中华创伤杂志
中華創傷雜誌
중화창상잡지
Chinese Journal of Traumatology
2010年
2期
165-171
,共7页
李沫%吴邦耀%胡学昱%黄景辉%张永光%罗卓荆
李沫%吳邦耀%鬍學昱%黃景輝%張永光%囉卓荊
리말%오방요%호학욱%황경휘%장영광%라탁형
周围神经%胶原%生物相容材料%京尼平
週圍神經%膠原%生物相容材料%京尼平
주위신경%효원%생물상용재료%경니평
Peripheral nerves%Collagen%Biocompatible materials%Genipin
目的 从超微结构、孔隙率、溶胀率、降解率、交联度及细胞毒性等方面分别比较紫外线、京尼平及戊二醛交联后的壳聚糖复合Ⅰ型胶原蛋白人工神经支架材料的生物学特性. 方法 (1)材料按交联方法不同分为三组:紫外线组、京尼平组、戊二醛组.(2)经扫描电镜观察三组材料内部结构的排列规律及走形,测量其孔径大小、计算孔隙率及孔径分布等指标.(3)溶胀率与体外降解率:三组材料交联完后立即称重(W_0),然后在培养皿中加入10 ml无菌PBS,24 h后用无菌滤纸擦干水分称重(W_1).溶胀率(%)=(W_1-W_0)/W_0×100%.剩余材料于4,8,12周分别取出后称重(W_2).降解率(%)=(W_1-W_2)/W_1×100%.(4)检测交联度:每组取10根材料,其中5根加入碳酸氢钠和三硝基苯磺酸(TNBS),再加盐酸,在346 nm测吸光度(A)值(A_(三硝基苯磺酸)).另外5根先加盐酸,然后再加TNBS,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).再取一组10根未交联过的材料,以同样步骤测吸光度,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%.(5)细胞毒性试验:遵照GB/T16886/ISO 10993医疗器械生物学评价之体外细胞毒性试验原则,采用国际标准的两种试验方法,选用建系的L929小鼠成纤维细胞对改性后的支架材料进行体外细胞毒性试验. 结果 (1)未交联的材料为均匀圆柱状,内部为孔径均匀且平行排列的微观结构,其微孔直径为30～120 μm;交联后紫外线组孔径基本不变,京尼平、戊二醛两组孔径均变小.(2)京尼平和戊二醛组孔隙率差异无统计学意义,两者都要高于紫外线组;而溶胀率京尼平组高于戊二醛组,戊二醛组又高于紫外线组.(3)京尼平和戊二醛两组的交联度分别为55.3%和82.5%.(4)京尼平和戊二醛组在PBS中浸泡4,8,12周,体外降解率差异无统计学意义;而紫外线组材料降解明显高于前两者.(5)戊二醛组浸提液培养的细胞出现部分坏死现象,相反京尼平和紫外线组细胞生长良好. 结论 以京尼平交联壳聚糖复合Ⅰ型胶原蛋白制备出改进的人工神经支架,具有良好的生物稳定性和生物相容性,为神经组织工程领域提供了一种具有应用潜力的材料.
目的 從超微結構、孔隙率、溶脹率、降解率、交聯度及細胞毒性等方麵分彆比較紫外線、京尼平及戊二醛交聯後的殼聚糖複閤Ⅰ型膠原蛋白人工神經支架材料的生物學特性. 方法 (1)材料按交聯方法不同分為三組:紫外線組、京尼平組、戊二醛組.(2)經掃描電鏡觀察三組材料內部結構的排列規律及走形,測量其孔徑大小、計算孔隙率及孔徑分佈等指標.(3)溶脹率與體外降解率:三組材料交聯完後立即稱重(W_0),然後在培養皿中加入10 ml無菌PBS,24 h後用無菌濾紙抆榦水分稱重(W_1).溶脹率(%)=(W_1-W_0)/W_0×100%.剩餘材料于4,8,12週分彆取齣後稱重(W_2).降解率(%)=(W_1-W_2)/W_1×100%.(4)檢測交聯度:每組取10根材料,其中5根加入碳痠氫鈉和三硝基苯磺痠(TNBS),再加鹽痠,在346 nm測吸光度(A)值(A_(三硝基苯磺痠)).另外5根先加鹽痠,然後再加TNBS,其餘步驟相同,測得吸光度取平均值作為對照(A_對),交聯後吸光度值為:A_(交聯後)=A_(三硝基苯磺痠)-A_(對).再取一組10根未交聯過的材料,以同樣步驟測吸光度,得到交聯前吸光度(A_(交聯前)).交聯度=(A_(交聯前)-A_(交聯後))/A_(交聯前)×100%.(5)細胞毒性試驗:遵照GB/T16886/ISO 10993醫療器械生物學評價之體外細胞毒性試驗原則,採用國際標準的兩種試驗方法,選用建繫的L929小鼠成纖維細胞對改性後的支架材料進行體外細胞毒性試驗. 結果 (1)未交聯的材料為均勻圓柱狀,內部為孔徑均勻且平行排列的微觀結構,其微孔直徑為30～120 μm;交聯後紫外線組孔徑基本不變,京尼平、戊二醛兩組孔徑均變小.(2)京尼平和戊二醛組孔隙率差異無統計學意義,兩者都要高于紫外線組;而溶脹率京尼平組高于戊二醛組,戊二醛組又高于紫外線組.(3)京尼平和戊二醛兩組的交聯度分彆為55.3%和82.5%.(4)京尼平和戊二醛組在PBS中浸泡4,8,12週,體外降解率差異無統計學意義;而紫外線組材料降解明顯高于前兩者.(5)戊二醛組浸提液培養的細胞齣現部分壞死現象,相反京尼平和紫外線組細胞生長良好. 結論 以京尼平交聯殼聚糖複閤Ⅰ型膠原蛋白製備齣改進的人工神經支架,具有良好的生物穩定性和生物相容性,為神經組織工程領域提供瞭一種具有應用潛力的材料.
목적 종초미결구、공극솔、용창솔、강해솔、교련도급세포독성등방면분별비교자외선、경니평급무이철교련후적각취당복합Ⅰ형효원단백인공신경지가재료적생물학특성. 방법 (1)재료안교련방법불동분위삼조:자외선조、경니평조、무이철조.(2)경소묘전경관찰삼조재료내부결구적배렬규률급주형,측량기공경대소、계산공극솔급공경분포등지표.(3)용창솔여체외강해솔:삼조재료교련완후립즉칭중(W_0),연후재배양명중가입10 ml무균PBS,24 h후용무균려지찰간수분칭중(W_1).용창솔(%)=(W_1-W_0)/W_0×100%.잉여재료우4,8,12주분별취출후칭중(W_2).강해솔(%)=(W_1-W_2)/W_1×100%.(4)검측교련도:매조취10근재료,기중5근가입탄산경납화삼초기분광산(TNBS),재가염산,재346 nm측흡광도(A)치(A_(삼초기분광산)).령외5근선가염산,연후재가TNBS,기여보취상동,측득흡광도취평균치작위대조(A_대),교련후흡광도치위:A_(교련후)=A_(삼초기분광산)-A_(대).재취일조10근미교련과적재료,이동양보취측흡광도,득도교련전흡광도(A_(교련전)).교련도=(A_(교련전)-A_(교련후))/A_(교련전)×100%.(5)세포독성시험:준조GB/T16886/ISO 10993의료기계생물학평개지체외세포독성시험원칙,채용국제표준적량충시험방법,선용건계적L929소서성섬유세포대개성후적지가재료진행체외세포독성시험. 결과 (1)미교련적재료위균균원주상,내부위공경균균차평행배렬적미관결구,기미공직경위30～120 μm;교련후자외선조공경기본불변,경니평、무이철량조공경균변소.(2)경니평화무이철조공극솔차이무통계학의의,량자도요고우자외선조;이용창솔경니평조고우무이철조,무이철조우고우자외선조.(3)경니평화무이철량조적교련도분별위55.3%화82.5%.(4)경니평화무이철조재PBS중침포4,8,12주,체외강해솔차이무통계학의의;이자외선조재료강해명현고우전량자.(5)무이철조침제액배양적세포출현부분배사현상,상반경니평화자외선조세포생장량호. 결론 이경니평교련각취당복합Ⅰ형효원단백제비출개진적인공신경지가,구유량호적생물은정성화생물상용성,위신경조직공정영역제공료일충구유응용잠력적재료.
Objective To compare biological properties of ehitosan composite artificial neural type Ⅰ collagen scaffold material cross-linked with ultraviolet rays (UV), genipin (GP) and glutaraldehyde (GTA) in aspects of uhrastrueture, porosity, swelling rate, degradation rate, crosslinking degree and cytotoxicity. Methods (1) According to different cross-linking methods, biomaterials were divided into three groups, ie, UV group, GP group and GTA group. (2)The mierostrueture of three groups was observed under scanning electron microscope (SEM) to measure pore size, porosity rate and pore-size distribution. (3)Swelling rate and in vitro degradation rate:the biomaterials were weighed (W_0) after crosslinking and then immersed in culture medium containing 10 ml aseptic phosphate buffer solution (PBS). The samples were drawn from the culture medium after 24 hours, wiped with filter paper to remove excess liquid and weighed (W_1). Swelling rate(%) = W_1-W_0/W_0×100%. The remaining sampies from each group were weighed (W_2) at 4, 8, 12 weeks with the same procedure. Degradation rate (%) = W_1-W_2/W_1×100%. (4)Determination of cross-linking index: 10 samples were prepared from each group, five samples from which were reacted with trinitro-benzen-sulfonic acid(TNBS)and sodium bicarbonate and then were hydrolyzed with hydrochloric acid. The absorbance of the diluted solution was measured at 346 nm. The other five samples were prepared by the same procedure, except for hydrochloric acid was added before addition of TNBS, when the absorbance was measured as control (A_(control)). The absorbance after crosslinking:A_(after)=ATNBS-A_(control). Another 10 samples without any crosslinking were detected with the same procedure to measure the absorbance before crosslinking (A_(before)). Crosslinkiag index = (A_(before)-A_(after))/A_(before)×100%. (5) Determination of cytotoxicity : two international standard experimental methods were adopted in the study according to experimental principle of GB/T 16886-ISO 10993 on medical apparatus. L929 fibroblasts of mouse were used for in vitro experimental study of cytotoxicity of modified scaffold. Results The biomaterials without any cross-linking were circular cylinder, with parallel arranged microscopic channel and uniform pore size of 30-120 μm. The pore size of UV group remained basically unchanged, while the pore size in GP group and GTA group was smaller than that in UV group. (2) The porosity rate in GP group and GTA group was higher than that in UV group, but there was no statistical difference between GP group and GTA group. The swelling rate of GP group was higher than that GTA group, which was higher than UV group. (3)The crosslinking index of GP group and GTA group were 55.3% and 82.5%. (4) No statistical difference was found in regard of in vitro degradation rate after GP group and GTA group were put in PBS for4, 8 and 12 weeks, respectively. But in vitro degradation rate in UV group was significantly higher than that in GP group and GTA group. (5) Cell culture in GTA group presented partial necrosis, while cells cultured in GP group and UV group grew well. Conclusion Collagen/chitosan scaffolds cross-linked with GP have sound biostability and good biocompatibility and hence are potential alternatives for nerve tissue engineering.
