烧伤性异位骨化早期跟腱局部巨噬细胞募集及极化研究

Study on recruitment and polarization of local Achilles tendon macrophages in the early stage of burn heterotopic ossification

目的模拟临床烧伤性异位骨化(HO)的发生,探索该疾病的发生机制。方法将24只C57BL/6小鼠随机分为对照(HO)组(n=12)与研究(HO+Burn)组(n=12)。HO组建立HO模型,HO+Burn组建立HO+烫伤模型。记录两组小鼠存活率及跟腱部位手术伤口愈合情况,以及HO+Burn组小鼠背部烫伤皮肤恢复情况。术后1周和3周,Micro-CT检测手术部位HO的生成情况;HE染色观察组织学特点;茜素红染色观察钙结节的生成情况;免疫荧光染色观察巨噬细胞浸润特征及极化特点。结果术后3周,各组小鼠跟腱部位均出现创伤性HO,跟腱部位可见明显的片状高密度影;HO组骨密度、骨体积分数、HO面积低于HO+Burn组,差异均有统计学意义(P<0.05)。术后1周、3周,HO+Burn组茜素红免疫荧光染色荧光信号强度均高于HO组,差异均有统计学意义(P<0.05)。术后1周、3周,HO+Burn组M1型巨噬细胞和M2型巨噬细胞表达均高于HO组,差异均有统计学意义(P<0.05)。术后3周,HO+Burn组成骨细胞标志物OCN荧光信号强度高于HO组,差异有统计学意义(P<0.05)。结论烧伤会显著加重创伤局部跟腱HO的生成,其机制可能与跟腱受损部位免疫活动的增强及巨噬细胞的募集和极化有关。

Objective To simulate the occurrence of clinical burn heterotopic ossification(HO)and to explore the mechanisms of the disease.Methods A total of 24 C57BL/6 mice were randomly divided into control group(HO group,n=12)and study group(HO+Burn group,n=12).HO model was established in HO group,and HO+Burn model was established in HO+Burn group.The survival rate and healing of the surgical wound at the Achilles tendon site were recorded in both groups,as well as the recovery of the scalded skin on the back of the mice in HO+Burn group.At 1 and 3 weeks after surgery,Micro-CT was performed to detect the HO at the surgical site.HE staining was performed to observe the histological characteristics.Alizarin red staining was performed to observe the formation of calcium nodules.Immunofluorescence staining was performed to observe the infiltration characteristics of macrophages and polarization characteristics.Results Three weeks after surgery,traumatic HO appeared in the Achilles tendon of all groups,and obvious patchy high-density shadows were observed in the Achilles tendon.Bone mineral density,bone volume/total volume in HO group were lower than those in HO+Burn group,and the differences were statistically significant(P<0.05).At 1 and 3 weeks after surgery,the fluorescence signal intensity of alizarin red immunofluorescence staining in HO+Burn group was higher than that in HO group,with statistical significance(P<0.05).The expressions of M1-type macrophages and M2-type macrophages in HO+Burn group were higher than those in HO group 1 and 3 weeks after surgery,with statistical significance(P<0.05).At 3 weeks after surgery,the intensity of OCN fluorescence signal of bone cell marker composed by HO+Burn was higher than that in HO group,the difference was statistically significant(P<0.05).Conclusion Burns significantly exacerbate the production of HO in the traumatized local Achilles tendon,and the mechanism may be related to increased immune activity at the damaged site of the Achilles tendon and the recruitment and polarization of macrophages.