Research at Penn Dental Medicine by Penn Dental Medicine

RESEARCH

Penn Dental Medicine

| 2024

ADVANCING HUMAN HEALTH

I Mark S. Wolff, DDS, PhD Morton Amsterdam Dean

Dana T. Graves, DDS, DMSc Vice Dean for Scholarship and Research

n 1889, Willoughby Dayton Miller (1853-1907) published a book called Microorganisms of the Human Mouth. A graduate of Penn Dental Medicine’s inaugural class of 1879, Miller was the first to establish the current bacterio-chemical theory of dental caries. He also helped confirm the relationship between oral sepsis and systemic disease. It was landmark research that set the course for dentistry to move from being simply reparative in nature, to a discipline aimed at biologically preventing and reversing disease, be it in the oral cavity or other locations in the body. This issue of Research at Penn Dental Medicine continues telling the story of our 130-year history of discovery and invention. Today, our School’s research enterprise spans multiple scientific health disciplines with a persistent drive to translate new knowledge into clinical therapies that positively impact the understanding of both oral and systemic diseases. We applaud all the research teams at Penn Dental Medicine for their intellectual curiosity, fervor, and devotion to this work. In recent years, along with growth in extramural support, we have continued to build on the strength and depth of our investigations through the recruitment of highly successful research faculty. The faculty and graduate students at Penn Dental Medicine, collaborating with faculty throughout our University and the world, are making vital contributions to understanding a host of diseases and conditions that limit human potential, including cancer, diabetes, autoimmune diseases, afflictions related to developmental disorders and trauma, or related to inevitable human aging. In addition, they are moving scholarship forward with an increase in publications across clinical and basic science disciplines. This work is often done in collaboration with other schools at Penn, partnerships we value profoundly.

Over the last ten years, Penn Dental Medicine has transformed our clinical facilities and greatly expanded our community outreach. Looking ahead, our goal is to similarly transform our research enterprise through exciting new facilities, recruitment of additional world-class scientists, adding new postgraduate educational opportunities, and by more fully leveraging partnerships with others at Penn and beyond. We will seek to connect with individuals who embrace Penn’s recently published strategic framework, In Principle and Practice, which calls for us to be The Inventive University, addressing the great challenges and opportunities of our time.

We applaud all the research teams at Penn Dental Medicine for their intellectual curiosity, fervor, and devotion. In the words of Dr. Eric Stoopler, Professor of Oral Medicine (page 36), “It is often the dentist who makes the connection between oral and systemic conditions, which starts the patient down the path toward effective management.” Penn Dental Medicine continues its commitment to improving the health of humanity globally, seeking solutions to complex social and physical health-related problems. The foregoing discussion of our research is only the beginning of the many discoveries Penn Dental Medicine will contribute to the goal of advancing human health. Remember, in the words of former Surgeon General C. Everett Koop, “you’re not healthy without good oral health!”

INSIDE Centers

Antiviral Therapeutics

4 Bone & Soft Tissue Healing

Anti-inflammation Therapeutics

10 Student Research

Dental Diagnosis & Treatment

RESEARCH AT PENN DENTAL MEDICINE: Vol. 1, No. 1 University of Pennsylvania School of Dental Medicine www.dental.upenn.edu

Research at Penn Dental Medicine is published by the Office of Institutional Advancement.

Dean: Mark S. Wolff, DDS, PhD

Contributing Writers: Beth Adams, Katie Cottingham, Judy Hill, Elizabeth Ketterlinus, Wynne Parry

Vice Dean for Scholarship and Research: Dana T. Graves, DDS, DMSc Vice Dean of Institutional Advancement: Elizabeth Ketterlinus

Director, Publications: Beth Adams

ON THE COVER: Zooming in on a sample of human saliva reveals a cluster of microorganisms, including the yeast Candida albicans (blue) and the bacterium Streptococcus mutans (green), which secrete polymers called α-glucans (red). Penn Dental Medicine’s Dr. Zhi Ren and Dr. Michel Koo have shown that in samples from children with severe tooth decay, clusters such as this thrive on sugar, and are capable of ‘walking’ along tooth-like surfaces using the yeast’s long filaments as ‘legs.’ As part of Koo’s lab, Ren developed a new way to study oral pathogens using real-time microscopy. This image was selected by the Nature photo team as one of the “sharpest science images” in its September 2023 issue. (From the lab of Dr. Michel Koo, see story p. 26)

Centers Research is fundamental to Penn Dental Medicine’s mission of advancing oral health locally, globally, and nationally. The School’s distinguished Centers play a unique role in linking both internal and external collaborators to the innovative research underway at Penn.

Care Center for Persons with Disabilities Nearly one in four Americans live with a cognitive, physical, or developmental disability. The Care Center for Persons with Disabilities was established to provide hands-on opportunity for every DMD student to learn how to accommodate patients living with a variety of disabling conditions, from autism and Alzheimer’s to movement disorders and a host of medical complexities. The Center is also a site for clinical research. Embedded within the space is the Colgate Innovation Laboratory, designed to collaborate with Colgate scientists on the design and implementation of products to enhance oral care in people with disabilities. With patient and caregiver consent, scientists have the opportunity to observe how their products are used and receive feedback that can inform design refinements or new inventions. Faculty are also exploring ways to measure and reduce anxiety in patients through cognitive behavioral therapy and by using specialized equipment to capture and alert providers to biometric indicators of stress in their patients.

Center for Integrative Global Oral Health Oral diseases such as caries, periodontal disease, oral cancer, and craniofacial deformity are among the world’s most prevalent, preventable and/or treatable non-communicable diseases impacting the social and economic wellbeing of millions of people. The Center for Integrative Global Oral Health (CIGOH) seeks creative solutions to address these unmet oral health needs by providing critical infrastructure to develop and test effective interventions and policy at both the individual and health-system level. The Center conducts groundbreaking research on topics of integrative oral health, including implementation science, disease prevention, care delivery systems, and cost reduction. Through its novel graduate degree program, CIGOH is building research capacity in these topics here and around the world. The Center is promoting oral health literacy and improved care locally, globally, and nationally through entities like the Cochrane Oral Health Collaborating Center at Penn Dental Medicine. Cochrane is a global independent not-for-profit network that produces systematic reviews across all areas of medicine. Cochrane has more than 37,000 contributors from over 190 countries working together to produce up-to-date, accessible health information that is free from commercial sponsorship and other conflicts of interest to help inform decisions about healthcare. The Collaborating Center at Penn Dental Medicine engages researchers from around the globe in systematic reviews summarizing the best available evidence on oral health topics to help clinicians, policymakers, patients, and caregivers make well-informed decisions.

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Center for Clinical and Translational Research The Center for Clinical and Translational Research (CCTR) provides centralized support to researchers within Penn Dental Medicine who conduct human subject research, advancing the growth of clinical research studies within the School while also mentoring junior faculty and students to excel as clinician scientists. The CCTR team works with faculty through all phases of clinical research, from study design and IRB submission to recruitment, retention, and compliance with study protocols. CCTR has facilitated dozens of clinical studies in areas such as caries prevention and control and pain management, as well as projects that aim to improve outcomes for patients living with HIV, cancer, autoimmune disease, and other serious medical conditions. Located in the Robert Schattner Center, the CCTR includes dental bays for seeing patients in active studies, a wet lab, a DEA Schedule I substance compliant research pharmacy room, and the CCTR team offices.

Center for Innovation & Precision Dentistry This vibrant community of scientists is a joint enterprise between Penn Dental Medicine and Penn’s School of Engineering & Applied Sciences. Through this cross-disciplinary research partnership, the Center for Innovation & Precision Dentistry (CiPD) aims to accelerate discovery and translation of new therapies, diagnostics, and devices to address unmet needs in oral health. From developing new biomedical tools, to finding precise means to study and prevent oral-craniofacial disorders, the CiPD fosters innovation in dental medicine, engineering, and the applied sciences through research, entrepreneurship, and education. The CiPD is training the next generation of dentists, scientists, and engineers through an NIH/NIDCR-sponsored postdoctoral training program as well as fellowships from industry. Faculty and trainees are exploring innovations in areas such as nanotechnology and microrobotics, host immunity and tissue regeneration, complex microbiomes, and low-cost chloroplast therapeutics. Through these technologies, the CiPD is striving to make oral healthcare more effective, more affordable, and in turn, more accessible to patients here and around the world.

Digital Dentistry Centers Penn Dental Medicine is squarely on the leading edge of the digital age of dentistry. The School’s two state-of-the-art centers — the Digital Design and Milling Center and the Center for Virtual Treatment Planning — serve as both the cornerstones and building blocks of Penn Dental Medicine’s commitment to the future of digital dentistry, as the School continues to enhance its global leadership and expertise in the field. The centers provide a full range of technology and equipment to digitally plan restorations, develop virtual treatment plans for complex cases, and manufacture restorations on-site, opening up new avenues for education, research, and patient care. There is little doubt that digital technology will continue to play an outsized role in the future of dentistry, and research at Penn Dental Medicine is helping to determine what that future may look like. Companies in this arena continue to turn to Penn Dental Medicine for feedback on dental materials and the CAD/CAM equipment and software moving the field forward.

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Antiviral Therapeutics With the ability to target specific viral components or interfere with essential viral processes, antiviral therapeutics play a pivotal role in both the treatment and prevention of viral diseases. Their development involves a deep understanding of viral biology, host-pathogen interactions, and the identification of vulnerable targets. Research in this realm not only reveals the molecular intricacies of viruses but also paves the way for innovative therapeutic interventions, which researchers at Penn Dental Medicine are moving forward as its relates to SARS-CoV-2, herpes simplex virus-2, a viral skin disease, and more. Two antiviral therapies developed through research from Penn Dental Medicine are currently in clinical trials. In addition, researchers at the School are studying how some antiretroviral therapies for HIV are impacting other aspects of patients’ health, from their oral health to neurocognitive impairment.

Neutralizing SARS-CoV-2 Using Plant-Based Platform

he plants growing in Penn Dental Medicine’s greenhouse on the Penn Center for Innovation campus are far from your garden-variety greens. Packed with human genes, these specially engineered plants offer a novel platform for producing and delivering life-saving medicines, including antivirals. “I grew up in a developing country and saw people die because they couldn’t afford drugs or vaccines,” says Henry Daniell, ViceChair and W.D. Miller Professor in the Department of Basic & Translational Sciences, who developed this innovative process and has over 150 related patents. “For me, affordability and global access to healthcare are the foundation for my work.” This game-changing plant-based drug production platform introduces a gene of interest into lettuce cells, prompting them to express that gene and eventually produce its protein in lettuce plants. The leaves can then be harvested, freeze-dried, and packaged into capsules, or potentially even added to gum, for oral therapy. This process is vastly different from the traditional way of making many current drugs and vaccines, which involves growing molecules and viruses in microbial cells or eggs, expensive processes requiring many steps and a low temperature for transportation and storage. Daniell’s method eliminates the need for costly complex laboratory equipment and results in a product that is shelf-stable at room temperature.

