温敏凝胶制备及其在体内的生物相容性评价

Preparation and in vivo biocompatibility of a novel thermosensitive hydrogel

学术背景:凝胶材料在体温条件下能以凝胶形式稳定存在,是其作为医学植入物的必备条件,故需要将温敏型凝胶的低临界溶解温度调节到超过人体体温的水平。目的:制备低临界溶解温度超过37℃的温敏凝胶材料聚-(N-异丙基丙稀酰胺/N-羟甲基丙烯酰胺)P(NIPAAm-co-NHMPA),并初步评价其作为医学植入物的安全性。设计:随机、非盲法、分组对照动物实验。单位:华中科技大学同济医学院附属协和医院骨科。材料:实验于2007-01/10在华中科技大学同济医学院附属协和医院中心实验室、武汉大学化学系医用高分子材料教育部重点实验室完成。NIPAAm和NHMPA单体购自Aldrich公司、交联剂N,N'-亚甲基双丙烯酰胺(MBAAm)购自Fluka公司、促进剂过硫酸铵(APS)和四甲基乙二胺(TEMED)购自Sigma公司。方法:①温敏凝胶的制备:以APS和TEMED为氧化还原引发体系,MBAAm为交联剂制备P(NIPAAm-co-NHMPA),反应体系中添加一定质量分数的NHMP,溶涨率法测定低临界溶解温度。②安全性评价:进行过敏实验、急性全身毒性实验、遗传毒性实验、植入实验等一系列体内生物相容性动物实验以评估植入物的安全性。主要观察指标:过敏实验记录激发部位红斑水肿;急性全身中毒实验观察记录注射后4,24,48和72h动物的一般状态;遗传毒性实验于注射6h后在显微镜下计数鼠骨髓多染红细胞微核;植入实验将制得的切片进行光镜下观察。结果:①合成的凝胶材料符合预期的温敏特性要求,低临界溶解温度为38℃。②在过敏实验皮内注射浸提液及生理盐水组皮肤无红斑水肿;急性全身毒性实验显示腹腔注射浸提液及生理盐水组无毒性表现;遗传毒性实验中实验组和阴性对照组鼠骨髓多染红细胞的微核出现率无差异;体内植入实验表明材料周围炎性反应轻微而局限。结论:温敏凝胶P(NIPAAm-co-NHMPA)的生物相容性良好,是一种有潜力的医学植入物。

BACKGROUND： Thermosensitive hydrogel materials present stability at human body temperature, which is necessary for its application as a medical implant, thus the lower critical solution temperature （LCST） of thermosensitive hydrogel should be beyond the human body temperature by adjustment. OBJECTIVE： To prepare a thermosensitive hydrogel poly-N-isopropylacrylamide-co-N-hydroxymethylacrylamide P（NIPAAm-co-NHMPA） with over 37℃ LCST, and primarily appraise its safety as a medical implant in vivo. DESIGN： Random, non-blind, group control, animal experimental study. SETTING： Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science ＆ Technology（HUST）. MATERIALS： The experiments were carded out in the Central Laboratory, Union Hospital, Tongji Medical College, HUST and the Key Laboratory of Biomedical Polymer Materials of the Ministry of Education, College of Chemistry, Wuhan University between January and October in 2007. NIPAAm monomer and NHMPA monomer were purchased from Aldrich Company, crosslinking agent N, N＇-methylene bisacrylamide from Fluka Company, and initiator ammonium persulfate and accelerating agent tetramethyl ethylene diamine from Sigma Company. METHODS： 1.Taking ammonium persulfate and tetramethyl ethylene diamine as oxidation-reduction initiation system, while N, N＇-methylene bisacrylamide as cross-linking agent, P-NIPAAm-co-NHMPA was prepared with the addition of NHMPA in the reaction system. LCST was determined by shrinking tests.2.A series of biocompatibility tests such as sensitization test, acute systemic toxicity test, genetic toxicity test and implantation test were conducted in several experimental animals to evaluate the safety of the implant. MAIN OUTCOME MEASURES： The erythema and edema of stimulated lesions were recorded in sensitization test; the general state of each animal in acute systemic toxicity test were recorded 4, 24, 48 and 72 hours after injection; in genetic toxicity test, mouse bone marrow polychromatic erythrocyte （PEC） micronucleus was counted six hours after injection under microscope; sections after implantation were observed under light microscope. RESULTS： 1.Synthesized hydrogel showed thermosensitive character and the LCST was 38℃.2.In sensitization test, there was no erythema and edema occurred after leaching liquor and saline were injected; acute systemic toxicity test result revealed no symptom of toxicity; the genetic toxicity test suggested no difference of PEC frequency between experimental group and negative control group; in vivo implantation test, the inflammation around the material was mild and limited. CONCLUSION： P（NIPAAm-co-NHMPA） shows good biocompatibility and can be potentially used as an implant material.