间充质干细胞(MSCs)和巨噬细胞共同促进损伤后的骨再生。然而,MSCs与炎性巨噬细胞(M1)相互作用的详细机制尚不清楚.使用氯膦酸盐脂质体(12.5mg/kg/小鼠,腹膜内)或生理盐水注射(对照)上颌第一磨牙拔除前。在拔牙后第1、3、5、7和10天处死小鼠(n=4)。再生骨量评估拔牙槽(TES)和组织化学分析CD80+M1,CD206+M2(抗炎巨噬细胞),PDGFRα+MSC,和TNF-α+细胞。体外,有或没有TNF-α刺激的分离的MSCs(10ng/mL,24h,n=3)是大量RNA测序(RNA-Seq)以鉴定TNF-α刺激特异性MSC转录组。第7天的显微CT和HE染色显示,氯膦酸盐组的平均骨体积显着降低(氯膦酸盐与对照组:0.01mm3对0.02mm3,p<0.0001)和每总TES中再生骨面积的平均百分比(41.97%对54.03%,p<.0001)。Clodronate组显示CD80+的平均数显着减少,TNF-α+,PDGFRα+,和第5天的CD80TNF-α细胞(306.5vs558.8,p<.0001;280.5vs543.8,p<.0001;365.0vs633.0,p<.0001,29.0vs42.5,p<.0001),而这些细胞在第7天显著恢复(493.3对396.0,p=.0004;479.3对384.5,p=.0008;593.0对473.0,p=.0010,41.0对32.5,p=.0003)。RNA-Seq分析表明,TNF-α刺激后的15个基因(|log2FC|>5.0,log2TPM>5)是调节MSC免疫调节能力的候选基因。在体内,Clec4e和Gbp6参与炎症和骨形成。Clec4e,Gbp6和Cxcl10敲低可在体外增加MSCs的成骨分化。暂时性巨噬细胞消耗后,产生TNF-α的M1巨噬细胞和MSC的时间减少明显恢复,表明在TES愈合过程中TNF-α激活了MSC。体外模拟TNF-α对MSC的作用表明有15个候选MSC基因用于调节免疫调节能力。
Mesenchymal stem cells (MSCs) and macrophages collaboratively contribute to bone regeneration after injury. However, detailed mechanisms underlying the interaction between MSCs and inflammatory macrophages (M1) remain unclear. A macrophage-depleted tooth extraction model was generated in 5-wk-old female C57BL/6J mice using clodronate liposome (12.5 mg/kg/mouse, intraperitoneally) or saline injection (control) before maxillary first molar extraction. Mice were sacrificed on days 1, 3, 5, 7, and 10 after tooth extraction (n = 4). Regenerated bone volume evaluation of tooth extraction socket (TES) and histochemical analysis of CD80+M1, CD206+M2 (anti-inflammatory macrophages), PDGFRα+MSC, and TNF-α+ cells were performed. In vitro, isolated MSCs with or without TNF-α stimulation (10 ng/mL, 24 h, n = 3) were bulk RNA-sequenced (RNA-Seq) to identify TNF-α stimulation-specific MSC transcriptomes. Day 7 micro-CT and HE staining revealed significantly lower mean bone volume (clodronate vs control: 0.01 mm3 vs 0.02 mm3, p<.0001) and mean percentage of regenerated bone area per total TES in clodronate group (41.97% vs 54.03%, p<.0001). Clodronate group showed significant reduction in mean number of CD80+, TNF-α+, PDGFRα+, and CD80+TNF-α+ cells on day 5 (306.5 vs 558.8, p<.0001; 280.5 vs 543.8, p<.0001; 365.0 vs 633.0, p<.0001, 29.0 vs 42.5, p<.0001), while these cells recovered significantly on day 7 (493.3 vs 396.0, p=.0004; 479.3 vs 384.5, p=.0008; 593.0 vs 473.0, p=.0010, 41.0 vs 32.5, p=.0003). RNA-Seq analysis showed that 15 genes (|log2FC| > 5.0, log2TPM > 5) after TNF-α stimulation were candidates for regulating MSC\'s immunomodulatory capacity. In vivo, Clec4e and Gbp6 are involved in inflammation and bone formation. Clec4e, Gbp6, and Cxcl10 knockdown increased osteogenic differentiation of MSCs in vitro. Temporal reduction followed by apparent recovery of TNF-α-producing M1 macrophages and MSCs after temporal macrophage depletion suggests that TNF-α activated MSCs during TES healing. In vitro mimicking the effect of TNF-α on MSCs indicated that there are 15 candidate MSC genes for regulation of immunomodulatory capacity.