整合KGN的壳聚糖-透明质酸水凝胶用于髓核修复的研究

Preparation of Kartogenin-Conjugated Chitosan-Hyaluronic Acid Hydrogel for Nucleus Pulposus Regeneration

可注射水凝胶在退变髓核组织再生修复中有着重要的意义。研究以磷酸甘油(glycerol phosphate, GP)作为交联剂,利用壳聚糖(chitosan,CS)和透明质酸(hyaluronicacid,HA),制备不同体积比的水凝胶(CS:GP:HA,6:3:1,5:3:2,4:3:3,3:3:4,2:3:5,1:3:6,V:V:V),并整合小分子物质kartogenin(KGN)。检测水凝胶的成胶时间,形态学特征,溶胀度,压缩模量,生物降解度,KGN累积释放量及细胞生物相容性。然后研究KGN,TGF-β及KGN+TGF-β对促进人脂肪干细胞(human adipose-derived stem cells,ADSCs)分化为髓核样细胞的影响。结果表明含高浓度HA的水凝胶成胶时间短,溶胀度高,生物降解及KGN释放速率快;其中体积比为5:3:2(CS:GP:HA)的水凝胶更适于脂肪干细胞增殖和分化;免疫荧光及实时定量PCR显示KGN,TGF-β及KGN+TGF-β诱导Ⅱ型胶原及蛋白聚糖表达量与对照组相比具有显著性差异,但各组间差异无统计学意义。研究成功建立可缓释KGN的可注射CS/HA/KGN水凝胶体系,该体系能促进脂肪干细胞增殖并分化为髓核样细胞,为临床椎间盘髓核退变微创手术后髓核再生修复提供一个简单有效的方法。

Injectable constructs for in vivo gelation have many advantages in the regeneration of degenerated nucleus pulposus.Injectable hydrogel consisting of chitosan(CS)and hyaluronic acid(HA)crosslinked by glycerol phosphate(GP)(with different CS:GP:HA ratios:6:3:1,5:3:2,4:3:3,3:3:4,2:3:5,1:3:6,V:V:V)was developed for kartogenin(KGN)delivery.In vitro gelation time,morphology,swelling ratio,weight loss,compressive modulus,cumulative release of KGN and biocompatibility were studied.Human adipose-derived stem cells(ADSCs)were encapsulated in the hydrogel to study the effects of KGN,TGF-βand KGN+TGF-βon stem cell proliferation and differentiation into nucleus pulposus-like cells.The results show that hydrogel with high HA concentration has shorter gelation time,higher water uptake,faster mass loss and KGN release.KGN-conjugated hydrogel with CS:GP:HA ratio of5:3:2show optimal cell proliferation and differentiation.PCR results show significant difference between hydrogel with and without KGN/TGF-β.However,there is no significant difference for nucleus pulposus induced by KGN and/or TGF-β.In conclusion,a KGN conjugated CS-HA hydrogel(5:3:2)for sustained release of KGN is developed for promoting ADSCs proliferation and nucleus pulposus differentiation,which is useful for the repair of degenerative NP tissues after minimally invasive surgery.