Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix


Cells are transplanted to regenerate an organs’ parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/β–catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure–mechanical equivalency to native dentin. Thus, the Alx3–Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Alx3 immunomapping and tissue reconstitution.
Fig. 2: Alx3 signalling pathways.
Fig. 3: Transplanted Alx3-restored adult human dental-pulp MSCs regenerated both parenchyma and stroma with enhanced Wnt signalling and improved cell survival.
Fig. 4: Alx3-restored adult MSC-induced angiogenesis and VEGF signalling.
Fig. 5: Parenchymal and stromal regeneration orthotopically in a preclinical, large animal model (total of 58 teeth in 10 minipigs).
Fig. 6: Structural and mechanical properties of regenerated dentin and overall schematic of Alx3/Wnt/VEGF cascades.

Data availability

Data supporting the findings of this study are available within the article and from the corresponding authors upon reasonable request.


  1. 1.

    Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S. & Blau, H. M. Self-renewal and expansion of single transplanted muscle stem cells. Nature 456, 502–506 (2008).

    CAS  Article  Google Scholar 

  2. 2.

    Shiba, Y. et al. Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts. Nature 538, 388–391 (2016).

    CAS  Article  Google Scholar 

  3. 3.

    Fox, I. J. et al. Stem cell therapy. Use of differentiated pluripotent stem cells as replacement therapy for treating disease. Science 345, 1247391 (2014).

    Article  Google Scholar 

  4. 4.

    Simmons, P. J., Przepiorka, D., Thomas, E. D. & Torok-Storb, B. Host origin of marrow stromal cells following allogeneic bone marrow transplantation. Nature 328, 429–432 (1987).

    CAS  Article  Google Scholar 

  5. 5.

    Mao, J. J. & Prockop, D. J. Stem cells in the face: tooth regeneration and beyond. Cell Stem Cell 11, 291–301 (2012).

    CAS  Article  Google Scholar 

  6. 6.

    Schworer, S. et al. Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals. Nature 540, 428–432 (2016).

    Article  Google Scholar 

  7. 7.

    Tao, G. et al. Pitx2 promotes heart repair by activating the antioxidant response after cardiac injury. Nature 534, 119–123 (2016).

    CAS  Article  Google Scholar 

  8. 8.

    Wei, K. et al. Epicardial FSTL1 reconstitution regenerates the adult mammalian heart. Nature 525, 479–485 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Fuchs, Y. et al. Sept4/ARTS regulates stem cell apoptosis and skin regeneration. Science 341, 286–289 (2013).

    CAS  Article  Google Scholar 

  10. 10.

    Nacu, E. et al. FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb regeneration. Nature 533, 407–410 (2016).

    CAS  Article  Google Scholar 

  11. 11.

    Mokalled, M. H. et al. Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish. Science 354, 630–634 (2016).

    CAS  Article  Google Scholar 

  12. 12.

    ten Berge, D. et al. Mouse Alx3: an aristaless-like homeobox gene expressed during embryogenesis in ectomesenchyme and lateral plate mesoderm. Dev. Biol. 199, 11–25 (1998).

    Article  Google Scholar 

  13. 13.

    Mallarino, R. et al. Developmental mechanisms of stripe patterns in rodents. Nature 539, 518–523 (2016).

    CAS  Article  Google Scholar 

  14. 14.

    Song, J. J. et al. Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat. Med. 19, 646–651 (2013).

    CAS  Article  Google Scholar 

  15. 15.

    Nakao, K. et al. The development of a bioengineered organ germ method. Nat. Methods 4, 227–230 (2007).

    CAS  Article  Google Scholar 

  16. 16.

    Jiang, N. et al. Exosomes mediate epithelium–mesenchyme crosstalk in organ development. ACS Nano 11, 7736–7746 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Thesleff, I. & Tummers, M. Tooth Organogenesis and Regeneration (StemBook, 2008).

  18. 18.

    Kaukua, N. et al. Glial origin of mesenchymal stem cells in a tooth model system. Nature 513, 551–554 (2014).

    CAS  Article  Google Scholar 

  19. 19.

    Endo, Y. et al. Wnt-3a and Dickkopf-1 stimulate neurite outgrowth in Ewing tumor cells via a Frizzled3- and c-Jun N-terminal kinase-dependent mechanism. Mol. Cell. Biol. 28, 2368–2379 (2008).

    CAS  Article  Google Scholar 

  20. 20.

    Lakhwani, S., Garcia-Sanz, P. & Vallejo, M. Alx3-deficient mice exhibit folic acid-resistant craniofacial midline and neural tube closure defects. Dev. Biol. 344, 869–880 (2010).

    CAS  Article  Google Scholar 

  21. 21.

    Hu, K. & Olsen, B. R. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. J. Clin. Invest. 126, 509–526 (2016).

