UW Regenerative Dentistry Partnership 

Co-culture image of stem cell-derived Ameloblast organoid and dental pulp stem cell-derived Odontoblasts secrete Enamelin (yellow) and Odontoblasts secrete DSPP (red). The nuclei of all cells is labeled with DAPI (blue)

Hannele Ruohola-Baker, PhD leads a team of researchers who are interested in the most fundamental mysteries of human development. For years, the lab has been studying how a single fertilized cell is able to differentiate and proliferate into multiple cell types, which in turn organize into the complex tissues, organs, and other structures that make us human, leading to discoveries that are relevant to everything from fertility to cancer. 

One of those structures, of course, is a tooth. Dr. Ruohola-Baker, who is a professor of biochemistry and an associate director of the University of Washington Institute for Stem Cell and Regenerative Medicine (ISCRM), realized years ago that it might be possible to apply her lab’s expertise in stem cell biology to an area of health that is essential to our wellbeing: dentistry. 

But where could a research team get their hands on cells to study how teeth form? One possibility occurred to Ruohola-Baker while her son was enduring an unpleasant rite-of-passage for many young adults – wisdom tooth removal. Not only are extracted wisdom teeth a potential source of useful tooth cells, these late arrivals in our mouths also mark the last appearance of the cells (known as ameloblasts) that form enamel. 

The possibility became a reality when a team led by Dr. Ruohola-Baker and ISCRM faculty member Julie Mathieu, PhD, an Assistant Professor of Comparative Medicine, successfully mined stem cells from donated wisdom teeth and grew them into dental pulp stem cells. That project, which was supported by a grant from the Brotman Baty Institute, helped inform a 2019 study, published in Scientific Reports, that described in new detail how dental pulp stem cells age, an important early step for regenerative dentistry. 

Ruohola-Baker and Mathieu knew that any undertaking related to oral health would need a clinical perspective. They did not have to look far. The scientists enlisted the support of Thomas Dodson, DMD, MPH, Philip Worthington Professor & Chair, Oral and Maxillofacial Surgery in the UW School of Dentistry. Dr. Dodson was immediately receptive to the clinical upsides of using stem cells to regenerate the tissues that make up our teeth. 

Left to right: Thomas Dodson, DMD, MPH, Hai Zhang, DMD, PhD, and Robert Cornell, PhD, Professor of Oral Health Sciences

The possibilities of regenerative dentistry soon attracted another partner in the School of Dentistry, Hai Zhang, DMD, PhD. Dr. Zhang is a Professor of Restorative Dentistry and a practicing prosthodontist, a specialty that brings him face-to-face with patients who have lost a tooth, or multiple teeth, to injury, wear-and-tear, or disease. Intrigued by the potential clinical implications, Dr. Zhang eagerly joined the project, which happened to involve three PhD students in the School of Dentistry: Ammar Alghadeer, Sesha Hanson-Dury, and YanTing (Blair) Zhao. 

Scientists at the Brotman Baty Institute and the Institute for Protein Design would soon become key collaborators on the expanding regenerative dentistry team. The ability to pull together such a mosaic of partners has a lot to do with geography, according to Ruohola-Baker. “I believe this collaboration between a stem cell institute, a dental school, and experts in genetic sequencing and protein design could only be possible in a few places, and one of them is in Seattle. This is definitely a Pacific Northwest story.” 

The Trouble with Enamel

Tooth decay is a common part of the human experience. Acidic or sugary food and drinks, medical issues, genetics, and a lifetime of chewing all wear down our teeth. While routine cavities can be treated with fillings and similar procedures, many people suffer from more serious dental conditions and millions of others do not have access to regular dental care. 

The root of the problem, so to speak, is that the tissues that form our teeth do not regenerate like skin or hair. That’s one reason dentists caution patients not to brush too vigorously. Enamel may be the hardest material in the human body, but once it’s gone, it’s gone. Without it, the dentin that makes up most of our teeth, and the pulp, which contains the nerves and blood vessels, are more vulnerable to damage, increasing the likelihood of cavities, tooth loss, and other complications. 

The premise of regenerative dentistry is that it might be possible to regrow enamel and dentin, with an assist from technology. One challenge is figuring out how to kick start the production of ameloblasts – the cells that are responsible for enamel formation early in life, but no longer present once a tooth erupts from the gums. Achieving that would require learning to harness the biological processes that naturally drive ameloblasts. 

Before the researchers could begin to think about growing incisors or molars or canines from stem cells, they had to answer a set of fundamental questions about the biology of our teeth. What kinds of stem cells exist in teeth? What are the characteristics of these stem cells? What genes are involved in the formation of dentin and enamel? And could these processes be controlled carefully enough to someday treat patients? 

The Building Blocks of Our Teeth

In this lab image of a developing incisor, colors identify which genes are being expressed at each stage of development.

Working together, the UW scientists turned their attention to dental pulp stem cells (DPSCs), which are known to repair injured dentin. The Ruohola-Baker Lab compared tissue from wisdom teeth donated by more than 300 people through the School of Dentistry and discovered details about the factors that contribute to the development of dental pulp stem cells. Among the revelations was the identification of a pathway that, when upregulated, is linked to more rapid aging in dental pulp stem cells. 

The findings were detailed in a 2019 paper published in Scientific Reports. “The 2019 paper gave us insights we needed to generate the cells that secrete dentine,” explains Ruohola-Baker. “But we still didn’t know how to make ameloblasts, which are necessary for the production of enamel – the other critical component of a tooth. 

Last fall, the Ruohola-Baker Lab, Dr. Zhang in the School of Dentistry, and collaborators from the Institute for Protein Design and BBI cast new light on those mysteries. That study, published in September 2023 in Developmental Cell, combined stem cell, designed protein, and single-cell sequencing technologies to map the signaling pathways that are necessary for ameloblast and dentin growth and described a tooth organoid that can be used for disease modeling now while pointing to the future of dental care. 

The first author of that paper is Ammar Alghadeer, a PhD student in the School of Dentistry’s Department of Oral Health Sciences and a member of the Ruohola-Baker Lab. ISCRM faculty members Julie Mathieu, PhD, Benjamin Freedman, PhD, David Baker, PhD, and Jay Shendure, MD, PhD are also authors of the paper. 

Ruohola-Baker points to three key advances in the paper: the first single cell sequencing atlas of the developing tooth, the first induced pluripotent stem cell-derived tooth organoid, and the first designed protein application in regenerative dentistry.  “Here we’ve discovered the pathways that need to be activated to do that. By putting lessons from the two studies together, we were able to create the first tooth organoid.” 

Crucial Contributions from Dental Students 

At the nexus of the research was a cohort of young scientists with a unique stake in the future of regenerative dentistry.

“Our goal was to create an organoid system that has the same cellular profile as human teeth,” says Ammar Alghadeer, at the time a PhD student in the School of Dentistry and a member of the Ruohola-Baker Lab. “We wanted to know whether the cells that grow in the organoid produce the same proteins they would produce in a developing tooth, to test the function of those proteins, and to assess the possibility of someday generating a complete tooth organoid that can be transplanted into a patient.” 

Dr. Sesha Hanson-Drury, a graduate of the UW School of Dentistry dual DDS/PhD program, also contributed to the research as a graduate student in the Ruohola-Baker Lab. She emphasizes the clinical significance behind the study.  

“As a dental student, I was seeing patients in the clinic who were having recurrent decay under crowns, leading to the need for further interventions,” says Dr. Hanson-Drury.  “Sometimes there’s not enough tooth structure to save, or the decay has reached the pulp of the tooth, requiring a root canal.  Much of our patient population can’t afford that procedure, which means we’re talking about removing the tooth. Because of all this, I’m interested in how we can apply stem cell tools to naturally produce replacement structures.”

Anjali Patni joined the Ruohola-Baker Lab in early 2022 and is a PhD student in the Graduate Program in Oral Health Sciences. With support from a state-funded ISCRM Fellowship, Patni, who is also an author of the Developmental Cell paper, is now making crucial contributions to the ongoing regenerative dentistry efforts. The goal of her fellowship project is to identify the signaling factors that promote the maturation of secretory ameloblasts. In the broader scope of oral health, she hopes her research yields insights that lead to new treatments for ailments like amelogenesis imperfecta. 

Protein Design, Genomic Sequencing, and Teeth

The single-cell sequencing work was performed in partnership with the Brotman Baty Institute, led by Dr. Jay Shendure. In this dimension of the research, the investigators wanted to know which cell populations were present in the tissues they were studying, how those cell populations communicated with each other, and which signaling mechanisms caused the cells to differentiate into mature cell types. 

Samples collected at five time points of human fetal development helped the researchers create the clearest picture yet of the process that ultimately leads to the production of enamel. A computational modeling tool designed by Alghadeer shed additional light on the pathways that activate genes that play a role in ameloblast development.  Understanding the regulatory mechanisms at work is key to being able to control these processes for therapeutic purposes. 

Dr. Hanson-Drury explains the significance of the findings in the September 2023 paper. 

“Once we were able to identify what signaling pathways are most active as cells transition from a progenitor to a more mature state, we used small molecules to activate those pathways in vitro on a plate in the lab. And we have now seen that co-culturing iPSC-derived ameloblasts with dental pulp stem cell-derived odontoblasts leads to even more mature tissues because these cell types develop parallel to each other in vivo. The clinical hope is that we’ll eventually use these methods to make an implantable tooth organoid.” 

Stem cells and single-cell sequencing were not the only cutting-edge tools used by the investigators. Designed protein technology also played a critical role. In their efforts to generate dentin tissue from human induced-pluripotent stem cells rather than dental pulp stem cells, the researchers found that a scaffold of six designed protein mini-binders supercharged a pathway, known as FGF, that helps give rise to odontoblasts. The mini-binders, which were developed through a partnership between the Ruohola-Baker Lab and the Institute for Protein Design, are particularly precise, meaning they all but eliminate the concern of leading to unwanted cell growth or other off-target effects. That research was detailed in a 2023 paper in Frontiers in Dental Medicine, first authored by Dr. Hanson-Drury

Healing the Whole Mouth  

The partnership between and the School of Dentistry is growing in other important areas of oral health. Along with ISCRM affiliate faculty member Dr. Robert Cornell, Professor of Oral Health Sciences, Dr. Mathieu and Dr. Ruohola-Baker recently received an NIH MPI grant to study the regulatory networks and genes which may be associated with congenital birth defects that affect the teeth, lip, and salivary glands, including cleft lip and cleft palate. 

Grant funding will allow the trio to use genetic sequencing technologies to better understand the developmental biology of structures vulnerable to dental deformities. The researchers hope that filling in knowledge gaps about the genetic origins of these abnormalities will expedite the design of preventative or regenerative therapies. 

The Future of Regenerative Dentistry

As the researchers continue to refine and test their ground-breaking work on tooth regeneration, Ammar Alghadeer reflects on the team’s progress. “The fact that we’re able to make the first ameloblasts in tissue culture is amazing. It’s a big step toward being able to regenerate whole teeth or correct genetic problems. This is why I came to the United States. It’s very exciting to be at this point.” 

“This is an amazing achievement by this group,” says Hai Zhang from the School of Dentistry. “From a clinician’s point of view the next step is to create an environment in the lab that will allow the cells to form enamel that is strong enough to be used to repair or recreate entire teeth that can be transplanted into patients.” 

Those cells, says Zhang, could be sourced from patients themselves. Cells obtained from a cheek swab, for example, could be differentiated in the lab to become the cell types that would produce enamel, dentine, and other tissues required to grow a whole replacement tooth or fill a deep cavity. 

According to Zhang, this approach would be the basis for a new generation of implants that could mean less time in the dentist’s chair and shorter recovery periods for patients who need dentures or crowns. “I would say it represents personalized dentistry,” says Zhang. “We can customize the shape or the age of the tooth to fit the individual patient. Another benefit is that the stem cell-based implants will feel more natural than current implants, which are made from synthetic materials.”

Zhang imagines a future in which replacement teeth could actually grow in a patient’s mouth. “Our goal is to someday be able to embed those engineered cells or buds into the gums and then form naturally in a controlled way.” 

The real-world value of regenerative dentistry was also apparent to ISCRM investigators with less direct connections to the clinic. Dr. Mathieu, PhD was a postdoc in the Ruohola-Baker Lab when the dental research began. Mathieu helped the team work out the nuts and bolts of differentiating stem cells into ameloblasts. 

“We wanted to know how the support cells communicate, which pathways are involved in each step of ameloblast development, and what comes from each cell type. It was exciting for me to realize that this was more than just a new way to fix cavities. We could actually help patients with diseases that were affecting the health of their whole bodies.” 

Regenerative Dentistry in the News

September 22, 2023
How University of Washington scientists use stem cells to regenerate tooth enamel
Watch the story here

Oregon Public Broadcasting
November 13, 2023
University of Washington research could lead to ‘living’ dental fillings made from real enamel
Listen to the story here

The Economist (Requires Subscription)
August 16, 2023
Scientists want to fix tooth decay with stem cells
Read the story here