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Trapping, Neutralizing SARS-CoV-2 One recent target of Daniell’s plant-based technology is SARS-CoV-2. In collaboration with Penn Medicine, a clinical trial is now underway to evaluate a gum designed to trap SARS-CoV-2 in the saliva, potentially blocking transmission of COVID-19 from one person to another. The technology behind this experimental treatment has moved from the laboratory bench toward the beginning of clinical application, all within Penn Dental Medicine, a first for the school. Prior to the pandemic, one of the proteins Daniell had explored was ACE2 for its impact on pulmonary hypertension. As the pandemic began, ACE2 began receiving attention for another reason — its receptor provides the docking station for the spike protein of SARS-CoV-2. Daniell had already been working on protein drug delivery via a chewing gum, and, given that the SARS-CoV-2 virus replicates in the salivary glands, delivering ACE2 to the mouth with a gum appeared to him to be a potentially powerful approach. The experimental gum now in trial contains plant-derived material genetically engineered to contain ACE2. In experimental models, chewing the gum released the embedded ACE2, blocking the interaction of the chewer’s own ACE2 receptor with the viral spike protein. In effect, the gum is designed to trap and neutralize SARS-CoV-2 in the saliva and diminish the amount of virus left in the mouth. A preclinical study found that the gum neutralized the virus in patient samples and reduced the viral load to nearly undetectable levels. It is hoped that less virus would mean a lower likelihood of passing the infection on to others. The gum could be particularly helpful for people in countries that don’t have the resources to benefit as broadly from the current vaccines and other therapies. The team also is working on another gum that contains a protein for targeting a broader range of respiratory viruses, including influenza.

An FDA-approved freeze drier in the Daniell lab prepares the ACE2 plants for incorporating into the gum tablets.

Booster Vaccines, Insulin and More Daniell’s plant-based platform is being applied to a host of other conditions as well. His lab has grown proteins to develop booster vaccines against polio, tuberculosis, malaria, and cholera. In addition, animal models showed promise for delivering proteins to degrade the plaque of Alzheimer’s disease and to speed healing of bone fractures. The team also has grown insulin in lettuce for oral delivery. Insulin currently requires cold storage and an injection, which clinical studies show can cause the hormone to reach the bloodstream so quickly that hypoglycemia, or low blood sugar levels, can result. In addition, the insulin that patients have been using for decades is actually missing one of the three peptides found in naturally occurring insulin. The Daniell lab created a plant-based insulin that not only can be ingested orally, but also contains all three peptides. In a recent study using diabetic mice, their insulin regulated blood sugar within 15 minutes of ingestion, similar to how naturally secreted insulin works. This platform is advancing care for hemophilia as well. Patients with this disease regularly receive injections of a clotting factor to prevent excessive bleeding, but 20 to 30 percent of them develop antibodies against it. Daniell encapsulated a therapy in plant cells that prevents the formation of these harmful antibodies. Zea Biosciences has licensed Daniell’s hemophilia patents and is conducting toxicology studies in preparation for seeking FDA approval. “If you have something that saves lives, you have an obligation to make it available to everyone,” says Daniell on his mission to lower drug costs and improve drug accessibility globally. “We won’t solve the high cost of pharmaceuticals unless we solve the cost of production and delivery.”

“We won’t solve the high cost of pharmaceuticals unless we solve the cost of production and delivery.” — Dr. Henry Daniell Daniell's plant-based platform introduces a gene of interest into lettuce cells prompting them to express the gene and produce the protein in the lettuce leaves.

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Antiviral Therapeutics

Treating Disease by Exploiting a Viral Vulnerability Molluscum is a viral skin disease that can appear as lesions on the eyelids, mostly in children and immunecompromised people.

ometimes, researchers start out pursuing a line of investigation out of pure curiosity, and later find themselves on the path to a new therapeutic. Such is the case in current work underway on a potential drug to treat molluscum contagiosum, a skin condition with no specific antiviral approved treatment. Molluscum is a viral skin disease that appears as lesions on the face and body, including the eyelids and conjunctiva, mostly in children and immune-compromised people. With a curiosity for processivity factors — key molecules present in every cell of virtually every living organism and pathogen — Robert Ricciardi, Chair and Professor of Basic & Translational Sciences, hit upon a potential way to treat a variety of viral diseases, including molluscum. These molecules are essential for DNA replication, and without them, a virus can’t multiply and cause infection. Ricciardi realized that drugs that specifically inhibit viral processivity factors — and not the human versions of the protein — could attack the infection. The Ricciardi lab and collaborators from Fox Chase Therapeutics Diversity, Inc. (FCTDI) teamed to develop a therapeutic strategy using processivity factors to target molluscum. A major hurdle in developing a therapeutic for the disease was that the virus couldn’t be grown in culture, so the team engineered a surrogate virus containing the molluscum processivity factor. By taking a structure-guided approach, based on biophysics and mutational analysis, they synthesized a small molecule that binds to the molluscum processivity factor. A dermatology company licensed the compound in 2022 and is now performing preclinical testing with the goal of producing the very first approved drug in the form of a topical therapeutic, which is specifically aimed at blocking the molluscum virus. Ricciardi and his FCTDI collaborators are also working on applying the approach of blocking virus’s activity via their processivity factor to ocular herpes keratitis, the most common form of infectious corneal blindness.

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A curiosity for processivity factors — molecules present in every living cell — hit upon a potential way to treat viral diseases, including molluscum, a viral skin disease.

3-D structure of Herpes simplex virus glycoprotein gD, the major component of the vaccine in clinical trial.

Bringing Years of Discovery to a Herpes Vaccine Now in Clinical Trial According to the World Health Organization, about 500 million people globally are affected by genital infection caused by herpes simplex virus-2 (HSV-2), with painful genital lesions, an increased risk for HIV, and high level of emotional distress. Once acquired, HSV persists for a patient’s entire life with recurring symptomatic outbreaks. Currently available HSV therapies only reduce the severity and frequency of symptoms, and there is no vaccine for the disease — that could soon change. A first-in-human Phase 1 clinical trial of an mRNA vaccine to prevent genital lesions caused by HSV-2 is now underway. In collaboration with Penn Medicine’s Harvey Friedman and Drew Weissman and BioNTech, Penn Dental Medicine’s Gary Cohen, Professor in the Department of Basic & Translational Sciences, was involved in conducting the discovery science that helped lead to the launch of the BioNTech-Penn trial. Still in its early stages, it has been dosing patients since late 2022. mRNA-based vaccines gained notoriety during the COVID-19 pandemic as the fastest way to produce a vaccine against a virus that was quickly causing a global public health crisis. Although researchers had been working on this Nobel Prize-winning

technology for years, Cohen remembers that 2020 was the year that these vaccines really entered the spotlight. “You could not have made a vaccine with protein in any timeframe that could have made a dent in this pandemic,” he says. Cohen knows a thing or two about making vaccines with proteins — he has devoted much of his career to studying HSV-1 (oral disease) and HSV-2 (genital disease) proteins with the ultimate goal of producing a prophylactic vaccine against these two viral infections. He also knows how tough it can be. To be used in a cost-effective vaccine, a protein must be easily and inexpensively made in vast amounts and be very pure, which is a tall order. However, animals injected with a small amount of mRNA against the same protein will do all that work on their own, he says. Their cells will translate the nucleic acid sequence and produce large amounts of pure protein against which the immune system can then mount a response. After years of intense work, including 10 years of support through the National Institutes of Health’s highly selective MERIT Award, Cohen’s team determined that four viral glycoproteins, called gD, gB, gH, and gL, are responsible for getting HSV into cells

through their interactions with a few different receptors. In close collaboration with Friedman, the team determined that three HSV-2 viral glycoproteins, gD, along with proteins called gC and gE, would be a good combination for use in a vaccine. After showing positive results in mice and guinea pigs, these three proteins are now represented as mRNAs in the vaccine currently being tested in the clinical trial.

The mRNA vaccine for genital herpes currently in a Phase 1 clinical trial includes three of the four proteins identified by Cohen’s lab as responsible for getting the herpes virus into cells. For Cohen, the work just keeps going. He is currently studying the antibodies that are produced during a natural infection and vaccination to see which specific viral proteins the body is responding to. “We’re dissecting the antibodies generated to each one of these glycoproteins,” he says, “in an attempt to ask which ones are important for neutralization and for protection.” 2 024 | RESEARCH AT PENN DENTAL MEDICINE

Antiviral Therapeutics

At the Intersection of HIV and Oral Health

or people living with HIV, antiretroviral therapy (ART) — a combination of medications that suppress the virus and improve immune function — is lifesaving, but like all medications, it has side effects that may be compromising health in other ways, including oral health. Current research at Penn Dental Medicine on the potential impact of these therapies on oral health is being led by Temitope Omolehinwa, Assistant Professor of Oral Medicine — a study that sprang directly from her own clinical observations. Within the School’s Care Center for Persons with Disabilities, which also takes care of the dental needs of patients with medical complexities and infectious diseases, Omolehinwa cares for many patients who are living with HIV. Within this group, she found that she was seeing more systemic conditions — hypertension, diabetes, hyperlipidemia — than in the general population. She also saw a higher rate of dental caries (cavities). “That piqued my interest,” says Omolehinwa. “Day in and day out I was asking, ‘Why am I seeing these trends? What’s the link between what’s going on in the general body system and what’s going on in the mouth?’” The quest for answers led to her current longitudinal study on the association between oral and systemic health in patients with HIV on ART. Omolehinwa’s hypothesis was that patients with noninfectious comorbid conditions will present with higher rates of oral diseases, such as caries and periodontitis, as well as higher degrees of salivary gland hypofunction, leading to changes in the quality and quantity of saliva produced. Now entering its fourth year, the study follows each participant for two years. Patients are tracked for dental caries, periodontal health, and oral precancerous lesions or oral cancer. Preliminary analysis shows that dental caries is indeed more prevalent in patients with HIV, and those with comorbid conditions are more likely to have caries. Omolehinwa is not finding a similar associa-

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tion so far with periodontitis and analysis on soft tissue lesions has yet to be completed. Omolehinwa hopes to extend the study for another year and also to expand it to more African American/African communities. “If you look at the statistics,” she says, “about 38 million people worldwide are living with HIV and out of those, about 28 million are in sub-Saharan Africa. If I look at my population here in Philadelphia, 60 to 70 percent of my patients are Black American males. It’s worth exploring further what is happening in that population.” In another line of study, Omolehinwa is exploring the potential association between the oral and mental health status of people with HIV who are on ART. Dr. Temitope Omolehinwa is This pilot study just recently began studying the association between oral and systemic health of HIV recruiting participants. At the end of the day, she says, the patients on antiretroviral therapy. purpose of research is to change lives. “I look at my HIV population and they want answers to why they are experiencing these changes to their bodies. Even though they’re living longer, all these other non-HIV-associated comorbid conditions that they’re developing are affecting their quality of life.” Finding answers, she says, could influence policy around treatment modalities and ultimately lead to prioritizing medications that have less side effects on systemic health. Even as she pursues her research, “I am forever a clinician,” says Omolehinwa. “I love my patients and I love being able to give them answers and see them get better. They motivate me to do my research.”

