不同交联透明质酸复合凝胶在体内的宿主反应及基质金属蛋白酶9表达

背景:利用羟丙基甲基纤维素分子内的羟基与透明质酸中的羟基、羧基发生相互作用,可降低透明质酸的酶解,提高其作为皮肤填充材料的可使用性。目的:观察不同交联方式透明质酸复合凝胶体内植入后的宿主反应及基质金属蛋白酶9表达变化。方法:取新西兰大耳白兔(购自北京隆安实验动物养殖中心)12只,在其脊柱两侧皮下共注射10个点,左侧4点注射化学交联透明质酸钠-羟丙基甲基纤维素凝胶(化学交联组),右侧4点注射物理混合透明质酸钠-羟丙基甲基纤维素凝胶(物理交联组),左右侧各一点注射已上市注射用修饰透明质酸钠凝胶(对照组)。注射后1,4,12周分别切取包括植入材料在内的皮下组织,苏木精-伊红染色观察炎症反应和纤维化程度,Masson染色观察胶原纤维形成状况,免疫组织化学染色观察基质金属蛋白酶9表达变化。实验经天津市医药科学研究所动物伦理委员会批准(批准号:IMPS-EAEP-H-2017030)。结果与结论:①苏木精-伊红染色:化学交联组注射后1,4周时的炎症反应较对照组明显,囊壁及微囊壁较对照组增厚,注射后12周时炎症反应及纤维化程度与对照组类似;物理交联组注射后1周时可见少量炎细胞浸润,纤维包裹及微囊壁形成较对照组稍明显,注射后4周时纤维包裹及微囊壁较对照组增厚,注射后12周时炎症反应及纤维化程度与对照组类似;②Masson染色:化学交联组与物理交联组注射后1,4周时的胶原纤维较对照组增多,12周时胶原纤维进一步增加,与对照组无差异;③免疫组织化学染色:化学交联组注射后1周的基质金属蛋白酶9阳性表达低于对照组,其余时间点与对照组无差异;物理交联组注射后1周的基质金属蛋白酶9阳性表达低于对照组,注射后4周的阳性表达高于对照组,注射后12周与对照组无差异;④结果表明:两种交联透明质酸钠复合凝胶皮下植入后12周内的生物相容性良好,胶原纤维生成和基质金属蛋白酶9表达逐步增加,有利于组织重塑的进行,并且物理交联的样品植入早期组织反应更小,中期基质金属蛋白酶9表达明显增多,更利于组织修复和重塑。

Host response of different cross-linked hyaluronic acid composite gels and matrix metalloproteinase-9 expression

BACKGROUND:The interaction between hydroxyl in hydroxypropyl methylcellulose and hydroxyland carboxyl in hyaluronic acid can reduce the enzymatic hydrolysis of hyaluronic acid and increase the usability when used as a skin filling material.OBJECTIVE:To investigate the host response of different cross-linked hyaluronic acid composite gels after they were implanted in vivo and matrix metalloproteinase-9 expression.METHODS:Twelve New Zealand white rabbits(purchased from Beijing Longan Laboratory Animal Breeding Center)were injected with cross-linked hyaluronic acid composite gels through 10 points in subcutaneous tissue at both sides of the spine.On the left side,chemically cross-linked sodium hyaluronate-hydroxypropyl methylcellulose gel was injected via 4 points(chemical cross-linking group).On the right side,physically mixed sodium hyaluronate-hydroxypropyl methylcellulose gel was injected via 4 points(physical cross-linking group).The modified sodium hyaluronate gel which was marketed was injected via 1 point on each side(control group).Subcutaneous tissues including implant material was excised at 1,4,and 12 weeks after injection.Hematoxylin-eosin staining was performed to evaluate the degree of inflammation and fibrosis.Masson staining was performed to observe the formation of collagen fibers.Immunohistochemical staining was performed to detect the expression of matrix metalloproteinase-9.This study was approved by the Animal Ethics Committee of Tianjin Institute of Medical and Pharmaceutical Sciences(approval No.IMPS-EAEP-H-2017030).RESULTS AND CONCLUSION:Hematoxylin-eosin staining:In the chemical cross-linking group,at 1 and 4 weeks after injection,inflammatory reaction was more obvious,and the capsule wall and the microcapsule wall were thicker compared with the control group.At 12 weeks after injection,the degree of inflammation and fibrosis were similar between chemical cross-linking and control groups.At 1 week after injection,in the physical cross-linking group,a small amount of inflammatory cells were observed,and the fiber wrap and microcapsule wall formation were slightly more obvious than those in the control group.At 4 weeks after injection,the fiber wrap and microcapsule wall were thicker in the physical cross-linking group compared with the control group.At 12 weeks after injection,the degrees of inflammatory reaction and fibrosis in the physical cross-linking group were similar to those of the control group.Masson staining:In the chemical cross-linking group and physical cross-linking group,at 1 and 4 weeks after injection,the collagen fibers were increased compared with the control group and they further increased at 12 weeks,and at this time,there was no significant difference between chemical cross-linking and physical cross-linking groups.Immunohistochemical staining:in the chemical cross-linking group,matrix metalloproteinase-9 expression in the chemical cross-linking group was lower than that in the control group at 1 week after injection,and there was no significant difference at other time points.At 1 week after injection,matrix metalloproteinase-9 expression in the physical cross-linking group was lower than that in the control group.At 4 weeks after injection,matrix metalloproteinase-9 expression in the physical cross-linking group was higher than that in the control group.At 12 weeks after injection,there was no significant difference in matrix metalloproteinase-9 expression between physical cross-linking and control groups.These results showed that the biocompatibility of the two cross-linked sodium hyaluronate composite gels was good in 12 weeks after subcutaneous implantation.The expression of collagen fiber and matrix metalloproteinase-9 was gradually increased.All these are conducive to tissue remodeling.In the physical cross-linking gel,tissue reaction is smaller at the early stage,and matrix metalloproteinase-9 expression is significantly increased at the middle stage,which are more conducive to tissue repair and remodeling.