. Controlled and targeted drug delivery strategies towards intraperiodontal pocket diseases. Journal of clinical pharmacy and therapeutics, 2000, 25: 21-42.
51. Pussinen PJ, Nyyssonen K, Alfthan G, et al. Serum antibody levels to Actinobacillus actinomycetemcomitans predict the risk for coronary heart disease. Arteriosclerosis, thrombosis, and vascular biology, 2005, 25: 833-838.
52. Pussinen PJ, Tuomisto K, Jousilahti P, et al. Endotoxemia, immune response to periodontal pathogens, and systemic inflammation associate with incident cardiovascular disease events. Arteriosclerosis, thrombosis, and vascular biology, 2007, 27: 1433-1439.
53. Pussinen PJ, Alfthan G, Jousilahti P, et al. Systemic exposure to Porphyromonas gingivalis predicts incident stroke. Atherosclerosis, 2007, 193: 222-228.
54. Pussinen PJ, Alfthan G, Rissanen H, et al. Antibodies to periodontal pathogens and stroke risk. Stroke; a journal of cerebral circulation, 2004, 35: 2020-2023.
55. Offenbacher S, Katz V, Fertik G, et al. Periodontal infection as a possible risk factor for preterm low birth weight. Journal of periodontology, 1996, 67: 1103-1113.
56.李德懿.牙周病微生物学.天津:天津与科技翻译出版公司, 1994.
57. Iqbal Z, Jain N, Jain GK, et al. Dental therapeutic systems. Recent patents on drug delivery & formulation, 2008, 2: 58-67.
58. Slots J, Bragd L, Wikstrom M, et al. The occurrence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius in destructive periodontal disease in adults. Journal of clinical periodontology, 1986, 13: 570-577.
59. Dzink JL, Tanner AC, Haffajee AD, et al. Gram negative species associated with active destructive periodontal lesions. Journal of clinical periodontology, 1985, 12: 648-659.
60. Mayrand D, Holt SC. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiological reviews, 1988, 52: 134-152.
61.曹采方.牙周病学.北京:人民卫生出版社, 2003.
62.黄世光,潘剑波,张宁.四环素药物对牙周炎治疗作用的研究进展.中国病理生理杂志, 1999, 15: 767.
63. Slots J, Rams TE. Antibiotics in periodontal therapy: advantages and disadvantages. Journal of clinical periodontology, 1990, 17: 479-493.
64. Walker CB, Gordon JM, Cornwall HA, et al. Gingival crevicular fluid levels of clindamycin compared with its minimal inhibitory concentrations for periodontal bacteria. Antimicrobial agents and chemotherapy, 1981, 19: 867-871.
65. Genco RJ. Antibiotics in the treatment of human periodontal diseases. Journal of periodontology, 1981, 52: 545-558.
66. Gates KA, Grad H, Birek P, et al. A new bioerodible polymer insert for the controlled release of metronidazole. Pharmaceutical research, 1994, 11: 1605-1609.
67. Pitcher GR, Newman HN, Strahan JD. Access to subgingival plaque by disclosing agents using mouthrinsing and direct irrigation. Journal of clinical periodontology,1980, 7: 300-308.
68. Slots J, Pallasch TJ. Dentists' role in halting antimicrobial resistance. Journal of dental research, 1996, 75: 1338-1341.
69. Sanai Y, Persson GR, Starr JR, et al. Presence and antibiotic resistance of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in children. Journal of clinical periodontology, 2002, 29: 929-934.
70. Walker CB. The acquisition of antibiotic resistance in the periodontal microflora. Periodontology 2000, 1996, 10: 79-88.
71. Loesche WJ. Antimicrobials in dentistry: with knowledge comes responsibility. Journal of dental research, 1996, 75: 1432-1433.
72. Vandekerckhove BN, Quirynen M, van Steenberghe D. The use of tetracycline-containing controlled-release fibers in the treatment of refractory periodontitis. Journal of periodontology, 1997, 68: 353-361.
