摘要
目的探究预混有地塞米松(Dex)的高分子自交联透明质酸(cHA)水凝胶(cHA-Dex水凝胶)抑制软骨细胞凋亡缓解早期创伤性骨关节炎(PTOA)的作用机制。方法取2月龄雄性SD大鼠140只分两组,70只进行前交叉韧带切断(ACLT)手术制造PTOA动物模型,以假手术组(70只)为对照,在术后不同时间点(1~14 d,每组每个时间点5只大鼠)检测关节灌洗液中白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)、基质金属蛋白酶3(MMP-3)及MMP-13等炎性指标的变化趋势。将cHA-Dex(0.5 mg/ml)水凝胶(实验组,n=70)及预混有Dex的普通低分子透明质酸(HA)水凝胶,即HA-Dex(0.5 mg/ml)水凝胶(对照组,n=70)注入PTOA大鼠关节腔,检测每个时间点(1~14 d,每组每个时间点5只大鼠)大鼠关节液中的Dex的释放量及累计释放量,揭示cHA-Dex水凝胶的缓释机制。取2020年1月至2022年12月在山西医科大学第二医院关节科进行膝关节置换术后的骨关节炎(OA)患者(胫骨平台上Outbridge评分1~2分,n=18)膝关节软骨进行体外组织块培养,将预混有0.1、0.2、0.5 mg/ml三种梯度Dex浓度的cHA-Dex水凝胶处理软骨组织块后,检测关节软骨组织块中IL-1β、IL-6、TNF-α、MMP-3以及MMP-13的mRNA表达水平变化。运用酶解法分离出OA软骨细胞并进行体外原代培养(来自18例OA患者,分6次重复实验,每次3例患者的软骨细胞提取后混合均匀培养),软骨细胞贴壁后分为4组,分别进行下列干预:生理盐水干预组、cHA水凝胶干预组、Dex(0.5 mg/ml)干预组、cHA-Dex(0.5 mg/ml)水凝胶干预组,检测不同干预对软骨细胞增殖及凋亡的影响。结果大鼠PTOA组和假手术组膝关节番红O固绿染色国际骨关节炎协会(OARSI)评分分别为(3.34±0.35)分和(1.17±0.21)分(P=0.010);PTOA大鼠膝关节骨赘Meachim评分相对于假手术组增高[(2.66±0.41)分比(0.22±0.17)分,P=0.010],大鼠PTOA造模成功。与PTOA大鼠关节内炎症因子早期出现峰值变化规律相对应的cHA-Dex水凝胶中Dex也在早期释放,并在后期长期缓释。预混0.1、0.2、0.5 mg/ml Dex的cHA-Dex水凝胶处理OA关节软骨组织块后,组织块中的IL-1β、IL-6、TNF-α、MMP-3及MMP-13的mRNA表达均下降(均P<0.05)。体外培养OA软骨细胞,与Dex(0.5 mg/ml)水凝胶干预组相比,cHA-Dex(0.5 mg/ml)水凝胶干预组凋亡率明显减少(0.60±0.07比6.63±0.98,P=0.010)。相对生理盐水干预组或单纯cHA水凝胶干预组,cHA-Dex(0.5 mg/ml)水凝胶干预组细胞增殖明显,各时间点差异均有统计学意义(均P<0.05)。结论针对PTOA的早期炎症,cHA-Dex水凝胶可能通过缓释Dex,既可抑制软骨炎症,又可逆转Dex导致的软骨细胞凋亡增加、增殖率降低,最终缓解PTOA进展。
Objective To explore the mechanism of cross-linked hyaluronic acid-dexamethasone hydrogel(cHA-Dex)in inhibiting chondrocyte apoptosis and alleviating early post-traumatic osteoarthritis(PTOA).Methods To generate PTOA model,anterior cruciate ligament transection(ACLT)was performed on SD rats(n=70),and the sham surgery group(n=70)was set as control.The changes in inflammatory indicators such as interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-3(MMP-3),and matrix metalloproteinase-13(MMP-13)in the joint lavage fluid were measured at different time points(1-14 days,5 rats at each time point)after surgery.The cHA-Dex(0.5 mg/ml)hydrogel(experimental group,n=70)and ordinary low-molecular-weight hyaluronic acid(HA)hydrogel premixed with Dex,that was,HA-Dex(0.5 mg/ml)hydrogel(control group,n=70)were injected into the joint cavity of PTOA rats,and the release amount and cumulative release amount of Dex in the joint fluid of rats at each time point(1-14 days,5 rats at each time point)were detected to reveal the release mechanism of cHA-Dex hydrogel.The cartilage of knee joint of patients with osteoarthritis(OA)who underwent knee arthroplasty in the Second Hospital of Shanxi Medical University from January 2020 to December 2022 was taken for in vitro tissue block culture(Outbridge score=1 or 2,n=18).After the cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1,0.2,and 0.5 mg/ml Dex,the mRNA expression levels of IL-1β,IL-6,TNF-α,MMP-3,and MMP-13 in the articular cartilage tissue block were detected.OA chondrocytes were isolated from cartilage samples using enzymatic hydrolysis and cultured in vitro(n=18).Chondrocytes were divided into 4 groups:saline,cHA hydrogel,Dex(0.5 mg/ml),and cHA-Dex(0.5 mg/ml)hydrogel group.The effects of different interventions on chondrocyte proliferation and apoptosis were tested.Results The Osteoarthritis Research Society International(OARSI)score of safranine O-solid green staining in PTOA group was 3.34±0.35,and it was 1.17±0.21 in Sham group(P=0.010).The Meachim score of knee joint osteophytes in PTOA rats was significantly higher than that in the Sham group(2.66±0.41 vs 0.22±0.17,P=0.010),indicating PTOA model in rat was established successfully.The cHA-Dex hydrogel,which corresponded to the peak changes of inflammatory factors in the joints of PTOA rats in the early stage,was also released in the early stage and sustained-released in the late stage.After the OA articular cartilage tissue block was treated with cHA-Dex hydrogel premixed with 0.1,0.2,and 0.5 mg/ml Dex,the mRNA expression levels of IL-1β,IL-6,TNF-α,MMP-3,and MMP-13 in the tissue block were reduced significantly(all P<0.05)and in a dose-dependent manner.Compared with Dex(0.5 mg/ml)alone group,the apoptosis rate of cHA-Dex(0.5 mg/ml)hydrogel group was significantly reduced(0.60±0.07 vs 6.63±0.98,P=0.010).Compared with the normal saline or the cHA hydrogel alone group,the cHA-Dex(0.5 mg/ml)hydrogel group had significant cell proliferation,and the difference at each time point were all significant statistically(all P<0.05).Conclusion For the early inflammation of PTOA,cHA-Dex hydrogel can not only inhibit cartilage inflammation,but also reverse the increased apoptosis and decreased proliferation rate of chondrocytes caused by Dex,and finally alleviate the progress of PTOA by releasing Dex.
作者
张志伟
赵静雅
冯毅
尹崑
李鹏翠
卫小春
Zhang Zhiwei;Zhao Jingya;Feng Yi;Yin Kun;Li Pengcui;Wei Xiaochun(Shanxi Key Laboratory of Bone and Soft Tissue Injury Repair,Department of Orthopedics,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China;Department of Endocrinology,Taiyuan People′s Hospital,Taiyuan 030001,China)
出处
《中华医学杂志》
CAS
CSCD
北大核心
2024年第9期695-703,共9页
National Medical Journal of China
基金
国家自然科学基金(U21A20353,82172503)
山西省应用基础研究计划青年项目(20210302124192)
山西省教育厅高等学校教学改革创新项目(J20220335)
山西省中医药管理局科研课题(2023ZYYC2038)
山西医科大学第二医院青年基金(202202-05)
关键词
骨关节炎
创伤性骨关节炎
地塞米松高分子自交联透明质酸水凝胶
软骨
炎症因子
凋亡
增殖
Osteoarthritis
Post-traumatic osteoarthritis
Cross-linked hyaluronic acid-dexamethasone hydrogel
Cartilage
Inflammatory factors
Apoptosis
Proliferation
