Macrophages are a normal constituent of the developing root

The authors first mapped immune cells in developing mouse first mandibular molars from postnatal day 0.5 to 15.5. Imaging mass cytometry and flow cytometry showed that F4/80+CD68+ macrophages were the predominant immune population and increased in abundance as roots formed, colonizing the dental pulp and periodontal ligament. Most of these macrophages carried an M2-like CD206+ signature. CD68+F4/80- multinucleated osteoclasts were present in the dental follicle and alveolar bone, distinct from the pulp and periodontal-ligament macrophages (Lei et al. 2026, Fig. 1 and Fig. S1).

Removing macrophages shortens roots and widens the periodontal ligament

To test function, the team injected clodronate liposomes at PN3.5, the onset of root morphogenesis, and compared them with PBS liposomes. The treatment cut local F4/80+ macrophages without changing body weight, body length, tibia length, or spleen index. Crown morphology looked normal, but micro-CT at PN15.5 showed shorter roots. Histology at PN15.5 and PN21.5 revealed a disorganized, widened periodontal ligament, and periostin staining was reduced, pointing to defective periodontal-ligament maturation (Lei et al. 2026, Fig. 2; n=5 for root length, p<0.01; n=3-4 for periodontal-ligament width).

Gli1+ progenitors proliferate but do not differentiate

Lineage tracing in Gli1-CreER; B6-G/R mice showed that macrophage depletion increased the number of Gli1+ cells in the apical root region and raised Ki67+ Gli1+ cells, indicating expanded proliferation. Yet expression of the odontoblast markers Dspp and Nestin was unchanged, and periostin expression fell. The phenotype is therefore not a broad failure of odontoblast differentiation but a lineage-commitment block: more progenitors, less mature periodontal output (Lei et al. 2026, Fig. 3).

Macrophage-derived TGF-β is the candidate driver

Single-cell RNA sequencing of PN15.5 molars and surrounding tissue identified 10 dental mesenchymal subclusters. Macrophage depletion changed 1,050 genes; gene-set enrichment showed downregulation of osteogenic programs, ECM-receptor interaction, focal adhesion, and TGF-β signaling. CellChat predicted macrophages as a major source of growth-factor signals to mesenchymal subpopulations. In vitro, conditioned medium from human PBMC-derived macrophages or mouse M2 bone-marrow macrophages increased osteogenic markers (RUNX2, SP7, COL1A1) and mineralization in human DPSCs and mouse molar MSCs; a TGF-β neutralizing antibody blunted the effect (Lei et al. 2026, Figs. 4-5).

Boundary and why it matters for root organoids

The study is developmental, not regenerative: it removes macrophages acutely in healthy neonatal mice and asks what goes wrong, not whether adding macrophages back repairs an injury. Clodronate liposomes are not clinically translatable, and the analysis stops at PN21.5, before functional eruption or load-bearing performance can be assessed. Nevertheless, the work places a new constraint on the root-organoids route: root-like constructs made from postnatal dental stem cells may need to recapitulate immune-mesenchymal crosstalk, specifically macrophage-derived TGF-β, if they are to produce organized dentin, cementum, pulp, and periodontal ligament in the right proportions.

Provenance: Primary source read in full at Cell Death & Disease; analysis written for the daily pass. See /method/ for the site’s evidence standards.