73. Goodson JM, Haffajee A, Socransky SS. Periodontal therapy by local delivery of tetracycline. Journal of clinical periodontology, 1979, 6: 83-92.
74. Greenstein G. Local drug delivery in the treatment of periodontal diseases: assessing the clinical significance of the results. Journal of periodontology, 2006, 77: 565-578.
75.杨禾.常用局部缓释剂及其在牙周病治疗中的应用.国外医学口腔医学分册, 2001, 28: 224-226.
76. Kornman KS. Controlled-release local delivery antimicrobials in periodontics: prospects for the future. Journal of periodontology, 1993, 64: 782-791.
77.欧阳翔英, Mombelli A, Lang N.甲硝唑棒在深牙周袋病损治疗中的作用.口腔医学纵横, 2001, 17: 220-222.
78. Sakellari D, Goodson JM, Kolokotronis A, et al. Concentration of 3 tetracyclines in plasma, gingival crevice fluid and saliva. Journal of clinical periodontology, 2000, 27: 53-60.
79.中岛光一,水道裕久,江口彻.牙周病的抗生素治疗1,抗生素的选择.日本牙周病学会刊1987, 29: 463.
80.顾晶晶,胡纯贞,束蓉.派丽奥(PERIOCLINE)治疗牙周炎临床疗效分析.口腔材料器械杂志, 2000, 9: 20.
81. Soory M, Tilakaratne A. The effect of minocycline on the metabolism of androgens by human oral periosteal fibroblasts and its inhibition by finasteride. Archives of oral biology, 2000, 45: 257-265.
82. Soory M, Virdi H. Implications of minocycline, platelet-derived growth factor, and transforming growth factor-beta on inflammatory repair potential in the periodontium. Journal of periodontology, 1999, 70: 1136-1143.
83.吴勇,于晓霞,金艳.米诺环素软膏局部治疗慢性牙周炎的临床疗效现代口腔医学杂志, 2005, 19: 31-32.
84. Noyan U, Yilmaz S, Kuru B, et al. A clinical and microbiological evaluation of systemic and local metronidazole delivery in adult periodontitis patients. Journal of clinical periodontology, 1997, 24: 158-165.
85.唐志辉,曹采方,沙月琴.超声洁治和局部用甲硝唑治疗轻中度种植体周围炎.中华口腔医学杂志, 2002, 37.
86.安媛,孙德江.奥硝唑口腔膜剂的制备及质量控制.中国药房, 2005, 16: 908-909.
87.王海龙,杰罗,苏文泰, et al.替硝唑棒局部应用治疗牙周炎的细菌学研究华西口腔医学杂志, 2001, 19: 41-42.
88. Goffin G. Efficacy of sustained local delivery of chlorhexidine Periochip as an adjuvant to scaling and root planning in the treatment of chronic periodontal disease. Int Dent Rev, 1998, 18: 1-18.
89. Lindhe J, Heijl L, Goodson JM, et al. Local tetracycline delivery using hollow fiber devices in periodontal therapy. Journal of clinical periodontology, 1979, 6: 141-149.
90. Goodson JM, Offenbacher S, Farr DH, et al. Periodontal disease treatment by local drug delivery. Journal of periodontology, 1985, 56: 265-272.
91. Coventry J, Newman HN. Experimental use of a slow release device employing chlorhexidine gluconate in areas of acute periodontal inflammation. Journal of clinical periodontology, 1982, 9: 129-133.
92. Goodson JM, Holborow D, Dunn RL, et al. Monolithic tetracycline-containing fibers for controlled delivery to periodontal pockets. Journal of periodontology, 1983, 54: 575-579.
93. Tonetti M, Cugini MA, Goodson JM. Zero-order delivery with periodontal placement of tetracycline-loaded ethylene vinyl acetate fibers. Journal of periodontal research, 1990, 25: 243-249.
94. Michalowicz BS, Pihlstrom BL, Drisko CL, et al. Evaluation of periodontal treatments using controlled-release tetracycline fibers: maintenance response. Journal of periodontology, 1995, 66: 708-715.
95. Goodson JM, Cugini MA, Kent RL, et al. Multicenter evaluation of tetracycline fiber therapy: II. Clinical response. Journal of periodontal research, 1991, 26: 371-379.
96. Addy M, Langeroudi M. Comparison of the immediate effects on the sub-gingival microflora of acrylic strips containing 40% chlorhexidine, metronidazole or tetracycline. Journal of clinical periodontology, 1984, 11: 379-386.
97. Addy M, Hassan H, Moran J, et al. Use of antimicrobial containing acrylic strips in the treatment of chronic periodontal disease. A three month follow-up study. Journal of periodontology, 1988, 59: 557-564.
98. Larsen T. In vitro release of doxycycline from bioabsorbable materials and acrylic strips. J Periodontol, 1990, 61: 30-34.
99. Noguchi T, Izumizawa K, Fukuda M, et al. New method for local drug delivery using resorbable base material in periodontal therapy. The Bulletin of Tokyo Medical and Dental University, 1984, 31: 145-153.
100. Taner IL, Ozcan G, Doganay T, et al. Comparison of the antibacterial effects on subgingival microflora of two different resorbable base materials containing doxycycline. The Journal of Nihon University School of Dentistry, 1994, 36: 183-190.
101. Kimura S, Toda H, Shimabukuro Y, et al. Topical chemotherapy in human periodontitis using a new controlled-release insert containing ofloxacin. I.Microbiological observation. Journal of periodontal research, 1991, 26: 33-41.
102. Deasy PB, Collins AE, MacCarthy DJ, et al. Use of strips containing tetracycline hydrochloride or metronidazole for the treatment of advanced periodontal disease. The Journal of pharmacy and pharmacology, 1989, 41: 694-699.
103. Maze GI, Reinhardt RA, Agarwal RK, et al. Response to intracrevicular controlled delivery of 25% tetracycline from poly(lactide/glycolide) film strips in SPT patients. Journal of clinical periodontology, 1995, 22: 860-867.
104. Paolantonio M, D'Angelo M, Grassi RF, et al. Clinical and microbiologic effects of subgingival controlled-release delivery of chlorhexidine chip in the treatment of periodontitis: a multicenter study. Journal of periodontology, 2008, 79: 271-282.
105. Friedman M, Golomb G. New sustained release dosage form of chlorhexidine for dental use. I. Development and kinetics of release. Journal of periodontal research, 1982, 17: 323-328.
106. Barat R, Srinatha A, Pandit JK, et al. Ethylcellulose inserts of an orphan drug for periodontitis: preparation, in vitro, and clinical studies. Drug delivery, 2007, 14: 531-538.
107. Hirasawa M, Takada K, Makimura M, et al. Improvement of periodontal status by green tea catechin using a local delivery system: a clinical pilot study. Journal of periodontal research, 2002, 37: 433-438.
108. Soskolne WA. Subgingival delivery of therapeutic agents in the treatment of periodontal diseases. Crit Rev Oral Biol Med, 1997, 8: 164-174.
109. Medlicott NJ. Delivery systems for the administration of the drugs to the periodontal pocket. Advanced drug delivery reviews, 1994, 13: 181-203.
110. Minabe M, Takeuchi K, Tamura T, et al. Application of local drug delivery system to periodontal therapy. 3. The duration of therapeutic effect after administration of the collagen film immobilized tetracycline. Nippon Shishubyo Gakkai kaishi, 1988, 30: 576-582.
111. Golomb G, Friedman M, Soskolne A, et al. Sustained release device containing metronidazole for periodontal use. Journal of dental research, 1984, 63: 1149-1153.