Protecting Brain Health in HIV Infection People living with HIV run the risk of developing neurocognitive problems ranging from difficulty with attention to full-on dementia. Both the virus and the life-preserving drugs used to suppress it may contribute to this deterioration. Kelly Jordan-Sciutto, Professor in the Department of Oral Medicine, investigates how HIV infection and antiviral medication provoke inflammatory responses that harm brain cells. Findings from her studies point toward ways to protect the brain. In one such recent project, she and colleagues identified a mechanism by which the HIV drug bictegravir interferes with the fatty insulation that neurons need to efficiently send signals. This research, conducted in collaboration with Lindsay Festa, a research associate in the Department of Oral Medicine, and Judith Grinspan, at Children’s Hospital of Pennsylvania, revealed

“Most disease-causing mutations have already been discovered, but we still need to understand the minor variations that make different populations uniquely vulnerable.” — Kelly Jordan-Sciutto

that bictegravir impaired the development of oligodendrocytes, brain cells that produce this insulation, which is known as myelin. This impairment, Jordan-Sciutto explains, may be responsible for some of the cognitive problems that people with HIV may experience. The team’s experiments indicated that bictegravir, like other antiretroviral drugs, reduces the acidity in lysosomes, membranebound structures that contain digestive enzymes. This change, their findings suggest, interferes with the process required to insulate and protect neurons. “If we could maintain the drug’s ability to suppress the virus and distribute properly within the body, while preserving the pH of the lysosome, that would provide a powerful benefit to people with HIV,” Jordan-Sciutto says. Her lab is working on just such an approach. The unintended consequences of the antiretrovirals aren’t the only problem. The viral infection itself also blocks the maturation of oligodendrocytes. In this case, it does so indirectly. Although HIV doesn’t infect oligodendrocytes, it does invade immune cells within the brain. These infected cells release inflammation-promoting signals and other substances, which activate an enzyme known as PERK. PERK governs a stress response pathway, and one variation in the gene that encodes it has been linked to cognitive impairment in people with HIV. Jordan-Sciutto’s lab is now investigating the mechanism

TOP: Co-culturing stem cell-derived astrocytes, microglia, and neurons in one dish provides an effective model to study intercellular impact of HIV and other diseases of the nervous system.

ABOVE: Mature oligodendrocytes

(magenta) have lysosomes (green) in the cell body and to a lesser extent in the processes. Nuclei in blue.

behind this change. They have so far learned that it alters PERK’s activity in certain brain cells, rendering them less resistant to chronic stress. “Most disease-causing mutations have already been discovered, but we still need to understand the minor variations that make different populations uniquely vulnerable,” she says. Such research could help identify those people prone to suffer from HIV-related cognitive decline before it sets in. It could potentially do the same for other disorders that involve this stress response pathway, such as diabetes and the neurological effects of COVID-19. In other research, Jordan-Sciutto is studying how certain cannabinoids, compounds found in the cannabis plant, may have anti-inflammatory or antiviral properties that prevent neurodegeneration. She and her lab are also investigating the neurocognitive risk faced by young people who take antiretrovirals to prevent HIV infection. The results of this project could suggest the safest approaches for these preventative regimens. In addition, her group is working to better understand within which brain cells the virus hides out while someone is on antiretroviral therapy, and its level of activity and impact within them.

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Anti-inflammation Therapeutics Inflammation, a complex biological response to harmful stimuli, is part of the body's defense mechanism. While acute inflammation serves as a protective and reparative process, chronic inflammation has emerged as a critical factor underlying a wide spectrum of health issues, including autoimmune conditions, neurodegenerative ailments, and more. As a result, research on inflammation has the potential to have profound implications on overall health. Understanding the molecular pathways and cellular mechanisms involved in inflammation holds the key to developing targeted therapeutic interventions, and Penn Dental Medicine researchers are contributing to that understanding through diverse lines of study, ranging from periodontal disease and degenerative eye disease to cancer and fibrosis, among others. Their work not only provides insight into disease etiology but also lays the groundwork for translating science into innovative therapeutic strategies.

Targeting Inflammatory Processes in Periodontal Disease and Beyond

n periodontal disease, certain bacteria in the mouth kick off an inflammatory response that can destroy the gums and connective tissue. In its most severe form, known as periodontitis, out-of-control inflammation can cause the loss of underlying bone and teeth. A drive to understand the inflammatory mechanisms involved in periodontal disease is leading George Hajishengallis, the Thomas W. Evans Centennial Professor in the Department of Basic & Translational Sciences, to new insights that could advance treatment not only of this common oral disease, but many others associated with inflammatory processes. In one line of study, Hajishengallis and colleagues discovered that the bacterium long believed to be the main culprit in periodontal disease — Porphyromonas gingivalis — is in fact a “keystone pathogen,” meaning that its presence sets the stage for other pathogens to flourish. To survive and feed, it exploits the functioning of complement, a part of the innate immune system, by hijacking certain receptors on white blood cells. This subversion of the immune system benefits not just P. gingivalis but allows a wide range of other bacteria living in the mouth to multiply, causing inflammation to go wildly out of control.

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To intervene, he and his team targeted the C3 component of complement, which is a main player in signaling pathways that trigger inflammation and activate the immune system. In experiments with nonhuman primates, the team found they could interfere with the inflammation and the bone loss associated with periodontitis by blocking C3 with an inhibitor developed by collaborator John Lambris of Penn Medicine. This research became the basis for an experimental treatment. A refined version of the C3 blocker, called AMY-101, is now showing promising results in clinical trials. Most recently, in a small safety and efficacy study, three locally administered doses of AMY-101 within two weeks reduced gum inflammation and signs of tissue destruction. These benefits persisted for at least three months after initiating treatment. Blocking the C3 component of complement, part of the immune system, interfered with the inflammation and bone loss associated with periodontitis.

Training the Immune System, for Good and Ill

A Protective Factor in the Mouth and Beyond

Periodontitis sometimes brings company. This inflammatory condition may increase risk for others, including cardiovascular disease, diabetes, rheumatoid arthritis, and Alzheimer’s disease. Hajishengallis’ research has also uncovered a connection via an overlooked type of immunological memory — a discovery that has wide-ranging implications for addressing many conditions. The adaptive arm of the immune system, which contains T and B cells, is well known for forming memories of pathogens and using them to launch targeted responses should those germs show up again. Only within roughly the past decade have researchers, including Hajishengallis, shown that its counterpart, the innate immune system, which includes the inflammatory response, can also remember, albeit without the same specificity and for shorter periods. Working with colleagues in Germany, his group pinpointed where these memories resided, tracing them to the bone marrow, where the stem cells that generate blood cells reside. They also “trained” the innate immune system with β-glucan, a compound derived from fungus. Exposure to β-glucan primed cells known as neutrophils to prevent or attack malignant tumors in an animal model. A similar training approach could potentially be used to boost protection against newly emerged pathogens for which no vaccine is available, according to Hajishengallis. While some potential therapies could enhance trained immunity, others would seek to restrain it, he says. “Nothing in the immune system is 100 percent protective. There is always a dark side.” To illustrate this, he and colleagues gave mice bone marrow transplants from animals that had periodontitis. When subjected to experimental inflammatory arthritis, these transplant recipients developed more severe disease than their counterparts did. Their work demonstrated that periodontitis had trained the immune system to overreact, which increases the risk for other inflammation-related conditions. “Trained immunity is the reason why we have comorbidities, that is, inflammatory diseases occurring together, most often in older people,” says Hajishengallis.

His study of periodontitis has also led Hajishengallis to another factor shared with other diseases: the protein DEL-1. While at the University of Louisville, he found that DEL-1 became much less abundant in the gums of older animals, who are more prone to periodontitis. Later at a conference, he met a future collaborator, Triantafyllos Chavakis, present work showing DEL-1 restrained the recruitment of inflammatory cells. In the years since, he and Chavakis, now at Germany’s Technical University Dresden, have shown that DEL-1 protects against periodontitis, multiple sclerosis and rheumatoid arthritis, not only by inhibiting the initiation of inflammation, but also by helping to resolve this response after it flares up. “DEL-1 is a molecule that helps us understand a lot of the biology,” he says, “not only in periodontal disease, but also in other diseases.”

“Trained immunity is the reason why we have inflammatory diseases occurring together, most often in older people.” — George Hajishengallis

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Anti-inflammation Therapeutics

Intervening in Inflammation and Cellular Aging Dr. Esra Sahingur (right) is studying the mechanisms linking inflammation and aging in the mouth.

Image showing the gum tissue from an older mouse lacking TLR9 immune receptor.

Inflammation and cellular aging go hand in hand, with one feeding the other. This reciprocal relationship shows up in periodontal disease: Nearly 60% of people 65 and older have some form of this inflammatory condition, which affects the soft tissue and bone around teeth. Exploring the connection between these two processes has led researchers to a possible means for intervening, and stopping the damage that can lead to bone and tooth loss. A team led by Esra Sahingur, Associate Professor in the Department of Periodontics, is exploring the mechanisms linking inflammation and aging in the mouth, as well as testing natural products known as flavonoids against both processes, in the hopes of improving oral health, and perhaps, well-being in general. Widely found in fruits and vegetables, flavonoids can exert a broad spectrum of health-promoting effects. Sahingur’s study of flavonoids has arisen from her research on an immune pathway that activates — then calms — the inflammatory response. An immune sensor known as toll-like receptor 9 (TLR9), emits “danger signals” that trigger this pathway when it detects foreign RNA and DNA, a sign of stress. While the connection to inflammation was already well established, experiments in Sahingur’s lab have established a link between TLR9 and aging too. Mice lacking TLR9 did not display the same age-associated inﬂammatory response and bone loss resulting from periodontal disease as did mice with TLR9. Meanwhile in humans, older patients with gum disease had elevated expression of TLR9. Over time, the effects of cellular aging and inflammationpromoting danger signals build up, leading TLR9 to become more active and increase inflammation.

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“This is how we age, right?” Sahingur says. “Alterations to our metabolism, organ dysfunction, everything associated with aging is because of the accumulated wear and tear.” However, cells possess the means to bring inflammation under control as well, and this is where her team identified a flavonoid to support that process. Her lab has shown that an enzyme named A20, part of cells’ system for regulating several key biological functions, puts the brakes on inflammation initiated by TLR9 and other immune sensors in gum tissue, A search for compounds that boost A20 led them to a flavonoid called quercetin, a plant pigment with anti-oxidant and antiinflammatory properties. When the team fed quercetin to mice with periodontitis, they found it reduced inflammation and bone loss. Studies have also linked malfunctions in A20 to many conditions, including cancer. In ongoing research, her group continues to study how A20 functions, and what goes wrong with it during periodontal disease and other related conditions. They also uncovered a positive feedback loop between the disease and cellular aging: Periodontitis appeared to drive accelerated cellular aging in gum tissue while aging-related changes contributed to the disease. In addition to quercetin, the team is testing the ability of other flavonoids either alone or in combination with pharmaceutical agents, to protect periodontal health by reducing inflammation and the number of aged cells in gum tissues. The inflammation that comes with periodontitis can have consequences elsewhere in the body, a connection her group is exploring. In one of their studies, treating periodontitis improved measures related to cirrhosis, an inflammatory and degenerative liver condition. Sahingur notes that the flavonoids her lab is studying also show promise for potential therapeutics for cirrhosis and other diseases linked to periodontitis, such as Alzheimer’s and arthritis.

Leveraging Far-Flung Taste and Smell Sensors

he tastebuds of the tongue contain bundles of specialized cells bearing sensors that detect chemical attributes of food. Known as taste receptors, these sensors were long believed to reside only on the tongue. Marco Tizzano, an Associate Professor in the Department of Basic & Translational Sciences, was among those who discovered them in many places elsewhere in and on the body. He has since documented taste receptors throughout the airways, especially in the nose, but also the trachea and the lungs, as well as the gums and even dental pulp. These sensors, he and others have found, detect bacterial pathogens and allergens, then alert the innate immune system. Tizzano’s research has shown that bitter sensing taste receptors in the nose respond to a compound bacteria use to communicate. These sensors then activate the trigeminal nerve, a cranial nerve that provides sensation to the face, leading to a significant inflammatory response. Bitter taste receptors also show up in the gums. In experiments, Tizzano’s research team demonstrated that in gum tissue these receptors actually offer some natural protection against periodontitis, the most severe form of gum disease. After sensing pathogenic bacteria, they trigger the production of antimicrobial peptides to kill the germs. Without these receptors, mice suffered from more damaging periodontitis, the researchers found. In another line of study, Tizzano’s lab is looking at odor-detecting receptors found on neurons in dental pulp. When these relatives of the taste sensors detect eugenol, an odor molecule from cloves, they numb the teeth. As someone who looks for connections between disciplines, Tizzano ultimately seeks to use naturally occurring compounds, such as eugenol, to stimulate these Mouse circumvallate papilla filled with taste buds that are receptors in ways that benefit expressing the biomarker GFP. health. Such applications could include a new form of pain relief for dental procedures or a therapy to stifle the microbial growth triggering the inflammation that drives periodontal disease. “I think we are still at the tip of the iceberg,” Tizzano says. “There is so much more to discover about these receptors.”