    Article  Google Scholar 

  22. 22.

    Song, J. et al. Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells. Proc. Natl Acad. Sci. USA 111, 7337–7342 (2014).

    CAS  Article  Google Scholar 

  23. 23.

    Rivkin, E. et al. The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis. Nature 498, 318–324 (2013).

    CAS  Article  Google Scholar 

  24. 24.

    Carmeliet, P. & Jain, R. K. Molecular mechanisms and clinical applications of angiogenesis. Nature 473, 298–307 (2011).

    CAS  Article  Google Scholar 

  25. 25.

    Lee, C. H. et al. Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study. Lancet 376, 440–448 (2010).

    CAS  Article  Google Scholar 

  26. 26.

    Diogenes, A., Ruparel, N. B., Shiloah, Y. & Hargreaves, K. M. Regenerative endodontics: a way forward. J. Am. Dent. Assoc. 147, 372–380 (2016).

    Article  Google Scholar 

  27. 27.

    Zhang, Y. D., Chen, Z., Song, Y. Q., Liu, C. & Chen, Y. P. Making a tooth: growth factors, transcription factors, and stem cells. Cell Res. 15, 301–316 (2005).

    CAS  Article  Google Scholar 

  28. 28.

    Satcher, D. Oral Health in America: A Report of the Surgeon General 1–13 (US Department of Health and Human Services, National Institute of Dental and Craniofacial Research, National Institutes of Health, 2010).

  29. 29.

    2005–2006 Survey of Dental Services Rendered (American Dental Association, 2007).

  30. 30.

    Ng, Y. L., Mann, V., Rahbaran, S., Lewsey, J. & Gulabivala, K. Outcome of primary root canal treatment: systematic review of the literature—part 1. Effects of study characteristics on probability of success. Int. Endod. J. 40, 921–939 (2007).

    Article  Google Scholar 

  31. 31.

    Hunter, D. J. et al. Wnt acts as a prosurvival signal to enhance dentin regeneration. J. Bone Miner. Res. 30, 1150–1159 (2015).

    CAS  Article  Google Scholar 

  32. 32.

    Arany, P. R. et al. Photoactivation of endogenous latent transforming growth factor-β1 directs dental stem cell differentiation for regeneration. Sci. Transl. Med. 6, 238ra269 (2014).

    Article  Google Scholar 

  33. 33.

    Langer, R. & Vacanti, J. P. Tissue engineering. Science 260, 920–926 (1993).

    CAS  Article  Google Scholar 

  34. 34.

    Lee, C. H. et al. Protein-releasing polymeric scaffolds induce fibrochondrocytic differentiation of endogenous cells for knee meniscus regeneration in sheep. Sci. Transl. Med. 6, 266ra171 (2014).

    Article  Google Scholar 

  35. 35.

    Franz, W. M., Zaruba, M., Theiss, H. & David, R. Stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet 362, 675–676 (2003).

    Article  Google Scholar 

  36. 36.

    Askari, A. T. et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet 362, 697–703 (2003).

    CAS  Article  Google Scholar 

  37. 37.

    Kavanagh, D. P. & Kalia, N. Hematopoietic stem cell homing to injured tissues. Stem Cell Rev. 7, 672–682 (2011).

    Article  Google Scholar 

  38. 38.

    Bottino, M. C. et al. Bioactive nanofibrous scaffolds for regenerative endodontics. J. Dent. Res. 92, 963–969 (2013).

    CAS  Article  Google Scholar 

  39. 39.

    Huang, G. T. et al. Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng. A 16, 605–615 (2010).

    CAS  Article  Google Scholar 

  40. 40.

    Iohara, K. et al. Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1. Tissue Eng. A 17, 1911–1920 (2011).

    CAS  Article  Google Scholar 

  41. 41.

    Iohara, K. et al. A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration. Stem Cells Transl. Med. 2, 521–533 (2013).

    CAS  Article  Google Scholar 

  42. 42.

    Conde, M. C. et al. Stem cell-based pulp tissue engineering: variables enrolled in translation from the bench to the bedside, a systematic review of literature. Int. Endod. J. 49, 543–550 (2016).

    CAS  Article  Google Scholar 

  43. 43.

    Nakashima, M. et al. Pulp regeneration by transplantation of dental pulp stem cells in pulpitis: a pilot clinical study. Stem Cell Res. Ther. 8, 61 (2017).

    Article  Google Scholar 

  44. 44.

    Xuan, K. et al. Deciduous autologous tooth stem cells regenerate dental pulp after implantation into injured teeth. Sci. Transl. Med. 10, eaaf3227 (2018).

    Article  Google Scholar 

  45. 45.

    He, L. et al. Regenerative endodontics by cell homing. Dent. Clin. North Am. 61, 143–159 (2017).

    Article  Google Scholar 

  46. 46.