112. Azoury R, Elkayam R, Friedman M. Nuclear magnetic resonance study of an ethyl cellulose sustained-release delivery system. I: Effect of casting solvent on hydration properties. Journal of pharmaceutical sciences, 1988, 77: 425-427.
113. Soskolne A, Golomb G, Friedman M, et al. New sustained release dosage form of chlorhexidine for dental use. II. Use in periodontal therapy. Journal of periodontal research, 1983, 18: 330-336.
114. Stabholz A, Sela MN, Friedman M, et al. Clinical and microbiological effects of sustained release chlorhexidine in periodontal pockets. Journal of clinical periodontology, 1986, 13: 783-788.
115. Stabholz A, Soskolne WA, Friedman M, et al. The use of sustained release delivery of chlorhexidine for the maintenance of periodontal pockets: 2-year clinical trial. Journal of periodontology, 1991, 62: 429-433.
116. Minabe M, Kodama T, Hori T, et al. Effects of atelocollagen on the wound healing reaction following palatal gingivectomy in rats. Journal of periodontal research, 1989, 24: 178-185.
117. Ozmeric N, Ozcan G, Haytac CM, et al. Chitosan film enriched with an antioxidant agent, taurine, in fenestration defects. Journal of biomedical materials research, 2000, 51: 500-503.
118. Ahuja A, Ali J, Rahman S. Biodegradable periodontal intrapocket device containing metronidazole and amoxycillin: formulation and characterisation. Die Pharmazie, 2006, 61: 25-29.
119. Agarwal RK. Development and characterization of tetracycline–poly(lactide/glycolide) films for treatment of periodontitis. J Control Release, 1993, 23: 137-146.
120. Webber WL, Mathiowitz E. Modulating tetracycline release from PLA/PG films. Proc Int Symp Control Release Bioact Mater, 1997, 24: 575-576.
121. Higashi K, Matsushita M, Morisaki K, et al. Local drug delivery systems for the treatment of periodontal disease. Journal of pharmacobio-dynamics, 1991, 14: 72-81.
122. Stolze K, Stellfeld M. Systemic absorption of metronidazole after applicationof a metronidazole 25% dental gel. J Clin Periodontol, 1992, 19: 693–697.
123. Paquette DW, Waters GS, Stefanidou VL, et al. Inhibition of experimental gingivitis in beagle dogs with topical salivary histatins. Journal of clinical periodontology, 1997, 24: 216-222.
124. Nakagawa T, Yamada S, Oosuka Y, et al. Clinical and microbiological study of local minocycline delivery (Periocline) following scaling and root planing in recurrent periodontal pockets. The Bulletin of Tokyo Dental College, 1991, 32: 63-70.
125. Hayashi K, Takada K, Hirasawa M. Clinical and microbiological effects of controlled-release local delivery of minocycline on periodontitis in dogs. American journal of veterinary research, 1998, 59: 464-467.
126. Pattnaik S, Panigrahi L, Murthy RS. Periodontal muco-adhesive formulations for the treatment of infectious periodontal diseases. Current drug delivery, 2007, 4: 303-323.
127. Polson AM, Stoller NH, Hanes PJ, et al. 2 multi-center trials assessing the clinical efficacy of 5% sanguinarine in a biodegradable drug delivery system. Journal of clinical periodontology, 1996, 23: 782-788.
128. Polson AM, Garrett S, Stoller NH, et al. Multi-center comparative evaluation of subgingivally delivered sanguinarine and doxycycline in the treatment of periodontitis. II. Clinical results. Journal of periodontology, 1997, 68: 119-126.
129. Esposito E, Cortesi R, Cervellati F, et al. Biodegradable microparticles for sustained delivery of tetracycline to the periodontal pocket: formulatory and drug release studies. Journal of microencapsulation, 1997, 14: 175-187.
130. Kong LX, Peng Z, Li SD, et al. Nanotechnology and its role in the management of periodontal diseases. Periodontology 2000, 2006, 40: 184-196.