Cdt, or cytolethal distending toxin, targets a signaling pathway within cells to cause distinct types of harm to the adaptive immune system and the outer layer of the tissue it infects.

Uncovering Bacterial Trickery More than 30 varieties of pathogenic bacteria rely on a particular chemical weapon to cause sustained infections and chronic, destructive inflammation in certain places within the body, such as the mouth. This bacterial toxin, known as Cdt, wreaks havoc by, among other things, turning the body’s defenses against it. To better understand how Cdt does its damage, a team led by Bruce Shenker, a Professor in the Department of Basic & Translational Sciences, is focusing on one such germ, Aggregatibacter actinomycetemcomitans (Aa). This bacterium causes periodontitis, in which an out-of-control inflammatory response attacks the gums and connective tissue of the mouth. Aa, like the other bacteria, produces Cdt, otherwise known as cytolethal distending toxin. This molecule targets a nearly universal signaling pathway within cells to cause distinct types of harm to the adaptive immune system and the outer layer of the tissue it infects. Not least of all, Cdt also disrupts the innate immune system, provoking inflammation and interfering with immune cells known as macrophages. Macrophages consume pathogens and other foreign matter, however, Cdt prevents them from killing the bacteria they have engulfed. In one line of research, Shenker and fellow Penn Dental Medicine faculty member Kathleen Boesze-Battaglia are investigating how Cdt achieves these effects, enabling Aa and other pathogens to survive the immune system’s onslaught. Shenker’s previous research has also shown that Cdt produced by periodontal pathogens attacks B and T cells, from the immune system’s adaptive arm. He and Boesze-Battaglia are building on this discovery by investigating how the toxin’s blockade of the signaling pathway leads these cells to self-destruct. In addition, they are looking for means to neutralize Cdt. These studies have the potential to shed light on the mechanisms at play in aggressive periodontitis and other chronic infectious and inflammatory disorders. In addition, their results could lead to the development of new therapies to protect against or intervene in disease caused by Cdt-producing bacteria. 2 024 | RESEARCH AT PENN DENTAL MEDICINE

Anti-inflammation Therapeutics

Exploring the Mysteries of Mast Cells Mast cells are found throughout the body close to blood vessels and are packed with granules containing the immune signaling compound histamine, and enzymes that attack pathogens and promote white blood cell recruitment. When triggered by a perceived threat, mast cells expel their contents. This activity protects against infection and aids healing, but it has also made mast cells infamous for driving allergic asthma and inflammatory skin disorders, such as rosacea and contact dermatitis. Despite their prominence, few researchers study these scarce and difficult-to-work-with cells. Hydar Ali, a Professor in the Department of Basic & Translational Sciences, belongs to a select group who focus on mast cells. His research explores how they function to protect the body and how things can go wrong. Mast cells (in red) are found throughout the body close to blood vessels (in green).

“These cells are relatively poorly understood, and yet we’ve been able to identify some of the most sought-after molecular targets to affect diseases like allergies and asthma that have the potential to kill,” Ali says. He and his colleagues discovered a receptor, known as MRGPRX2, that only appears on mast cells. This research led them to find that small proteins called antimicrobial peptides could activate mast cells through MRGPRX2 to harness their protective function and help clear pathogens. In the context of an allergic response, however, he has shown that this receptor drives problematic inflammation. “It’s two sides of the same coin,” Ali says. More recently, his team has investigated mast cells’ role in systemic allergic reactions that can lead patients to stop using the multiple sclerosis drug glatiramer acetate. Their experiments demonstrated that this medication activates MRGPRX2 receptors causing mast cells to expel histamine and their other contents, results suggesting MRGPRX2 inhibitors could treat these reactions and help patients remain on the medication. His lab is also investigating this receptor’s role in periodontal disease, an inflammatory condition that damages the gums and connective tissue of the mouth. Their experiments have shown, for example, that mast cells expressing MRGPRX2 become more abundant in chronic periodontitis, the more severe form of the condition. Ultimately, he hopes to study the potential for MRGPRX2 blockers to reduce inflammation in periodontitis.

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Watery Gels Shed Light on Inflammation

ydrogels, water-based gels with varied biomedical applications, have become a key focus of bioengineering research in recent years, with the burgeoning field transforming the way diseases are being studied and potentially treated. A dentist and engineer, Kyle Vining, Assistant Professor of Preventive & Restorative Sciences, is developing hydrogels in his lab to study inflammation. Specifically, his lab is investigating how physical cues control inflammation of white blood cells with the goal of developing new treatments for disease, ranging from cancer to fibrosis, the outcome of many chronic inflammatory diseases. Among his projects, he has developed a hydrogel matrix that mimics the physical properties of fibrosis to see how white blood cells interact with the surrounding tissue. “Fibrosis is a physical change in tissues that produces a scar-like matrix that can impair cancer treatment, inhibit healing, and in general is not compatible with tissue regeneration,” says Vining. “There’s been a lot of effort on antifibrotic drugs, but we’re looking at fibrosis differently. Instead of directly inhibiting fibrosis, we’re trying to understand its consequences for the immune system because the immune system can be hijacked and become detrimental for your tissues.” Through a better understanding of the feedback loop between fibrotic tissue and the immune system, Vining hopes to design interventions for treating head and neck cancer, including ways to boost efficacy of immunotherapies. This work also has possible applications for facilitating wound healing and tissue remodeling during restorative dental procedures. In other work, Vining and collaboraABOVE: A cryogenic scanning tors from Penn Engineering are gearing electron microscopy image of up to develop next-generation treata human white blood cell in a ments for tooth decay — a project hydrogel matrix. awarded the Center for Innovation & TOP: Dr. Kyle Vining Precision Dentistry and Penn HealthTech IDEA Prize in 2023. They are working on encapsulating mRNA inside lipid nanoparticles that can be delivered directly to dental pulp through its tubules, or pores. “Using mRNA, we can reprogram the cells to produce their own medication to help promote healing of the tooth and improve long-term survival of the tooth cells,” Vining says.

Protecting the Eye from Degeneration Our sense of sight relies on the retina, a layer of specialized cells at the back of the eye, which perceives light and transmits information about it. Age and disease can cause the retina to degenerate, leading to the loss of vision, with inflammation contributing to the deterioration, just as it does elsewhere in the body. Two Penn Dental Medicine faculty members, Kathleen BoeszeBattaglia and Claire Mitchell, take differing approaches to studying the inflammatory processes that can lead to degenerative eye conditions, such as age-related macular degeneration and glaucoma. Photoreceptors, the light-sensing cells within the retina, exchange nutrients with the layer of tissue, called the retinal pigment epithelium, behind them. The epithelium supplies the photoreceptors with glucose in return for fatty molecules and lactate, an energy rich compound involved in metabolism. As the eye ages, the fatty molecules, known as lipids, collect in the epithelium and elsewhere. These deposits can provoke a damaging inflammatory response that leads to age-related macular degeneration. Boesze-Battaglia, a Professor in the Department of Basic & Translational Sciences, studies the processes that prevent the harmful buildup of lipids. In one ongoing effort, her lab is examining how key enzymes and transporter proteins, which act as conduits across cell membranes, control this exchange. They are simultaneously investigating how the formation of particles of lipid-containing cholesterol

As the eye ages, the fatty molecules, known as lipids, collect in the epithelium and elsewhere. These deposits can provoke a damaging inflammatory response that leads to age-related macular degeneration. and their removal from cells might help control the deposit of toxic lipids between the epithelium and the photoreceptors and elsewhere. In both lines of research, she is looking at the consequences when something perturbs these processes, creating changes that can potentially lead to disease. Her research so far has pointed to two potential therapeutic strategies for reducing lipid levels. One, a small molecule, would enhance the rate at which cells burn the lipids. The other approach, an experimental gene

Neurons are indicated in red and microglia in green.

therapy, would increase the rate at which lipids are transported to cells’ internal garbage disposals, organ-like structures known as lysosomes, and how quickly lysosomes degrade them. Meanwhile, Mitchell, also a Professor in the Department of Basic & Translational Sciences, is exploring the link between inflammation and increased pressure within the eye during the development of glaucoma. The buildup of pressure, the cardinal risk factor for this condition, occurs when the clear liquid in the front of the eye can no longer drain properly. Research has linked the strain from the pressure to the activation of a neuronal receptor known as P2X7, which in turn leads to the release of a powerful inflammation-promoting signal. Known as cytokine IL-1β, this signal aggravates inflammation and as a result, the researchers believe, contributes to damaging or even killing neurons. She and her colleagues suspect that the cytokine’s involvement may help to explain how pressure causes the neural damage seen in glaucoma. Her studies also extend to lysosomes. Over the years, these organ-like structures accumulate waste and become prone to leakage. While this effect of aging occurs in long-lived neurons, it is particularly influential within some of their supporters, microglia, which are immune cells found in the brain. In microglia, the receptor P2X7 can cause lysosomes to leak and so initiate a harmful inflammatory response. Her lab is working to identify the best possible target for blocking P2X7, an effort that could one day lead to new therapeutics for glaucoma.

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Bone & Soft Tissue Healing Researchers at Penn Dental Medicine are studying how bone and tissue heal and the conditions that can interfere in those processes with the goal of developing therapies that support and enhance the body’s natural healing process. Advances in this area hold promise for addressing challenging clinical scenarios, such as slow-healing wounds in diabetes — one line of study at the School — and bone erosion in rheumatoid arthritis, another. Stem cells are also being harnessed for their regenerative properties, with stem cells from gum tissue showing promise in nerve repair and those of the jawbone demonstrating particularly strong regenerative properties. Another line of study is building our understanding of the unique healing ability of oral tissue with the hope of translating these healing properties to other parts of the body as well, while a new study model underway could pave the way for tissue engineering options for cleft palate patients.

Unraveling the Mechanisms in Diabetic Wound Healing

octors have long known that diabetics with high glucose levels are more likely to suffer from slowly healing and nonhealing wounds, but why this happens is only partially understood. Dana Graves, Professor in the Department of Periodontics and Vice Dean for Scholarship and Research, is helping to bring clarity to this field of research, which has the potential to improve the quality of life for the 38.4 million Americans currently living with diabetes. “The harm that non-healing wounds inflict on diabetic patients is alarming,” Graves explains. “Wounds that heal slowly can convert to non-healing wounds that are debilitating and a major cause of limb amputation.” Through his research, Graves has honed in on the cellular mechanisms underlying wound healing in diabetic conditions, uncovering a path to potentially alleviate this often-severe consequence of the disease. Graves and his colleagues found that a molecule called FoxO1 plays an unexpected role in the process. Earlier research had suggested that it was needed for normal healing in healthy animals, however, they saw the opposite result in diabetic wounds. In their studies, the team observed that the ability of diabetic wounds to heal was suppressed unless FoxO1 was genetically deleted in mice or blocked by a specific inhibitor in large animals, in which case this negative impact of diabetes was largely reversed. “In terms of a wound-healing response, it looks like FoxO1 might be one of the central regulators that is negatively affected by diabetic conditions,” Graves says. “This may make it a good drug target, which could possibly be administered locally to minimize systemic effects in diabetic wounds.”

