Department of Orthodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, 210029, China.
Jiangsu Province Key Laboratory of Oral Diseases, Nanjing, 210029, China.
J Transl Med. 2023 Mar 14;21(1):193. doi: 10.1186/s12967-023-04046-1.
In the repair of maxillofacial bone defects, autogenous craniofacial bone can often provide superior clinical results over long bone grafts. Most current studies have focused on the osteogenic differences between alveolar bone marrow (ABM) and long bone marrow (LBM), however, studies about the angiogenic differences between the two are currently lacking. We downloaded single-cell RNA sequencing (scRNA-seq) of mouse ABM and LBM respectively from the public database, and the data were processed by using Seurat package. CellphoneDB2 results showed that macrophages had the strongest interaction with mesenchymal stem cells (MSCs) and endothelial cells (ECs). ELISA results confirmed that ABM macrophages secreted a higher level of vascular endothelial growth factor A (Vegfa) compared to LBM macrophages, which further promoted angiogenesis of ECs and MSCs. Using SCENIC package, six key transcription factors (TFs) were identified to regulate the difference between ABM and LBM macrophages, and activating transcription factor 4 (Atf4) was confirmed to be more expressed in ABM macrophages by polymerase chain reaction (PCR) and western blot (WB), with predicted target genes including Vegfa. Besides, the result of scRNA-seq implied ABM macrophages more in M1 status than LBM macrophages, which was confirmed by the following experiments. From the results of another assay for transposase accessible chromatin sequencing (ATAC-seq) and RNA-seq about M1 macrophages, Atf4 was also confirmed to regulate the M1 polarization. So, we suspected that Atf4 regulated the different expression of Vegfa between ABM and LBM macrophages by activating M1 polarization. After knocking down Atf4, the expression of M1 polarization markers and Vegfa were downregulated and vasculogenic differences were eliminated, which were subsequently reversed by the addition of LPS/IFN-γ. Our study might provide a new idea to improve the success rate of autologous bone grafting and treatment of oral diseases.
在颌面部骨缺损的修复中，自体颅面骨往往比长骨移植物能提供更优的临床效果。目前大多数研究都集中在牙槽骨骨髓（ABM）和长骨骨髓（LBM）之间的成骨差异上，但目前缺乏关于两者之间血管生成差异的研究。我们从公共数据库中分别下载了小鼠 ABM 和 LBM 的单细胞 RNA 测序（scRNA-seq）数据，并使用 Seurat 包对数据进行了处理。CellphoneDB2 结果表明，巨噬细胞与间充质干细胞（MSCs）和内皮细胞（ECs）的相互作用最强。ELISA 结果证实，ABM 巨噬细胞分泌的血管内皮生长因子 A（Vegfa）水平高于 LBM 巨噬细胞，进一步促进了 ECs 和 MSCs 的血管生成。使用 SCENIC 包，鉴定出六个关键转录因子（TFs）来调节 ABM 和 LBM 巨噬细胞之间的差异，聚合酶链反应（PCR）和蛋白质印迹（WB）证实激活转录因子 4（Atf4）在 ABM 巨噬细胞中表达更高，预测的靶基因包括 Vegfa。此外，scRNA-seq 的结果表明，ABM 巨噬细胞比 LBM 巨噬细胞更倾向于 M1 状态，这一点通过以下实验得到了证实。另一项关于转座酶可及染色质测序（ATAC-seq）和 M1 巨噬细胞 RNA-seq 的实验结果也证实了 Atf4 通过调节 M1 极化来调节 Vegfa 在 ABM 和 LBM 巨噬细胞中的不同表达。敲低 Atf4 后，M1 极化标志物和 Vegfa 的表达下调，血管生成差异消除，随后加入 LPS/IFN-γ 后逆转。我们的研究可能为提高自体骨移植成功率和治疗口腔疾病提供新的思路。
