摘要
目的:从交联度、细胞毒性、溶胀率与降解率方面比较京尼平和戊二醛交联明胶的生物学特性。方法:实验于2005-06/2006-01在哈尔滨医科大学附属第二医院实验中心完成。①材料分为两组,京尼平组用10g/L京尼平交联明胶72h,戊二醛组用10g/L戊二醛交联明胶24h。②检测交联度:每组材料取8个样本,其中5个加入碳酸氢钠和三硝基苯磺酸,再加盐酸,在346nm测吸光度值(A三硝基苯磺酸)。另取3个样本先加盐酸,然后再加三硝基苯磺酸,其余步骤相同,测得吸光度取平均值作为对照(A对),交联后吸光度值为:A交联后=A三硝基苯磺酸-A对。再取11mg明胶加19μL水,不加京尼平,同样步骤测吸光度,得到交联前吸光度(A交联前)。交联度=(A交联前-A交联后)/A交联前×100%。③细胞毒性试验:按照1cm2/mL比例用培养液浸提5组材料制备浸提液,将中国仓鼠V-79细胞种植于96孔板,分为3组:阴性组(单纯培养液)、京尼平组(加京尼平明胶浸提液)、戊二醛组(加戊二醛明胶浸提液),48h后做四甲基氮唑蓝检测。细胞相对增殖率=浸提液组平均A值/阴性对照组平均A值×100%。各组的细胞相对增殖率转换成6级(0～5级)细胞毒性以评定材料的毒性程度。④溶胀度与降解率:材料交联完成后立即称重(W0)。然后在离心管中加2mL无菌磷酸盐缓冲液,24h后两组各取8个材料,用无菌滤纸擦干水分后称重(W1)。溶胀率=(W1-W0)/Wo×100%。其他材料3d换液1次,于1,2,3,4,8,12周分别取出两组材料各8个,用无菌滤纸擦干水分后称重(W2)。降解率=(W1-W2)/W1×100%。结果:①京尼平和戊二醛交联明胶材料的交联度分别为79.1%和52.3%。②京尼平材料的溶胀度高于戊二醛材料犤(166±38)%,(133±31)%,P<0.05犦。③京尼平和戊二醛材料在磷酸盐缓冲液中浸泡12周,降解率差异无显著性(14.1%,12.6%)。④戊二醛交联明胶的浸提液培养的细胞出现坏死现象,而京尼平材料浸提液培养的细胞生长良好。京尼平和戊二醛材料的细胞增殖度分别为96.6和23.1。结论:京尼平交联明胶材料与戊二醛交联明胶材料比较,交联度降低,溶胀度增加,但二者制成的材料在磷酸盐缓冲液中浸泡12周都仅有少量降解,差异无显著性。京尼平的细胞毒性明显低于戊二醛。
AIM： To compare the property between genipin and glutaraldehyde crosslinked gelatin from the aspects of crosslinking index, cytotoxicity, swelling ratio and degradation rate. METHODS： The experiment was carded out at the Experimental Center, Second Hospital Affiliated to Harbin Medical University from June 2005 to January 2006. （1）Biomaterials were divided into 2 groups： genipin group crosslinked gelatin with 10 g/L genipin for 72 hours, glutaraldehyde group crosslinked gelatin with 10 g/L glutaraldehyde for 24 hours. （2） Determination of crosslinking index： 8 samples were prepared from each group, 5 samples reacted with trinitrobenzensulfonic acid （TNBS） and NaHCO3, then they were hydrolysod with HCl, and extracted with ethyl ether. The absorbance of the diluted solution was measured at 346 nm, so we gain the ATNRS .The other 3 samples were prepared by the same procedure, except for HCl was added before the addition of TNBS; and the absorbance was measured as control （Acontrol）. The absorbance after crosslinking： Aafter = ATNRS -Arntral. The absorbance of 11 mg gelatin and 19 p.L withour genipin was detected in the same procedure and the absorbance before crosslinking（Abafare） was gained. Crosslinking index=（Abafore- Aafter）/ Abafore×100%.（3））Determination of cytotoxicity： The materials of each group were extracted with BPML 1640 medium at the ratio of 1 cm^2/mL. V-79 cells were seeded in a 96-well culture plate. After incubation for 24 hours, the wells with cells were divided into 3 groups： negative group （culture with 1640）, genipin material group （culture with 1640 and extracted ginipin material）, glutaraldehyde material group （culture with 1640 and extracted glutaraldehyde material）. After incubation in the atmosphere of 0.05 volume fraction of CO2 at 37℃ for 48 hours. Methylthizaolyl tetrazolium assay Was performed 48 hours later. The cellular relative growth rate （BGB） =Anasteial/Acontrol×100% .BGB in each group was scored into 6 grades （0-5 grade） to evaluate the cy- totoxicity of the materials. （4） Swelling ratio and degradation rate： The material was weighed （W0） after crosslinking, then immersed in an aseptic tube that contained 2 mL aseptic phosphate buffer solution. 8 samples were drawn from the aseptic tube in each group after 24 hours, wiped with filter paper to remove excess liquid, and weighed （W1）. Swelling ratio =（W1-W0）/W0×100%. The culture medium of the other materials was changed every 3 days. Eight samples were collected from each group after 1,2,3,4,8,12 weeks and weighed （W2） in the same procedure. Degradation rate= （W1-W2）/W1×100%. RESULTS： （1） The crosslinking index of genipin and glutaraldehyde crosslinked gelatin was 79.1% and 52.3%, respectively. （2） Swelling ratio was higher in genipin group than in （133±31）%,P 〈 0.05]. （3）Genipin glutaraldehyde group [（ 166±38）% vs and glutaraldehyde were put in phosphate buffer solution for 12 weeks; their degradation rate was 14.1% and 12.6%, respectively, without significant difference （4） Cells cultured with glutaraldehyde presented necrosis, while cells cultured with genipin grew well. The cellular growith rate of genipin group and glutaraldehyde group were 96.6 and 23.1 respectively. CONCLUSION： Genipin crosslinked gelatin＇s crosslinking index is decreased; swelling ratio is increased, in comparison of glutaraldehyde crosslinked gelatin. Two groups of materials degraded a little after 12 weeks in phosphate buffer solution, without significant difference. The cytotoxicity is obvious lower in genipin group than in glutaraldehyde group.
出处
《中国临床康复》
CAS
CSCD
北大核心
2006年第25期60-62,共3页
Chinese Journal of Clinical Rehabilitation