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Recently, Graves has been further investigating how diabetes converts FoxO1 from a pro-healing to an anti-healing factor. The team’s results indicate that diabetes causes epigenetic changes that alter FoxO1’s regulation of gene expression in a detrimental way. He explains that epigenetic changes determine how environmental factors, such as diet, exercise, or disease can modify the behavior of molecules and cells. When the team blocked epigenetic changes caused by diabetic conditions, they found that wound healing in diabetic mice was now similar to that in healthy mice. They will now be looking more closely at how diabetes-induced epigenetic changes affect wound healing to investigate targeting them as a therapeutic strategy.

Treatment with Epigenetic Inhibitor

When epigenetic changes caused by diabetic conditions were blocked, wound healing in diabetic mice was similar to healthy mice. Top to bottom: Normal Diabetic, Diabetic + Treatment.

Epigenetic Change in Diabetic Wounds Connecting Diabetes and Periodontal Disease

EP H3K4me3/ DAPI

In other studies, Graves’ lab has investigated the biological connection between diabetes and periodontal disease. To do so, they compared the oral microbiomes of diabetic mice and healthy mice and found that the bacterial composition changed once they developed hyperglycemia, or high blood sugar levels. The result was increased inflammation and greater risk of developing periodontitis. Transferring bacteria from diabetic to healthy mice reproduced the rapid bone loss and significant inflammation linked to diabetes-associated periodontitis, confirming that diabetes is indeed the factor that causes these shifts in the oral microbiome that triggers the onset of periodontal disease. These studies point to the need for particularly good oral hygiene and dental treatment in diabetic patients.

From top to bottom: normal, diabetic, and diabetic plus treatment.

A Serendipitous Link to Atopic Dermatitis

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Research can take unexpected paths with implications across disciplines, which was the case in another line of study from Graves’ lab. In a surprising twist, his team pinpointed a cascade of inflammatory signaling that occurs before skin ulcers appear in atopic dermatitis, a common skin condition, shedding light on its early stages and identifying potential therapeutic targets. The finding grew out of research from Graves’ lab that was exploring the role of inflammatory signaling in bone fracture healing in diabetes. As part of that work, the team used a mouse model in which IKKb, a molecule that activates inflammation, was deleted to see if that would improve bone healing in diabetic mice. While it did, something unexpected happened — the mice developed skin lesions — namely, atopic dermatitis, a type of eczema that affects millions of people. Drilling deeper with collaborators in dermatology from Penn Medicine and computational systems biology experts outside Penn, they were surprised to discover that fibroblasts — cells in the middle layer of skin that secrete collagen and give skin its structure — appeared to be responsible for the lesions. Without IKKb, the fibroblasts produced an inflammation-inducing molecule called CCL-11 that promotes inflammation by recruiting immune cells to the site; CCL-11 is also linked to eczema-like skin lesions in humans. In mice, an antibody that blocked CCL-11 reduced inflammation, suggesting that this pathway could be one to target to reduce atopic dermatitis-associated inflammation. Graves notes that the work not only points to a possible new approach to treatment, but also underscores a developing appreciation that fibroblasts play important roles in immune processes in the skin.

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“Wounds that heal slowly can convert to non-healing wounds that are debilitating and a major cause of limb amputation.” — Dana Graves

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Bone & Soft Tissue Healing

Coaxing Stem Cells from Gum Tissue to Repair Nerves

aced with repairing a major nerve injury to the craniofacial region, surgeons can use a nerve from an arm or leg to restore movement or sensation. This approach — known as an autograft — is the standard of care, but it can take a toll on a previously uninjured body part, and the procedure doesn’t always result in complete and functional nerve regrowth. Anh Le, Chair and Norman Vine Endowed Professor of Oral Rehabilitation in the Department of Oral and Maxillofacial Surgery, is pioneering a different approach. Le and collaborators are coaxing gingival mesenchymal stem cells (GMSCs) — stem cells from gum tissue — to produce nervesupportive cells that facilitate nerve regrowth. “We wanted to create a biological approach and use the regenerating ability of stem cells,” says Le. “To be able to recreate nervesupportive cells in this way is really a new paradigm.”

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“We wanted to create a biological approach and use the regenerating ability of stem cells.” — Anh Le For more than a decade, Le’s lab has explored the use of GMSCs to regenerate different types of craniofacial tissues and to treat osteonecrosis of the jaw that can occur when a patient takes bisphosphonate, a drug used to treat metastatic cancer or prevent bone loss in osteoporosis. Her lab team, led by Qunzhou Zhang, Research Assistant Professor, was able to apply their previous understanding of GMSCs to facilitate their conversion into Schwann-like cells, the pro-regenerative cells of the peripheral nervous system that make neural growth factors and myelin, the insulating layer around nerves.

To move the work forward, Le collaborated with bioengineer D. Kacy Cullen of Penn Medicine, an expert in creating and testing nerve scaffold materials. Together they showed that infusing a collagen scaffold with these cells and using them to guide the repair of facial nerve injuries in animals was just as effective as an autograft procedure. Although the repaired gap was small, the team is continuing to refine the method to repair larger ones that often result from trauma or tumor-removal surgeries. Le notes that this approach would give patients with oral cancer or facial trauma the opportunity to use their own tissue to recover motor function and sensation and to have cosmetic improvements following a repair. And while Le’s group focuses on the head and neck, further work on this model could translate to nerve repair in other areas of the body as well. “I’m hopeful we can continue moving this forward toward clinical application,” she says. Recently, Le has turned her attention to oral cancer, investigating how the complex ecosystem of cells surrounding a tumor (its microenvironment) contributes to disease development and how to manipulate it to improve treatment. Oral squamous cell carcinoma is the most common type of head and neck cancer, and the survival rate has stagnated, despite research advances. With investigations in a mouse model of tongue squamous cell carcinoma, Le’s team has found that blocking the production of an uncommon amino acid called hypusine and its post-translational modification of the protein eIF5A can stop tumor cells from proliferating, slow tumor growth, and inhibit infiltration of tumor-associated white blood cells. Thus, disrupting this modification of the eIF5A protein could be a viable drug target for oral cancer.

Stem Cells Could Hold Key to Osteoporosis and Head/ Neck Cancer Therapies During embryonic development, stem cells mature and form all of the tissues and organs in the body. These building blocks help maintain and repair tissues, but in some diseases, they can become impaired, die, or contribute to disease progression. To harness stem cells for therapies, researchers must understand how things go wrong and how to resolve them. Chider Chen, Assistant Professor in the Department of Oral and Maxillofacial Surgery and Pharmacology, is investigating this as it relates to osteoporosis and head and neck cancer. In one line of study, Chen’s team is looking at how inflammation in postmenopausal women destroys stem cells in osteoporosis, a degenerative disease in which bone is broken down faster than it is built. Patients with the disease have weakened bones that can fracture easily, resulting in pain and impaired mobility. In inflammatory diseases, activation of T cells — a type of white blood cell — leads to bone loss. A protein called mTOR, a master regulator of many processes such as metabolism, is involved in T cell activation, but it wasn’t clear how this related to osteoporosis. Preliminary data from Chen’s lab suggests that mTOR activates T cells, which in turn causes bone loss by killing mesenchymal skeletal stromal cells, a type of stem cell found in bone. In further studies, the team will explore exactly how this happens at the molecular level, which could lead to developing a novel molecule-based therapy for osteoporosis to test in mice. Another area of interest for the Chen team is head and neck squamous cell carcinomas (HNSCC). Most head and neck cancers are squamous cell carcinomas, which occur in the outermost layer of skin and tissues. To design effective treatments, researchers want a better understanding of how the cancer develops and progresses, but that’s challenging because so many different types of cells are involved. To cut through the noise, Chen’s team is studying gene expression in single cells in the craniofacial region.

In their work, they found that the Gli1+ subpopulation of stromal cells — cells that form connective tissue — function as progenitors, cells that are almost as flexible as stem cells at becoming more specialized. They also found that the levels of stromal cells are elevated in HNSCC, and they support tumor progression. The researchers are expanding on the findings of this study, looking for other unique stromal populations in mouse and human HNSCC samples to determine their molecular and pathway signals with the goal of discovering novel therapeutic avenues to manage HNSCC.

Chen’s lab is looking for unique stromal populations and their molecular and pathway signals with the goal of discovering novel therapeutic avenues. A microscope image of a mesenchymal stem cell.

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Bone & Soft Tissue Healing

Remedying Facial Birth Defects Craniofacial defects present at birth can encompass a variety of disorders — among some of the more prevalent are cleft lip and palate, in which the tissue in the baby’s face and mouth don’t fuse properly, and craniosynostosis, in which the soft spots of the skull close too early. In the U.S., about 1 in every 1,000 babies is born with cleft lip with or without cleft palate and about 1 in every 2,500 with craniosynostosis. Researchers are starting to make headway in gaining a better understanding of these conditions, laying the groundwork for more effective treatments. Cleft lip and palate (CLP) is the second most common congenital malformation. Children with CLP usually undergo corrective surgery early, but with very little tissue available, extensive scarring is a common complication that can affect the growth and development of the jaw. Chenshuang Li, Assistant Professor of Orthodontics, has embarked on a project to help tackle this issue. “Current animal models don’t properly mimic human cleft lip and palate development,” says Li. “Our project aims to develop a suitable model in young rats that replicates the craniofacial growth and development pattern observed in patients, with a special focus placed on extensive scarring after early cleft lip revision.” She notes that a successful model will pave the way for future studies on orthodontic and jaw treatment strategies, as well as tissue engineering options for CLP patients. With craniosynostosis, the head can become misshapen, or in more extreme cases, the brain may not have enough room to grow, resulting in pressure that can cause neurological damage. In collaboration with researchers from the ADA Forsyth Institute and UCLA, Li’s work on Nell-1, a protein originally identified in patients with craniosynostosis, has revealed that it also regulates neurological development and function. “Our study opens new avenues for understanding and treating craniofacial patients suffering from skeletal deformities and behavior, memory, and cognition difficulties by uncovering a novel bone-brain-crosstalk network,” she explains. Another Nell-1-related project of the team was selected as a NASA mission study, with findings showing that it also helps to prevent microgravity-induced bone loss, making it as a potential therapeutic agent against osteoporosis and other bone diseases.

1 in every 1,000 babies is born with cleft lip with or without cleft palate

Exploring the Regenerative

ave you ever wondered why scars rarely form in the mouth? Researchers at Penn Dental Medicine have pondered this very question, and they are on their way to finding the answer. “Oral tissues exhibit remarkably regenerative properties, unlike many other organs in the human body,” says Kang Ko, Assistant Professor in the Department of Periodontics. “The goal is to understand the cellular and molecular mechanism by which this occurs in the oral cavity, with the hope of employing therapeutics to enhance oral soft and hard tissue healing, as well as translating these unique healing properties to other body parts to promote regeneration over repair.” To get a better handle on the exceptionally accelerated wound healing in the oral cavity, Ko’s lab is investigating different populations of oral fibroblasts. These types of cells form connective tissue, and work from Ko’s studies is revealing they may play important roles in the wound healing process. In a recent paper, the researchers reported finding a unique subset of oral fibroblasts that is primed to promote rapid wound healing. The team studied what happens to

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Mucosal injury Resolution of inflammation

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Expedited healing

Properties of Oral Tissue wounds in mice in two different parts of the palate — the front, or anterior, region that heals rapidly and the back, or posterior, palate that heals more slowly. In the study, they identified a distinct population of cells called paired-related homeobox-1+ (Prx1+) fibroblasts in the anterior palate that are missing from the posterior section, and they showed that these cells are responsible for quick wound healing observed in the mouth. Transplanting these cells to the posterior palate sped up healing, and deleting the cells in the anterior section delayed the process. In human gingival samples, these Prx1+ fibroblasts were in the same locations and expressed the same genes. The results support an emerging concept that fibroblasts are actively involved in supporting the body’s immune response, contrasting with the traditional view that these cells are featureless building blocks for maintaining structure. “The potential of this finding is significant not only for periodontal regeneration — for instance, the ability to take grafts from tissues enriched with pro-healing fibroblasts — but also for its implications in other parts of body that heal sub-optimally,” says Ko. “Regeneration over scar formation in response to injury is a huge topic.”

