羧甲基壳聚糖-HA-聚乙烯醇共混膜片的制备及其理化性质研究

PREPARATION AND PHYSICOCHEMICAL PROPERTY OF CARBOXYMETHYL-CHITOSAN/HYALURONIC ACID/POLY(VINYL ALCOHOL) BLEND MEMBRANE

目的制备并评价羧甲基壳聚糖-HA-聚乙烯醇[carboxymethyl-chitosan/HA/poly(vinyl alcohol),CHP]共混膜片的理化性质、眼内生物相容性,探讨其用于青光眼滤过手术的可行性。方法取羧甲基壳聚糖、HA、聚乙烯醇按5∶4∶1(M/M)共混交联,用流延法制备CHP共混膜片。测定膜片吸水率、溶胀比、通透性及力学性能。取新西兰白兔结膜下组织,分离培养兔眼结膜下成纤维细胞,取第4代细胞以5×103个/mL密度接种于贴附CHP共混膜片(实验组)及不加膜片(对照组)的细胞培养板中培养,于培养24、48、72h取细胞行MTT检测。将6只新西兰白兔随机分为2组(n=3),将CHP共混膜片及SK胶分别植入兔眼前房及结膜下,作为实验组及对照组。术后第1、3、5、9、11、20、30、45、60天进行裂隙灯观察,记录双眼反应,第15天实验组取角膜组织行扫描电镜观察,以研究共混膜片生物相容性和降解性。结果CHP共混膜片吸水率为83.8%±1.3%,溶胀比为3.59±0.50,干、湿态时抗张强度分别为(20.59±1.73)、(0.51±0.13)MPa,断裂伸长率分别为10.69%±1.16%、53.15%±2.46%。CHP共混膜片对NaCl、L-酪氨酸均有良好的通透性。接种后24、48、72h实验组吸光度(A)值分别为0.207±0.083、0.174±0.080、0.181±0.048,对照组分别为0.284±0.011、0.272±0.083、0.307±0.056,组间比较差异均有统计学意义(P<0.05)。术后第1天实验组前房植入共混膜片周围有少量絮状物渗出;第3天渗出物吸收;第11天无明显炎性反应;第60天植入共混膜已大部分降解。术后15d,扫描电镜观察示角膜内皮细胞六边形形态完整,表面无降解颗粒附着。对照组因SK胶质地软,易断裂,未能成功植入至前房。术后第1天实验组结膜植入处结膜局部有轻度充血,第9天恢复正常,至第60天无角膜水肿、前房炎性反应。对照组观察结果与实验组相似。结论CHP共混膜片具有较好的理化性质及眼内生物相容性,在青光眼滤过手术中具有潜在应用价值。

Objective To prepare carboxymethyl-chitosan/hyaluronic acid/poly（vinyl alcohol） （CHP） blend membrane, evaluate its physicochemical properties and intraocular biocompatibility and to investigate its feasibility to be applied to glaucoma filtering surgery. Methods CHP blend membrane was prepared using solution casting method after blending carboxymethyl-chitosan, HA and poly（vinyl alcohol） in a proportion of 5 ： 4 ： 1 （M/M）. Its water absorption rate, swelling rate, permeability, and mechanical properties were detected. Subconjunctival fibroblasts separated from subconjuncitival tissue of New Zealand white rabbits were cultured, and the cells at passage 4 were cultured on cell culture plate with or without the CHP blend membrane, serving as the experimental group and the control group, respectively. Effect of the CHP blend membrane on the subconjunctival fibroblasts was tested by MTT method 24, 48, and 72 hours after culture. Six New Zealand white rabbits were randomly divided into two groups （n=3 rabbits per group）, and the CHP blend membrane and SK gel were implanted into the rabbits＇ subconjunctival space and anterior chamber in the experimental group and the control group, respectively. Slit lamp observation and binocular reaction record were conducted 1, 3, 5, 9, 11, 20, 30, 45, and 60 days after operation. Corneal tissue harvested from the experimental group was observed using scanning electron microscope 15 days after operation to study ophthalmic biocompatibility and biodegradability. Results The water absorption rate and the swelling rate of the CHP blend membrane was 83.8% ± 1.3% and 3.59 ± 0.50, respectively. The tensile strength of the dry and the wet CHP blend membrane was （20.59 ± 1.73） and （0.51 ± 0.13） MPa, respectively. The breaking elongation rate of the dry and the wet CHP blend membcane was 10.69% ±1.16% and 53.15%± 2.46%, respectively. The CHP blend membrane had good permeability to NaCl and L-tyrosine. Absorbance （A） value of the experimental group 24, 48, and 72 hours after breeding was 0.207 ± 0.083, 0.174± 0.080, and 0.181 ± 0.048, respectively, while the A value of the control group was 0.284 ±0.011, 0.272± 0.083, and 0.307 ±0.056, respectively. Significant difference was evident between two groups （P 〈 0.05）. In the experimental group, a small amount of floccus was exuded around the implanted membrane 1 day after operation; the fioccus was absorbed on the third day, and there was no obvious inflammatory reaction occurring on the eleventh day. Most of the membrane degraded on the sixtieth day. Scanning electron microscope observation showed that the hexagonal morphology of the corneal endothelial cells was intact, and no degradation particles adhered to the surface. In the control group, the implantation of SK gel into anterior chamber was unsuccessful because the SK gel was quite soft and easily broken. In the experimental group, mild hyperemia emerged around the implanted membrane 1 day after the subconjunctival implantation of the membrane, and it became normal on the ninth day. No corneal edema and inflammatory reaction of anterior chamber occurred till the sixtieth day. The results in the control group and the experiment group were similar. Conclusion Due to its good physicochemical properties and biocompatibility, the CHP blend membrane has potential applications in glaucoma filtering surgery.