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Updated: Jun 18, 2025
Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging
Published on: July 16, 2021
Macrophages modulate mesenchymal stem cell function via tumor necrosis factor alpha in tooth extraction model
Aung Ye Mun1, Kentaro Akiyama1, Ziyi Wang2
1Department of Oral Rehabilitation and Regenerative Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
Abstract:
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.
Insights
Depleting macrophages impairs bone regeneration by reducing mesenchymal stem cells (MSCs) and inflammatory cells. TNF-α activation of MSCs is crucial for tooth extraction socket healing.
Area of Science:
- Regenerative Medicine
- Immunology
- Oral Biology
Background:
- Mesenchymal stem cells (MSCs) and macrophages are key players in bone regeneration.
- The precise mechanisms of MSC-macrophage interaction, particularly with M1 macrophages, are not fully understood.
Purpose of the Study:
- To investigate the role of macrophages in bone regeneration after tooth extraction.
- To elucidate the interaction between MSCs and M1 macrophages during the healing process.
- To identify molecular mechanisms, including TNF-α signaling, involved in MSC-macrophage crosstalk.
Main Methods:
- A macrophage-depleted mouse model using clodronate liposomes was established.
- Histochemical analysis quantified M1, M2, MSC, and TNF-α+ cells in tooth extraction sockets.
- In vitro bulk RNA-sequencing (RNA-Seq) analyzed TNF-α-stimulated MSCs.
Main Results:
- Macrophage depletion significantly reduced bone regeneration and the number of M1, MSC, and TNF-α+ cells.
- A temporal reduction and subsequent recovery of these cells were observed after depletion.
- RNA-Seq identified 15 candidate genes in MSCs potentially regulating immunomodulatory capacity.
Conclusions:
- Macrophages, especially M1, are essential for effective bone regeneration after tooth extraction.
- TNF-α signaling plays a critical role in activating MSCs during socket healing.
- Specific genes like Clec4e, Gbp6, and Cxcl10 are implicated in inflammation and bone formation, influencing MSC osteogenic differentiation.