Oral mucosa in anterior palate: Enriched with Prrx1high fibroblasts ↑ Wnt-associated transcripts

Pro-resolving macrophages

A rare, rapidly growing tumor, ameloblastoma occurs in the jaw as a result of the cells that form tooth enamel growing uncontrollably.

Unique Properties of Jaw Stem Cells Could Help Repair Damage Among the bones of the body, the jaw is unique. During embryonic development, jawbones arise from different types of stem cells compared to bones of the leg or arm. Even some bone disorders have distinctive characteristics when they occur in the jaw. Now, researchers at Penn Dental Medicine are working to uncover the molecular reasons for these differences, with implications that could help in healing following surgery and in the treatment of defects in the maxillofacial region. “Stem cells isolated from the maxilla and mandible have much higher regenerative properties, grow much more rapidly than those that we have from the hip bone, and they need relatively less chemical stimulation for them to form new bone,” says Sunday Akintoye, Associate Professor and Director of the Oral Medicine Research Program in the Department of Oral Medicine, who is studying orofacial bone mesenchymal stem cells (MSCs) and their site-specific characteristics and therapeutic applications. “Jawbone stem cells are unique, whether you look at them in humans, large animals or small animals.” Having found that the MSCs in the jaw are key to these special properties, Akintoye’s team is working on elucidating the factors that distinguish these MSCs from those in other bones. This information could provide clinicians with tools to replace bone lost to surgery, trauma, and tumors, such as ameloblastoma, which Akintoye has just begun investigating for possible biological indicators of recurrence after surgery. A rare, but rapidly growing tumor, ameloblastoma occurs in the jaw as a result of ameloblasts — the cells that form tooth enamel — growing uncontrollably. The tumor often grows painlessly for a long time in the jaw until eventually, a very large and disfiguring mass is evident. The mass can severely damage the jawbone, and about 10% of the tumors recur, even after surgery. The racial demographics of ameloblastoma patients, as well as whether there are racial disparities in the progression and recurrence of the condition, have not been clear. Akintoye has recently begun to make headway, studying the epidemiology of the condition with an assessment of the literature. In a preliminary analysis, he says the data show that ameloblastoma occurs and recurs more frequently in Black patients than non-Black patients. His team would like to eventually apply their knowledge about oral MSCs to understand ameloblastoma’s behavior. “Incorporation of the jaw stem cells into a graft could help promote rapid healing,” says Akintoye, “and can help with jaw reconstruction after surgery.” 2 024 | RESEARCH AT PENN DENTAL MEDICINE

Bone & Soft Tissue Healing

Identifying the Factors of Bone Loss, Inflammation in Rheumatoid Arthritis

heumatoid arthritis (RA) affects about 18 million people around the world, according to the World Health Organization. The chronic autoimmune disorder causes painful inflammation with RA patients experiencing joint damage and swelling as the body mistakenly attacks its own healthy cells. Currently available medications don’t work that well and have many side effects because they impact a host of different processes in the body, but Shuying Yang, Professor in the Department of Basic & Translational Sciences, has a more specific target in her sights. “Medications for RA can affect many physiologic functions, and many patients are resistant to the current treatments,” explains Yang. “So, identifying new factors that control bone erosion and inflammation specifically for RA is urgent and would have significant impact.” Yang is aiming to do just that through her study of a protein named RGS12. Yang’s lab has been researching the role of RGS proteins on bone remodeling and diseases for more than a decade. RGS proteins, which are key players in immune responses, are a family of cellular proteins that negatively regulate the signaling of G proteins. Acting as molecular switches, G proteins transmit signals from outside a cell to its interior via G protein-coupled receptors. Through her research, Yang was the first to discover that RGS12 plays an essential role in bone formation and resorption. She found that mice with RGS12 deleted in cells that degrade bone had greater

“Identifying new factors that control bone erosion and inflammation specifically for RA is urgent and would have significant impact.” — Shuying Yang

bone mass and less age-related inflammatory bone loss. Her team also found that RGS12 is involved in regulating multiple signaling pathways for inflammation and bone erosion. In addition, a common RA medication changes the activity of RGS12, providing further evidence that the protein has a role in the disease. Armed with these insights, Yang’s team conducted experiments showing that the RGS12 protein is elevated in RA patients and in a mouse model of the disease. Importantly, deleting the gene in mice bred to have RA prevented the characteristic joint swelling and bone damage. Yang is now extending these studies to better understand this process, and she is testing whether the protein could be a therapeutic target for RA. “We hope to reveal a new mechanism by which RGS12 acts as an inflammatory factor and demonstrate that inhibiting RGS12 may represent an attractive new or complementary therapeutic approach to suppressing inflammation and bone resorption for RA patients,” says Yang. “Most importantly, we are developing an RGS12-mediated therapeutic system to inhibit inflammation, which has translational significance not only for RA, but also for periodontitis-caused bone loss, aging and diabetic osteoporosis, and other inflammatory-associated diseases.” In another line of study, Yang is investigating a different set of molecules — a signaling molecule called INPP5E and intraflagellar transport (IFT) proteins, which help construct cilia, antenna-like sensory organs that extend from cells, by transporting proteins from the cilia’s base to their tip and back again. Yang’s team found that those proteins play critical roles in protecting against bone disruption and inflammation by serving as an inhibitor of cells that degrade bone and cause inflammation. These features make them a valuable target for potential therapeutic intervention for diseases that progressively destroy bone tissue.

Student Research DMD Student Rosemary Do (D’24)

Investigating Wound Healing, Gum Disease When Rosemary Do (D’24) learned about the research of Kang Ko, an Assistant Professor of Periodontics at Penn Dental Medicine, she knew she wanted to join his lab as part of the school’s Summer Research Program. “My dad has periodontal disease,” says Do, an international student from Vietnam, who is now in her fourth year of the DMD program. “His struggle with it was something that originally motivated me to study dentistry, so when I realized Dr. Ko’s research was relatable to my dad’s condition, I was enthusiastic about joining his lab.” Do joined Ko’s lab in the summer of 2021 after her first year at Penn Dental Medicine and she has been involved with research there ever since. Her summer research project focused on the role of a molecule known as IL-33 in the wound healing process. Specifically, she investigated how the expression dynamics of IL-33 contributed to wound healing in mice over a period of two, four, and six weeks. This research has implications for dentistry since patients who have periodontal diseases and underlying medical conditions such as diabetes frequently experience delayed oral wound healing. “We’re trying to see if we can ascertain biological factors that could eventually contribute to developing medications that can help accelerate the healing process,” says Do. Working with Ko has been a valuable learning experience, she says, because “he let me explore various experiments,” while still being “in a safe zone under his supervision.” His mentorship, she says, has helped her not only to learn more about the research aspect of dental medicine, but also how to work effectively on her own. Do extended her research with Ko through the Basic & Translational Research Honors Program, which is open to students who have an interest in hypothesis-driven basic science and research. Through the program, she is carrying out data analysis on more advanced research that monitors the molecule over longer periods of time, as well as further experiments that involve deleting the gene that produces IL-33 in mice to observe how that affects wound healing. The hypothesis is that without this molecule, wound healing will be severely delayed. With the support of a Student Research Fellowship that Do was awarded by the AADOCR (American Association for Dental, Oral, and Craniofacial Research), she has been able to connect with fellow students interested in oral health research around the U.S. and to share the findings of her own research project. She presented her work at the 2023 AADOCR Annual Meeting, where she was

ABOVE: Rosemary Do

(D’24) and faculty mentor Dr. Kang Ko. She has conducted research in his lab since 2021.

LEFT: IL33 expression

dynamics through the oral wound healing process.

also a finalist in the 2023 AADOCR Hatton Competition, in which the best junior research investigators from throughout the country compete. Do intends to pursue a general practice residency program after graduation to deepen her knowledge of advanced and complex procedures and strengthen her hand skills before embarking on a private practice dental career. Ultimately, she says, her goal is “to provide prevention and comprehensive care to people who have similar conditions to my dad, especially those who have not had consistent access to oral healthcare from an early age.”

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BELOW: The DScD program has given Dr. Dennis Sourvanos (GD‘23, DScD’23) many opportunities to present on his research.

Student Research

DScD Student Dennis Sourvanos

Lighting the Way to Improved Healing After a tooth extraction, dental patients have historically had just a couple of options to ease their post-operative pain: They can take a combination of over-the-counter medications or have an opioid prescribed. Now, through a minimally invasive type of red-light therapy known as photobiomodulation (PBM), they could soon receive access to a third option for pain relief that will safely reduce the effects of inflammation and improve wound healing. Dennis Sourvanos, (GD‘23, DScD’23), who has investigated this approach to using PBM in dentistry, is optimistic about its potential applications. “We’re starting to develop enough preliminary research to show that this can be just as effective, if not more effective, as a safe alternative to prescribing narcotics for our patients,” says Sourvanos, who at press time was nearing completion of his DScD degree at Penn Dental Medicine, along with clinical postdoctoral training in periodontics. Sourvanos has been working on this PBM research with his DScD mentor Dr. Joe Fiorellini, Professor of Periodontics, and a group of collaborators led by Dr. Tim Zhu in Penn Medicine’s Radiation Oncology. The technique, which is already used in other areas of medicine, is low cost, and utilizes FDA-approved devices. An added bonus, says Sourvanos, is that the therapy can be applied by different tiers of practitioners, including registered dental hygienists (depending on the state jurisdiction). “This could have an immediate social impact on how we treat patients and provide dental care.” A PBM treatment session involves placing the device on the side of the face and delivering red or near-infrared light for 30 seconds to a minute immediately after surgery. The therapy works by increasing ATP production in the mitochondria (the energy needed to power cells) to improve wound healing. “When you have a dental extraction, you typically experience swelling for the first three days,” says Sourvanos. “With PBM, we can bring growth factors directly to the wound site, accelerate the inflammatory

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BOTTOM: Red-light therapy known as photobiomodulation can reduce the effects of inflammation and improve wound healing.