131. Bako J, Szepesi M, Marton I, et al. Synthesis of nanoparticles for dental drug delivery systems. Fogorvosi szemle, 2007, 100: 109-113.
132. Shefer A, Shefer SD, inventors. Biodegradable bioadhesive controlled release system of nano-particles for oral care products. US Pat. No. 6,565,873, 2003.
133. Pinon-Segundo E, Ganem-Quintanar A, Alonso-Perez V, et al. Preparationand characterization of triclosan nanoparticles for periodontal treatment. International journal of pharmaceutics, 2005, 294: 217-232.
134. Shahverdi AR, Fakhimi A, Shahverdi HR, et al. Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli. Nanomedicine, 2007, 3: 168-171.
135. Satishkumar R, Vertegel A. Charge-directed targeting of antimicrobial protein–nanoparticle conjugates. Biotechnol Bioeng 2008, 100: 403–412.
136. Jones MN, Kaszuba M. Polyhydroxy-mediated interactions between liposomes and bacterial biofilms. Biochimica et biophysica acta, 1994, 1193: 48-54.
137. Vyas SP, Sihorkar V, Dubey PK. Preparation, characterization and in vitro antimicrobial activity of metronidazole bearing lectinized liposomes for intra-periodontal pocket delivery. Die Pharmazie, 2001, 56: 554-560.
138. Hobdell MH, Oliveira ER, Bautista R, et al. Oral diseases and socio-economic status (SES). British dental journal, 2003, 194: 91-96; discussion 88.
139. Muzzarelli RA. Biochemical significance of exogenous chitins and chitosans in animals and patients. Carbohydr Polym, 1993, 20: 7-16.
140. Nishimura K, Minami S. Application of chitin and chitosan for biomaterials. Biotechnol Genet Eng Rev, 1995, 13: 383-420.
141. Sandri G, Rossi S, Bonferoni MC, et al. Buccal penetration enhancement properties of N-trimethyl chitosan: Influence of quaternization degree on absorption of a high molecular weight molecule. International journal of pharmaceutics, 2005, 297: 146-155.
142.张艳艳,马启敏,江志华.壳聚糖季铵盐的合成及性质研究.中国海洋大学学报, 2005, 35: 459-462.
143. Lim SH, Hudson SM. Synthesis and antimicrobial activity of a water-soluble chitosan derivative with a fiber-reactive group. Carbohydrate research, 2004, 339: 313-319.
144. Jia Z, shen D, Xu W. Synthesis and antibacterial activities of quaternary ammonium salt of chitosan. Carbohydrate research, 2001, 333: 1-6.
145.张艳艳,马启敏,江志华.壳聚糖季铵盐的合成及性质研究.中国海洋大学学报, 2005, 35: 459-462.
146. National Committee for Clinical Laboratory Standards. Methods for antimicrobial susceptibility testing for anaerobic bacteria; USA 2004; Approved Standard, 6th ed, NCCLS document M11-A6.
147. Milazzo I, Blandino G, Caccamo F, et al. Faropenem, a new oral penem: antibacterial activity against selected anaerobic and fastidious periodontal isolates. The Journal of antimicrobial chemotherapy, 2003, 51: 721-725.
148.薛毅,许丽华,史俊南.龈下菌斑中产黑色素杆菌群与牙周病变的关系.现代口腔医学杂志, 1995, 9: 141-142.
149.曹采芳.牙周病学.北京:人民卫生出版社, 2003.
150.肖晓蓉.口腔微生物学.北京:北京医科大学中国协和医科大学联合出版社, 1993.
151. Helander IM, Nurmiaho-Lassila EL, Ahvenainen R, et al. Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria. International journal of food microbiology, 2001, 71: 235-244.
152. Qi L, Xu Z, Jiang X, et al. Preparation and antibacterial activity of chitosan nanoparticles. Carbohydrate research, 2004, 339: 2693-2700.
