Tooth decay is one of the most widespread diseases in the world, perhaps only second to the common cold. While tooth decay can be prevented and managed, a cavity or a break in the enamel can be the beginning of the end for the tooth, leading to a progression of temporary or burdensome interventions like fillings and dentures. For patients who do not have access to adequate care, or who suffer from genetic disorders that impair enamel growth, the problem can be much more serious. Because enamel loss is irreversible, replacement, not regeneration, remains the standard of care.
Generating any kind of living tissue in the lab is complex. To grow a whole tooth, or even portions of one, scientists need a map of the genetic signaling pathways that must be activated to turn stem cells into cells like ameloblasts and odontoblasts (the cells that secrete enamel and dentin, respectively). They must also mature those building-block cell types to a point where they can produce clinical-grade qualities (and quantities) of these substances.
Not surprisingly, researchers at the forefront of regenerative dentistry are inventing, adopting, and adapting a variety of sophisticated tools and methods to solve these challenges in the most efficient way possible, all in hopes of providing dentists with new ways to help us keep our teeth healthy.
Dr. Hannele Ruohola-Baker is a University of Washington Professor of Biochemistry and a faculty member in the Institute for Stem Cell and Regenerative Medicine (ISCRM). Over the last few years, the Ruohola-Baker Lab has been at the helm of a multidisciplinary effort to usher in the age of living teeth using stem cell, genomics, and protein design technologies in partnership with the Institute for Protein Design (IPD), the Brotman Baty Institute (BBI), and the UW School of Dentistry.
In a series of breakthroughs stretching over much of the past decade, the Ruohola-Baker Lab has begun to build the blueprint for regenerating enamel, and even an entire tooth. Studies published by her team in 2019 and 2023 described the sequence of genetic signals that drive tissue generation in our teeth and created a 3D organoid capable of secreting an immature, but real, form of enamel.
“Maturation is the big bottle neck in any attempt to differentiate stem cells,” says Ruohola-Baker. “It’s exciting to know we can make ameloblasts. But the cells we saw in previous investigations were too young, and the enamel was not dense enough to be clinically useful. So, we asked whether there was a mystery signal that could allow us to mature the ameloblasts further.”
Now, the results of a search for that missing piece of the puzzle have been published in the International Journal of Oral Science (IJOS). Ruohola-Baker is the senior author of the new study. The first author is Anjali Patni, a PhD student in the Ruohola-Baker Lab. ISCRM faculty members Dr. Julie Mathieu, Dr. Benjamin Freedman, Dr. Ted Gross, Dr. Rob Cornell, and Dr. David Baker are also authors of the study, which was conducted in collaboration with Mary Regier, director of the ISCRM Genomics Core, and Dr. George Daley, Dean of Harvard Medical School.
In the most recent study, the researchers show that a soluble AI-designed signaling protein (known as a ligand) can be used to mature ameloblasts capable of secreting more mineralized enamel that is detectable via micro-CT imaging – the gold standard for evidence of successful maturation – representing another important step forward for regenerative dentistry.
Patni led the quest to identify the signal required to push ameloblasts to a state where the cells could secrete mineralized, organized enamel. The key candidate in her bioinformatic analysis turned out to be the Notch pathway.
The odontoblasts express a Notch ligand (which emits a signal) while ameloblasts have a Notch receptor, which grabs on to the ligand, like a hooked finger, to generate force. Recreating this action in the lab with natural proteins has not been possible. But the AI-designed protein with just the right structure did the trick. In fact, using the AI-designed protein in place of its natural counterpart was beneficial for additional reasons.
“To make a tooth, which we hope to do in the future, you need ameloblasts and odontoblasts,” explains Patni. “In nature, these two cell types develop in close proximity to each other and constantly signal back-and-forth. The reality, though, is that making these two cell types in a correct synchrony is very complicated. Now, with the soluble Notch activator, we can mature ameloblasts without the presence of odontoblasts, which is an important step, and simplifies the process for us.”
The soluble nature of the activator is a crucial feature, explains Dr. Ruohola-Baker. “Notch signaling is normally activated through direct cell–to-cell contact, as both Notch receptors and Delta ligands are membrane-bound proteins expressed on adjacent cells. Engagement between these membrane-tethered proteins generates the mechanical force required to induce a conformational change in Notch, exposing cleavage sites that permit proteolytic processing and release of the Notch intracellular domain, thereby initiating downstream signaling.”
The soluble Notch activator allows the pathway to be turned on without odontoblast-ameloblast contact. The engineered Notch activator scaffold in this study (C3-DLL4) was designed to enable simultaneous engagement of Notch receptors on neighboring cell surfaces, effectively recreating the mechanical tension required for Notch activation. By structurally mimicking the spatial constraints of membrane-bound ligands, this scaffold provides sufficient force generation to activate Notch signaling.
With help from the Freedman Lab, the ameloblasts were implanted into the kidney capsule, a tiny pocket adjacent to the kidney where the cells could mature into more tooth-like material. The Gross Lab conducted the microCT imaging – the most visible proof yet that the enamel had sufficient density and calcification to someday be useful in the clinic.
Meanwhile, down the hall, the Mathieu Lab was studying another piece of the regenerative dentistry puzzle – the gene Dlx3, which is also an important component of tooth development. Mutations in Dlx3 have been linked to higher susceptibility to cavities and to dental disease like amelogenesis imperfecta. Less well understood was the role Dlx3 plays in the later stages of ameloblast development.
Rachel Moore, a third-year resident in the Mathieu Lab, generated a Dlx3 gene knockout in human iPSC, which allowed Patni to study its function in mature ameloblasts. The team observed that the absence of DLX3, which acts as a gene regulator inside cells, impaired ameloblast development even when Notch was activated, indicating that the gene was indeed critical for the ameloblast maturation process.
For now, Patni speaks to the significance of the most recent findings. “For the first time, we have a useful model of ameloblast maturation. We have shown that we can use an AI-designed ligand to mature these cells independent of other cell types, and that we can generate enamel that is more mineralized and more dense than any results we have seen in prior studies, which means we are getting closer to the clinic than we have ever been.”
Acknowledgements
This work is supported by ISCRM Fellows Program (Anjali Patni) and grants from the National Institutes of Health DE033016 (J.M., R.A.C. and H.R-B.), 1P01GM081619, R01GM097372, R01GM083867, NHLBI Progenitor Cell Biology Consortium (U01HL099997; UO1HL099993) SCGE COF220919 (H.R-B), Molecule (J.M. and H.R-B.), and AHA 19IPLOI34760143, Brotman Baty Institute (BBI), DOD PR203328 W81XWH-21-1-0006 and Stem Cell Gift Funds for H.R-B.