“We are actively repurposing novel technologies from medicine to elevate the standard of treatment we provide to our patients in dentistry.” — Dennis Sourvanos, (GD‘23, DScD’23)

cascade, and potentially reduce a lot of the post-surgical side effects of inflammation like swelling, pain, and discomfort.” Sourvanos’s current research is focused on determining the amount of PBM dosage needed so he can establish a treatment protocol to be tested in a future clinical trial. “Despite hundreds of publications, no one really understands how to calculate PBM dosage, so I’m working with a team of internationally recognized medical physicists to translate key dosage concepts in medicine to dentistry,” says Sourvanos. Sourvanos describes the DScD program “as a training platform with access to tremendous resources and opportunities to engage with centers across the entire university ecosystem.” In his case, that has included groups in

Engineering, Medicine, Veterinary Sciences, the Office of Clinical Research, and the Mack Institute for Innovation Management. “As a DScD student, there are many avenues for cross-disciplinary collaboration,” says Sourvanos, who is also a fellow in the Center for Innovation & Precision Dentistry NIDCR Postdoctoral Training Program and a prior NIH fellow in Penn’s Institute for Translational Medicine, and Therapeutics. “We are actively repurposing novel technologies from medicine to elevate the standard of treatment we provide to our patients in dentistry,” he says. “By leveraging the niche expertise of my mentors, we are exploring new areas of science, technology, and innovation that will inevitably benefit both of our disciplines.”

LEFT: Dr. Marshall Padilla (right) has

collaborated with Dr. Qunzhou Zhang, from the lab of Dr. Anh Le, on oral cancer research.

BELOW: A cryogenic transmission electron microscopy image of the lipid nanoparticles.

CiPD NIDCR T90/R90 Fellow Marshall Padilla

Repurposing mRNA Technology

ith a PhD in chemistry, Marshall Padilla has always been drawn to interdisciplinary research. A fellowship with the Center for Innovation & Precision Dentistry (CiPD), Penn Dental Medicine’s collaborative research center with Penn Engineering, has enabled him to pursue that interest and discover ways that his chemical synthesis expertise can benefit dental medicine. In graduate school, Padilla says, he gravitated toward the field of drug delivery, figuring out “how to build vehicles or construct formulations that allow drugs to get to where they need to go in a timely and safe manner.” As a postdoc at Penn, Padilla joined the lab of Associate Professor of Bioengineering Michael Mitchell, and soon after that, the CiPD NIDCR T90/R90 Postdoctoral Training Program was launched, with Padilla joining the first cohort of fellows in 2022. The goal of the training program is to develop groups of cross-trained dentists, engineers, and scientists to address unmet needs in oral and craniofacial health; fellows are co-mentored by faculty from both Penn Dental Medicine and Penn Engineering. Historically, says Padilla, the dental sciences and chemistry have not worked closely together. That made him all the more eager to work with Anh Le, Professor and Chair of the Department of Oral and Maxillofacial Surgery, as his dental school mentor for the program. With Le, Padilla is working to reapply the mRNA COVID-19 vaccine technology to oral cancer. Since viruses often take the form of foreign mRNA, our bodies have evolved numerous defenses to prevent them from entering our cells. To bypass those defense systems, explains Padilla, the COVID-19 vaccines

package the mRNA in little (100 or even 1,000 times smaller than a cell) quasi-spherical vehicles called lipid nanoparticles. “I’m taking that technology,” he says, “and repurposing it by synthesizing new lipids that are very specific for oral cancer.” After such a diagnosis, patients typically have surgery scheduled on the part of the mouth impacted by the cancer. This is where Padilla envisions his repurposed technology coming into play. “We would inject the patient with the lipid nanoparticle containing mRNA that encodes tumor suppressor proteins,” he says. “This could help shrink the cancer and stop it from metastasizing, so that when they do have surgery, it’s less invasive. Even shrinking it a little bit helps in terms of quality of life, since oral cancer surgery can so dramatically hurt one’s ability to eat, speak, and breathe.” Padilla sees a chemist’s input as particularly useful in tackling complex research problems such as oral cancer that require a diverse skill set. “You need people — dentists, chemists, pathologists — who can communicate and understand how the chemistry relates to the cancer and how the drug delivery relates to the treatments,” he says. Padilla had the opportunity to learn more from researchers in the oral health field when he was selected to be part of the American Association for Dental, Oral, and Craniofacial Research (AADOCR)’s Mentoring an Inclusive Network for a Diverse Workforce of the Future (MIND the Future) program for 2022–2023. “It was a good learning experience,” says Padilla, whose mentor was oral cancer researcher and Dean of the University of Michigan’s School of Dentistry Dr. Jacques Nor. “When you’re a postdoc, often you’re really focused on the research and don’t always have the opportunities or time to engage with your peers, which is important to career development as well.”

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Dental Diagnosis & Treatment Research related to the treatment and diagnosis of oral disease is ushering in new approaches and discoveries to enhance patient care and outcomes. Penn Dental Medicine faculty are exploring innovations in dental care on a variety of fronts from developing the use of microrobots to clean teeth and treat fungal infections to engineering a next-generation implant that wards off bacteria with nanoparticles and phototherapy. Applying physics and material science, the esthetics and durability of restorative dental ceramics are being taken to new levels, while 3-D radiography and artificial intelligence are being applied to improve diagnosis. In addition, ongoing studies are shedding light on how to reduce dental fear and manage pain, advancing patient-centered care, and systematic reviews are improving evidence-based dentistry across disciplines, reaffirming the interconnected nature of oral and systemic health and elevating clinical care.

Disrupting Biofilms with Innovative Technology

o eliminate the disease-fostering biofilms that cause tooth decay and oral infections, Hyun (Michel) Koo, a Professor in the Department of Orthodontics and divisions of Community Oral Health and Pediatric Dentistry, seeks to better understand the dynamics within these microbial communities and devise innovative ways to disrupt them. Meanwhile, as co-founder and co-director of the Center for Innovation & Precision Dentistry (CiPD), a partnership with Penn Engineering, he is uniting other dental and engineering faculty and trainees to spur a myriad of oral healthcare innovations. In his own lab, a key focus of recent cross-disciplinary collaborations has involved microscale, nanotechnology-based strategies to clean teeth, treat fungal infections, and more. Koo and his collaborators have turned to swarms of nanoparticles that, when their motion is coordinated, act as microscale robots. To create these microrobots, they have created engineering-based systems that rely on iron oxide nanoparticles. These tiny particles possess two key traits: First, they respond readily to magnetism, which researchers can use to manipulate them. Second, they can catalyze, or kick off, chemical reactions.

“The big innovation here is that the robotics system can brush, floss, and rinse in a single, hands-free, automated way.” — Hyun (Michel) Koo

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A hands-free toothbrush In one application, Koo and Edward Steager of Penn Engineering created shapeshifting throngs of these nanoparticles that showed promise as future all-in-one toothbrushes, rinses, and dental floss. Using an electromagnetic field, they directed the motion and configuration of the iron oxide nanoparticles to form either bristle like structures that sweep away dental plaque from the broad surfaces of teeth, or elongated strings that can slip between teeth like a length of floss. In both instances, a catalytic reaction drives the nanoparticles to produce antimicrobials that kill harmful oral bacteria on site. Experiments using this system on mock and real human teeth showed the robotic assemblies can conform to a variety of shapes to nearly eliminate the sticky biofilms that lead to cavities and gum disease. “Routine oral care can pose challenges for many people, especially those with limited manual dexterity,” Koo says. “The big innovation here is that the robotics system can do all three — brushing, flossing, rinsing — in a single, hands-free, automated way.” The iron oxide nanoparticles they used have been FDA approved for other uses. To move this technology forward, the team is continuing to optimize the robots’ motions and considering different means of delivering the microrobots through mouthfitting devices.

In Dr. Michel Koo's (right) lab, Dr. Zhi Ren (left) has developed a new way to study oral pathogens using real-time microscopy.

Focusing on fungal infections

More nanozymes and a “superorganism”

More recently, Koo and Steager harnessed the catalytic activity of the nanoparticles, called nanozymes in this context, to create a system capable of rapid, targeted elimination of fungal pathogens. Infections caused by fungi, such as Candida albicans, pose a significant global health risk due to their resistance to existing treatments. In the mouth, Candida can flourish in biofilms, leading to difficultto-treat infections, Koo says. As with the tooth-cleaning system, the researchers used electromagnetic fields to control the shape and movements of the nanoparticles with great precision. Steager enhanced the nanozymes’ controllability and catalytic activity, which leads to the generation of high amounts of reactive oxygen species (ROS), compounds that have proven biofilmdestroying properties, at the site of infection. It turned out that the nanozymes accumulated precisely where the fungi reside and, consequently, targeted them with the ROS. Coupled with the nanozymes’ inherent maneuverability, this results in a potent antifungal effect. In their study, the team reported eradicating fungal cells within an unprecedented 10-minute window. “We’ve uncovered a powerful tool in the fight against pathogenic fungal infections,” Koo says, noting its possible application for treating other types of stubborn infections.

In other recent work, a study led by Koo found that an iron-oxide nanoparticle that is FDA-approved to treat anemia, called ferumoxytol, also holds promise for treating, preventing, and even diagnosing harmful oral biofilms. The study found that a twicedaily application of ferumoxytol, which activated hydrogen peroxide contained in a follow-up rinse, could significantly reduce the buildup of dental plaque and had a targeted effect on the bacteria largely responsible for tooth decay. Another study with ferumoxytol by his team revealed that the combination of nanoparticles and stannous fluoride, the compound used in toothpaste to prevent tooth decay, could point to a potent cavity-fighting solution. “Our combined treatment not only amplifies the effectiveness of each agent but does so with a lower dosage, hinting at a potentially revolutionary method for caries prevention in high-risk individuals,” Koo says. In other work from Koo’s lab, his team found that a crosskingdom partnership between bacteria and fungi can result in the two joining to form a “superorganism” with unusual strength and resilience. What’s more, the assemblages sprout “limbs” that propel them to “walk” and quickly spread on the tooth surface. Found in the saliva of toddlers with severe childhood tooth decay, these assemblages are stickier, more resistant to antimicrobials, and more difficult to remove than either the bacteria or the fungi alone, the team has reported. The “superorganism” discovery came about when using advanced microscopy that allows scientists to visualize the behavior of living microbes in real time, a technique that opens new possibilities to investigate the dynamics of complex biological processes. 2 024 | RESEARCH AT PENN DENTAL MEDICINE

Dental Diagnosis & Treatment

1 in 5 (over 53 million Americans) who go to the dentist have moderate to severe dental fear

Helping to Address Dental Fear, Anxiety to Improve Oral Health Outcomes Approximately 21% of individuals report fear, anxiety, or stress about going to the dentist with as many 11% reporting severe fear. In one study, 17% of patients stated their fear was so severe that they avoided seeing the dentist even when they were in pain. Indeed, dental fear and anxiety can cause patients to miss or cancel appointments — keeping them from getting the preventive care they need. Avoiding necessary dental care can result in worsening oral health outcomes and lead some patients to require extensive emergency care. A recent U.S. Surgeon General’s report identified that avoidance of oral healthcare leads to pain and suffering, which can impact an individual’s overall health and well-being and significantly diminish their quality of life.

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Mark S. Wolff, Dean and Professor in the Department of Preventive & Restorative Sciences, is working on ways to help address patients’ fears. While techniques to help reduce patient fear and anxiety are part of dental training for all dentists, Wolff notes that often in everyday practice they may not be part of routine dental care. “Adopting universal dental fear screening at the very first visit can be of tremendous benefit to patients and dental practices,” says Wolff. “If clinicians ask patients through a single question to rate their dental fear, it provides important clinical information, informs the approach to treatment and the clinician’s perspective on patient behavior, and demonstrates the practice has empathy for their patients.” On a zero to 10 scale, Wolff notes that four or above conveys significant fear, and patients with this level of anxiety are being recruited for a current study that Wolff is leading in collaboration with New York University (NYU) College of Dentistry. The study is a nationwide randomized controlled trial of a brief steppedcare approach to treat dental fear. As a stepped-care study, people only get as much of an intervention as they need.