    Zhou, C. et al. Lhx8 mediated Wnt and TGFbeta pathways in tooth development and regeneration. Biomaterials 63, 35–46 (2015).

    CAS  Article  Google Scholar 

  47. 47.

    Jiang, N. et al. Postnatal epithelium and mesenchyme stem/progenitor cells in bioengineered amelogenesis and dentinogenesis. Biomaterials 35, 2172–2180 (2014).

    CAS  Article  Google Scholar 

  48. 48.

    Kawamura, R., Hayashi, Y., Murakami, H. & Nakashima, M. EDTA soluble chemical components and the conditioned medium from mobilized dental pulp stem cells contain an inductive microenvironment, promoting cell proliferation, migration, and odontoblastic differentiation. Stem Cell Res. Ther. 7, 77 (2016).

    Article  Google Scholar 

  49. 49.

    Khoo, C. P., Micklem, K. & Watt, S. M. A comparison of methods for quantifying angiogenesis in the Matrigel assay in vitro. Tissue Eng. C 17, 895–906 (2011).

    CAS  Article  Google Scholar 

  50. 50.

    Li, R. et al. Human treated dentin matrix as a natural scaffold for complete human dentin tissue regeneration. Biomaterials 32, 4525–4538 (2011).

    CAS  Article  Google Scholar 

  51. 51.

    Zheng, Y. et al. Mesenchymal dental pulp cells attenuate dentin resorption in homeostasis. J. Dent. Res. 94, 821–827 (2015).

    CAS  Article  Google Scholar 

  52. 52.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  Article  Google Scholar 

Download references


We thank Q. Guo, P. Ralph-Birkett and Y. Tse for administrative and technical assistance. We thank F. Meijlink and J. Deschamps for gifts of Alx3 fragment plasmid, M. S. Shelanski for suggestions for two antibodies, J. E. Nör for sharing his insight on VEGF signalling, Y. Zhang for technical help with the Wnt3a luciferase promoter constructs and A. F. Fouad for citation of the Survey of Dental Services Rendered. The work is supported by NIH grants R01DE025643, R01DE023112, R01AR065023, R01DE025969 and R01DE026297, and an Osteo Science Foundation grant to J. J. Mao. The scholarly effort of several co-authors was supported by National Natural Science Foundation of China grants 81570939 and 81741106 and the Beijing Municipal Administration of Hospitals’ Youth Programme (QML20161503 to J.Zhou), National Natural Science Foundation of China grants 81271110 to Z.W., 81170932 to J.L., 81371136 to X.Z., National Science and Technology Support Program 2014DFA31990 to Z.W. and Program of International Science and Technology Cooperation 2014DFA31990 to L.Y.

Author information




L.H. and J.Zhou performed the technical design and conducted pivotal experiments, and collected and analysed data for Figs. 14 (L.H.), Fig. 6m (Y.N. and L.H.) and Figs. 5 and 6a–l (primarily J.Zhou). L.H., J. Zhou, M.C. and C.-S.L. generated all the displayed items including all the figures, tables and Supplementary Information. C.-S.L. performed the CRISPR/Cas9 experiments and produced the corresponding notes. S.G.K., J.Zhou and L.H. performed the clinical procedures in the minipig root canals. L.X., M.X. and S.X. assisted in in vitro experiments. H.Y., H.B., Y.N., C.S., P.G.C., T.G.B., B.H., N.T. and L.W. performed the scaffold preparation and mechanical, μCT and SEM analyses. J.Zhong and J.Wu analysed the microarray data. K.C., J.Wu, J.Wen and G.Y. performed the genomic pathway analyses. D.W.R and T.-J.S. provided the model for microarray analysis. G.C. participated in the tooth sample collection. J.P., J.C. and Sainan Wang assisted in molecular assays. Q.G. and J. Zheng assisted in the athymic mice in vivo experiments. B.C. performed the statistical analyses. W.G., D.M.O., M.S., D.P.T., W.Z., J.L. and M.F. participated in study design and manuscript discussion. D.J.Z., X.Z., J.L., Z.W., L.Y. and X.J. participated in study design and manuscript revision. A.R. participated in the large animal study design and surgery. S.W. participated in the manuscript discussion. J.L. mentored L.H. and participated in the study design and manuscript discussion. J.J.M. conceived and designed the experiments, and discussed data interpretation and finalized the manuscript with input from all the co-authors.

Corresponding authors

Correspondence to Junqi Ling or Jeremy Mao.

Ethics declarations

Competing interests

J.J.M. has co-founded Innovative Elements and Xinkewo with the goal to develop regenerative products. The other authors declare no competing interests in this article.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Notes, Supplementary Figs. 1–8 and Supplementary Tables 1 and 2.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

He, L., Zhou, J., Chen, M. et al. Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix. Nat. Mater. 18, 627–637 (2019).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing