Researchers use light to coax stem cells to regenerate teeth

Low-level light therapy confirmed
May 29, 2014

Regenerating a tooth. The team used high-resolution x-ray imaging and microscopy techniques to assess the formation of reparative (tertiary) dentin 12 weeks after low-power laser treatment. In the microscopy images shown here, the yellow hashtags (#) sit atop the newly-formed tertiary dentin; there is more tertiary dentin in the laser-treated teeth (below) than in the control (above). (Credit: Harvard’s Wyss Institute and SEAS)

A Harvard-led team is the first to demonstrate the ability to use low-power light to trigger stem cells inside the body to regenerate tissue.

The research, reported in Science Translational Medicine and led by Wyss Institute Core Faculty member David Mooney, Ph.D., lays the foundation for a host of clinical applications in restorative dentistry and regenerative medicine more broadly, such as wound healing, bone regeneration, and more.

Lasers trigger dentin formation

The team used a low-power laser to trigger human dental stem cells to form dentin, the hard tissue that is similar to bone and makes up the bulk of teeth. They outlined the precise molecular mechanism involved and demonstrated its prowess using multiple laboratory and animal models.

A number of biologically active molecules, such as regulatory proteins called growth factors, can trigger stem cells to differentiate into different cell types. Current regeneration efforts require scientists to isolate stem cells from the body, manipulate them in a laboratory, and return them to the body — efforts that face a host of regulatory and technical hurdles to their clinical translation.

But Mooney’s approach is different and, he hopes, easier to get into the hands of practicing clinicians. “Our treatment modality does not introduce anything new to the body, and lasers are routinely used in medicine and dentistry, so the barriers to clinical translation are low,” said Mooney, who is also the Robert P. Pinkas Family Professor of Bioengineering at Harvard’s School of Engineering and Applied Sciences (SEAS). “It would be a substantial advance in the field if we can regenerate teeth rather than replace them.”

The team first turned to lead author and dentist Praveen Arany, D.D.S., Ph.D., who is now an Assistant Clinical Investigator at the National Institutes of Health (NIH). At the time of the research, he was a Harvard graduate student and then postdoctoral fellow affiliated with SEAS and the Wyss Institute.

Arany took rodents to the laboratory version of a dentist’s office to drill holes in their molars, treat the tooth pulp that contains adult dental stem cells with low-dose laser treatments, applied temporary caps, and kept the animals comfortable and healthy. After about 12 weeks, high-resolution x-ray imaging and microscopy confirmed that the laser treatments triggered the enhanced dentin formation.

Low-level light therapy confirmed

Next the team performed a series of culture-based experiments to unveil the precise molecular mechanism responsible for the regenerative effects of the laser treatment. They found that the laser first induced reactive oxygen species (ROS), which are chemically active molecules containing oxygen that play an important role in cellular function. The ROS then activated a regulatory cell protein called transforming growth factor beta-1 (TGF-β1), which, in turn, differentiated the stem cells into dentin.

Nailing down the mechanism was key because it places on firm scientific footing the decades-old anecdotes about low-level light therapy (LLLT), also known as Photobiomodulation (PBM).

Since the dawn of medical laser use in the late 1960s, doctors have been accumulating anecdotal evidence that low-level light therapy can stimulate all kind of biological processes including rejuvenating skin and stimulating hair growth, among others. But the same laser can also be used to ablate skin and remove hair — depending on the way the clinician uses the laser.

The clinical effects of low-power lasers have been subtle and largely inconsistent. The new work marks the first time that scientists have gotten to the nub of how low-level laser treatments work on a molecular level, and lays the foundation for controlled treatment protocols.

“The scientific community is actively exploring a host of approaches to using stem cells for tissue regeneration efforts,” said Wyss Institute Founding Director Don Ingber, M.D., Ph.D., “and Dave and his team have added an innovative, noninvasive and remarkably simple but powerful tool to the toolbox.”

Next Arany aims to take this work to human clinical trials. He is currently working with his colleagues at the National Institute of Dental and Craniofacial Research (NIDCR), which is one of the National Institutes of Health (NIH), to outline the requisite safety and efficacy parameters. “We are also excited about expanding these observations to other regenerative applications with other types of stem cells,” he said.

This work was a collaboration between the Wyss Institute, Harvard SEAS, Harvard School of Dental Medicine, Leder Human Biology and Translational Medicine, National Institute of Dental and Craniofacial Research (NIDCR) and NIH, Wellman Center for Photomedicine at Massachusetts General Hospital, Harvard Medical School’s Department of Dermatology, Harvard-MIT Division of Health Science and Technology, Boston Children’s Hospital, and New York University School of Medicine. It was funded by the Wyss Institute, Harvard Presidential Scholarship, Harvard Catalyst, Harvard Clinical and Translational Science Center, NIDCR and NIH.

Abstract of Science Translational Medicine paper

Rapid advancements in the field of stem cell biology have led to many current efforts to exploit stem cells as therapeutic agents in regenerative medicine. However, current ex vivo cell manipulations common to most regenerative approaches create a variety of technical and regulatory hurdles to their clinical translation, and even simpler approaches that use exogenous factors to differentiate tissue-resident stem cells carry significant off-target side effects. We show that non-ionizing, low-power laser (LPL) treatment can instead be used as a minimally invasive tool to activate an endogenous latent growth factor complex, transforming growth factor–β1 (TGF-β1), that subsequently differentiates host stem cells to promote tissue regeneration. LPL treatment induced reactive oxygen species (ROS) in a dose-dependent manner, which, in turn, activated latent TGF-β1 (LTGF-β1) via a specific methionine residue (at position 253 on LAP). Laser-activated TGF-β1 was capable of differentiating human dental stem cells in vitro. Further, an in vivo pulp capping model in rat teeth demonstrated significant increase in dentin regeneration after LPL treatment. These in vivo effects were abrogated in TGF-β receptor II (TGF-βRII) conditional knockout (DSPPCreTGF-βRIIfl/fl) mice or when wild-type mice were given a TGF-βRI inhibitor. These findings indicate a pivotal role for TGF-β in mediating LPL-induced dental tissue regeneration. More broadly, this work outlines a mechanistic basis for harnessing resident stem cells with a light-activated endogenous cue for clinical regenerative applications.