153. Vishu Kumar AB, Varadaraj MC, Gowda LR, et al. Low molecular weight chitosans--preparation with the aid of pronase, characterization and their bactericidal activity towards Bacillus cereus and Escherichia coli. Biochimica et biophysica acta, 2007, 1770: 495-505.
154. No HK, Lee SH, Park NY, et al. Comparison of physicochemical, binding, and antibacterial properties of chitosans prepared without and with deproteinization process. Journal of agricultural and food chemistry, 2003, 51: 7659-7663.
155. ISO10993-1 Biological evaluation of medical devices-Part 1: Evaluation and testing, 1997.
156. GB/T168861 Biological evaluation of medical devices-Part 1: Evaluation and testing, 2001
157.中华人民共和国卫生部药政管理局.中药新药研究指南.
158.王乔治,袁伯俊.新药临床前安全性评价与实践.北京:军中医学科学出版社, 1997.
159. Chung YM, Simmons KL, Gutowska A, et al. Sol-gel transition temperature of PLGA-g-PEG aqueous solutions. Biomacromolecules, 2002, 3: 511-516.
160. ISO TR 10271 Dentistry-Determination of tarnish and corrosion of metals and alloys.Bern: printed in Switzerland, 3. 1993.
161. Wu J, Wei W, Wang LY, et al. A thermosensitive hydrogel based on quaternized chitosan and poly(ethylene glycol) for nasal drug delivery system. Biomaterials, 2007, 28: 2220-2232.
162. Senel S, Ikinci G, Kas S, et al. Chitosan films and hydrogels of chlorhexidine gluconate for oral mucosal delivery. International journal of pharmaceutics, 2000, 193: 197-203.
163.殷凯生,殷民生.实用抗感染药物手册.北京:人民卫生出版社, 2001.
164.丁俊清,周海孝,张远芳.奥硝唑用于口腔厌氧菌感染的临床研究.临床口腔医学杂志, 2001, 17: 127.
165.唐明,肖晓蓉,章锦才.奥硝唑对牙周病病原菌的体外抗菌作用.牙体牙髓牙周病学杂志, 2003, 13: 127-129.
166. Kamma JJ, Nakou M, Mitsis FJ. The clinical and microbiological effects of systemic ornidazole in sites with and without subgingival debridement in early-onset periodontitis patients. Journal of periodontology, 2000, 71: 1862-1873.
167.刘琨,侯本祥,杨圣辉, et al.壳聚糖体外抑菌试验研究.现代口腔医学杂志, 2007, 21: 281-283.
168. Leeuwenburgh SC, Jansen JA, Malda J, et al. Trends in biomaterials research: An analysis of the scientific programme of the World Biomaterials Congress 2008. Biomaterials, 2008, 29: 3047-3052.
169. Langer R, Peppas N. Advances in biomaterials, drug delivery and bionanotechnology. AIChE J 2003, 12: 2990-3006.
170. Williams DF. On the mechanisms of biocompatibility. Biomaterials, 2008, 29: 2941-2953.
171. Cho JH, Kim SH, Park KD, et al. Chondrogenic differentiation of human mesenchymal stem cells using a thermosensitive poly(N-isopropylacrylamide) and water-soluble chitosan copolymer. Biomaterials, 2004, 25: 5743-5751.
172. Chen FM, Zhao YM, Wu H, et al. Enhancement of periodontal tissue regeneration by locally controlled delivery of insulin-like growth factor-I from dextran-co-gelatin microspheres. J Control Release, 2006, 114: 209-222.
173. van de Weert M, Hennink WE, Jiskoot W. Protein instability in poly(lactic-co-glycolic acid) microparticles. Pharmaceutical research, 2000, 17: 1159-1167.
174. Hoemann CD, Sun J, Legare A, et al. Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society, 2005, 13: 318-329.