What they tested
A longstanding problem in the bioengineered tooth germ program is that inductive embryonic tooth germ cells lose their ability to trigger tooth formation once they are expanded in vitro. Birjandi and Sharpe asked whether the paracrine output of those cells, especially small extracellular vesicles and the broader secretome, could replace the living inductive mesenchyme in a classic reassociation assay (Birjandi and Sharpe 2025, Introduction). The work was done in CD-1 mouse embryos: E14.5 tooth germ mesenchyme served as the inductive source, E10.5 first-branchial-arch mesenchyme served as the non-inductive control, and E13.5 tooth germ epithelium was the responding tissue (Birjandi and Sharpe 2025, Materials and Methods).
Characterizing the vesicles and their protein cargo
The team purified sEV from E14.5 tooth germ mesenchyme using differential centrifugation followed by ultracentrifugation. Nanoparticle tracking gave a mean particle size of 190.3 ± 4.0 nm and a concentration of 2.39 × 10^9 ± 9.67 × 10^7 particles/mL, with CD63 and CFSE staining supporting an extracellular-vesicle identity (Birjandi and Sharpe 2025, Results). Label-free LC-MS/MS identified 1,186 proteins in the inductive mesenchymal cells and 403 proteins across secretome groups. Most sEV proteins were shared with the secretome, while 13 proteins were unique to sEV (Birjandi and Sharpe 2025, Results).
Culture conditions changed the cargo. sEV from 3D cultures contained more proteins than sEV from 2D cultures, and sEV from 24-hour cultures retained more inductive-signature proteins than sEV from 5-day-expanded cultures. Proteins uniquely present in short-cultured inductive sEV mapped to BMP, integrin, RIG-I, CD40, basement-membrane organization, and angiogenesis pathways (Birjandi and Sharpe 2025, Results).
Epithelial transcriptome changes
Treating E13.5 tooth germ epithelium with inductive sEV for 24 hours changed the transcriptome. Genes uniquely upregulated in i-sEV-treated epithelium overlapped with genes active in the inductive E10.5 branchial-arch epithelium and were associated with protein kinase C, PDGFR, FGFR, and BMP signaling (Birjandi and Sharpe 2025, Results). Five-day i-sEV treatment also increased Lef1 expression, a readout of Wnt pathway activity, compared with controls (Birjandi and Sharpe 2025, Results). Treatment with the whole secretome produced broader changes, including extracellular-matrix organization and integrin signaling, than sEV alone (Birjandi and Sharpe 2025, Results).
The functional test: no tooth induction
To see whether those signaling changes were enough, the authors recombined the treated epithelium with non-inductive E10.5 branchial-arch mesenchyme in an ex vivo collagen-gel assay. Positive controls using E14.5 inductive mesenchyme and E13.5 epithelium formed tooth germs; negative controls using E10.5 mesenchyme did not. Despite the transcriptomic shifts, both i-sEV-treated and i-secretome-treated epithelium failed to rescue tooth induction when paired with non-inductive mesenchyme (Birjandi and Sharpe 2025, Results and Discussion).
What it means for the field
For the bioengineered tooth germ program, the paper draws a useful boundary: a cell-free cocktail of sEV and secreted factors can nudge epithelial gene expression toward an odontogenic profile, but it is not sufficient to replace direct inductive mesenchyme in this mouse model. The authors suggest that intact tooth development probably depends on tightly orchestrated, direct cell-cell and cell-matrix interactions that paracrine signaling alone cannot reproduce (Birjandi and Sharpe 2025, Discussion). That reinforces why expanded inductive cells lose potency and why simply harvesting their secretome is unlikely to shortcut whole-tooth bioengineering.
What it does not show
The study is purely murine and embryonic. It does not test human cells, postnatal dental stem cells, or an in vivo jaw environment. The sample sizes are small: three independent sEV batches and standard recombination assays with 2 × 10^5 cells per pellet. It also does not rule out that a higher sEV dose, longer treatment, or engineered enrichment of specific factors might someday push the response further. The authors themselves note that sEV heterogeneity and co-purified contaminants remain practical hurdles (Birjandi and Sharpe 2025, Discussion). A publisher correction was issued on 11 February 2026; it does not alter the scientific conclusions reported here.
Provenance: grounded in the open-access full text of Birjandi AA, Sharpe P. The Secretome of the Inductive Tooth Germ Exhibits Signals Required for Tooth Development. Bioengineering. 2025;12(2):96. doi:10.3390/bioengineering12020096, retrieved from PMC11851894 and read in full. Method and sourcing standard at /method/.