For the study, Wolff and his NYU collaborators adapted long-established and tested in-person dental-fear counseling techniques that use cognitive behavioral therapy into an at-home intervention. This new treatment approach helps patients modify what they do, think, and feel to improve their dental experience by using a free app called Dental Fearless, which helps identify fears and creates a list of ways to reduce those fears. For example, these techniques can include discussing a signal with the dentist to convey, “please stop working,” (e.g., by raising a hand); discussing each phase of treatment before it starts or, alternatively, not discussing what the dentist is going to do if that makes someone feel more comfortable. Patients enrolled in the study are introduced to the use of the app and receive a virtual session with a dental fear specialist. The purpose of the study is to test if standard in-person dental fear treatment can be equally effective when done virtually, with the hope of enabling it to be broadly available to all patients. Managing the fear of dental care is not only important for maintaining patients’ oral and overall health, but it can also be effective in lowering dental expenditures by reducing emergent and serious issues that can be complicated to treat in the future.

“Adopting universal dental fear screening at the very first visit can be of tremendous benefit to patients and dental practices.” — Mark S. Wolff

Engineering a Next-Generation Dental Implant

very year, roughly 3 million Americans receive implants to replace lost teeth. While this technology has represented a leap of progress in dental care, over time, inflammation and gum disease — nurtured by microbial biofilms, or plaque — can affect the soft tissue and bone surrounding the implant and cause these tooth replacements to fail. An innovative new implant now on the horizon may one day address these issues, disrupting biofilms through its inherent antimicrobial and antiinflammatory properties. The design of this next generation implant is the work of Geelsu Hwang, an Assistant Professor in the Department of Preventive & Restorative Sciences, who has a background in engineering that he brings to his research on biofilms and their role in oral health. “The lack of a good seal between the implant structure and the surrounding gum, compared to a natural tooth, means that the risk of disease around the implant is quite high,” Hwang says. The new implant he and his collaborators are developing would interfere with biofilms and combat peri-implant infection in two ways: First, the crown, the artificial tooth atop the implant structure, will be infused with nanoparticles made of a chemical compound that naturally wards off bacteria. Hwang and his team have been experimenting with the compound barium titanate (BTO). In addition, the base of the crown will contain LEDs that deliver a regular dose of phototherapy to the surrounding gum tissue. The LEDs will be powered by a piezoelectric material in the crown (like the BTO) that converts the motion of chewing or toothbrushing to electrical energy. Hwang notes that this platform could one day be integrated not only into dental implants, but into other applications, such as joint replacements, as well.

“Phototherapy can address a diverse set of health issues,” Hwang says. “Once a biomaterial is implanted, it’s not practical to replace or recharge a battery. We use a piezoelectric material to supply power instead through natural oral motions, and our experiments show it can successfully protect gum tissue.” In 2023, the National Institutes of Health awarded Hwang a five-year grant to further advance the implant development. The NIH funding will support tests of the antibacterial properties of the new implant technology, using laboratory cultures of human gum tissue and, ultimately, test implants in mini pigs as a preparation for human clinical trials. “This is a very ambitious project, but we believe it represents a new paradigm for implant technology and for oral healthcare in general,” says Hwang. In related research, he is also studying a new piezoelectric dental composite material for fillings. The material would generate an enhanced electrical charge at the interface from the mechanical pressure of chewing, and this on its own would inhibit plaque from forming on the composite surface. “In principle, we can use piezoelectric materials for many applications in dentistry,” says Hwang, “including the generation of electricity to speed wound healing and bone regrowth, and even the powering of biosensors that monitor oral health.”

“This is a very ambitious project, but we believe the smart implant represents a new paradigm for implant technology and for oral healthcare in general.” — Geelsu Hwang

The crown of the smart implant will be infused with nanoparticles that ward off bacteria and the base will contain LEDs that deliver a regular dose of phototherapy to the surrounding tissue.

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Dental Diagnosis & Treatment

Building Brighter Smiles with Physics and Material Science

eramics, specifically zirconium dioxide, or zirconia for short, has become increasingly relied upon in dentistry. From crowns and bridges to other applications, the resulting products can restore function and beauty to patients’ smiles. While an attractive restorative material, particularly for its strength, zirconia isn’t without its shortcomings. Yu Zhang, Professor in the Department of Preventive & Restorative Sciences, is applying his background in physics and material science to improving dental ceramics, from their esthetics to their durability. In one line of study, Zhang has manipulated zirconia to achieve more desirable properties. To create a more natural appearance, the

Graded 100

Core 200

300

Distance from surface (µm)

TOP: Dr. Yu Zhang (left) and Sonaj Vardhaman setting up a fatigue test on a tooth motion simulator in his lab..

ABOVE: Graded zirconia crowns and bridge.

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conventional approach was to place a layer of porcelain over the zirconia, but when these two materials are fused, residual thermal stresses make porcelain more susceptible to fracture. Zhang’s solution was to create a product in which one side of the material was zirconia, and the other was porcelain, but the fractions of each gradually change across the interface – this gradient material has enhanced esthetic, mechanical, and bonding properties. In addition, applying the same gradient approach to calcium phosphate glass and zirconia, Zhang has produced a strong material that promotes bone integration while repelling microbes. To address the somewhat unnatural opaque appearance of zirconia, Zhang had another innovative approach. “The goal is to make the material translucent with respect to visible light,” explains Zhang. “If we shine the light to which the human eye is most sensitive through zirconia (green light at a wavelength of 555 nanometers) we’ll perceive it as translucent.” Based on this insight and work in classical physics light-scattering theory, he adjusted the microstructure of zirconia in the lab, making it translucent while maintaining its durability. Ceramics start as powders and are heated for hours at very high temperatures, or sintered, to form a solid. Zirconia is partially sintered to create the blocks used to craft a dental restoration, but hours of sintering are still required to finish it. Zhang’s team is working on novel ultrafast strategies to reduce this time to as little

as 60 seconds using Joule heating elements with more effective radiation and conduction heat transfer. Zhang is also addressing issues associated with fabrication and finishing technologies of dental ceramic restorations. Currently, ceramic restorative materials are ground with diamond burs, but these expensive burs have to be replaced frequently. In addition, grinding exerts stresses on the ceramic, potentially introducing tiny cracks that can serve as weak points. Zhang is taking an approach known as “ductile” grinding, a process used in the manufacture of semiconductors that avoids introducing these defects, achieving an accuracy of form and maintaining the integrity of the material while preserving diamond burs. He’s developing new protocols to apply this technology to the crafting of dental ceramic materials. Once a restorative material is applied, another question comes up. Because X-rays can’t penetrate zirconia, how can dentists determine whether decay has occurred under the material? Zhang’s team is hard at work exploring the development of a novel nearinfrared imaging system that can see through many types of dental ceramic materials to visualize what’s happening underneath, while preventing radiation exposure from dental X-rays. The system also could help technicians screen ceramic products for flaws and cracks before they are placed in the mouth.

Evaluating Restorative Materials, Adhesive Technologies Digital technologies — namely, computer aided design/ computer aided manufacturing (CAD/CAM) — and new all-ceramic material developments are pushing the boundaries of esthetic, biocompatible, and long-lasting dental restorations. Markus Blatz, Professor and Chair of the Department of Preventive & Restorative Sciences, and Assistant Dean for Digital Innovation and Professional Development, is helping to evaluate the efficacy of emerging materials and technologies in this arena to move the field forward. In his research, Blatz’s particular focus is on adhesives and ceramics with the goal of advancing more natural-looking restorations and less-invasive solutions. In a current study, Blatz is conducting a clinical product surveillance trial to evaluate the performance of posterior single-tooth crowns made from the latest multi-translucent zirconia ceramic crowns. After teeth are prepared for the procedure, his team scans them with an intraoral scanner. With the help of AI, the data are analyzed and used to digitally design crowns, which are milled from the material being tested. Finished crowns are cemented with a novel self-adhesive resin cement that provides superior physical and bonding properties to the tooth and the crown via a simplified application process. For two years, the team will evaluate clinical parameters, such as restoration fracture resistance, marginal adaptation, shade/color match, and any complications that arise. “Our goal is to help advance the profession with solid evidence-based techniques and technologies that are also less invasive, helping our patients retain their teeth longer,” says Blatz. “Our goal is to help advance “So, in all we do, our main focus is to use the profession with solid technology to better evidence-based techniques serve patients based on their specific and and technologies that individual needs.”

are also less invasive, helping our patients retain their teeth longer.”

— Markus Blatz

Automated CBCT segmentation by AI (left) into tooth structure (yellow), restorative materials (green), bone (blue), and apical lesion (red) in comparison to the original CBCT image (top, left) and the manual segmentation by a clinician (top, right).

Using AI to Detect and Diagnose Oral Disease Artificial intelligence (AI) technology is seeping into many aspects of life, and medicine and dentistry are no exception. In particular, a subdomain of AI called deep learning is now gaining traction in complex medical image analysis, assisting in disease detection and diagnosis and providing clinicians with additional tools to make well-informed decisions. This type of AI teaches computers to process data through artificial neural networks inspired by the human brain. With multiple layers, the networks “learn” from datasets incorporated into the AI system. In a study at Penn Dental Medicine, Frank Setzer, Associate Professor of Endodontics, and Mel Mupparapu, Professor of Clinical Oral Medicine, are putting deep learning to work. In collaboration with several other institutions, they are working on integrating deep learning algorithms into an advanced computer-aided detection platform for cone-beam computed tomography (CBCT) scans, with the goal of aiding clinicians in the detection and diagnosis of periapical (root tip/canal) lesions, as well as rare jaw lesions. CBCT is a radiographic imaging method that allows three-dimensional imaging of hard tissue structures, but the scans can be difficult to interpret, and lesions are often missed or the nature of them can be hard to determine. Setzer notes that interpreting dental CBCTs poses a particularly high bar for AI deep learning algorithms given the complexity of the jaw area. It is filled with teeth and bone, as well as restorative materials, depending on the procedures a patient has undergone, and the AI system must learn to distinguish all the structures from abnormalities. To date, the research team has worked on the automated detection of periapical lesions, developing tools to improve the accuracy of the deep learning algorithms and have achieved successful results in the sensitivity and precision of detection. They are now moving on to the detection of ameloblastoma and keratocystic tumors and have already implemented strategies to overcome obstacles in training AI algorithms on the limited datasets expected from these rare jaw lesions. 2 024 | RESEARCH AT PENN DENTAL MEDICINE

Dental Diagnosis & Treatment

Investigating the Pathogens

P CBCT Radiography Improving Skeletal Age Evaluation, Orthodontic Care Did you know skeletal age can vary from chronological age? Skeletal age is an indication of bone maturity, and hormone levels, nutrition, and genetics can affect bone maturation at different rates, causing individuals of the same chronological age to have differing skeletal ages. Now, researchers at Penn Dental Medicine are working on developing a method that can more accurately determine skeletal maturity, an important factor for knowing the appropriate timing of certain types of orthodontic or other bone- or tissue-related procedures. “Especially in orthodontics, we would like to know skeletal age so that, instead of focusing only on corrective treatments after an oral structure has fully grown, we could start treatments earlier — before they are difficult or impossible to correct,” says Mel Mupparapu, Professor of Clinical Oral Medicine. For example, a procedure to expand the hard palate should be done while the area is still growing, before the mid-palatal suture closes around puberty. Traditionally, orthodontists have determined skeletal age by noting when wisdom teeth emerge, assessing the maturity of vertebrae in the neck, and examining hand and wrist X-rays. These measures have ultimately proven to be unreliable, so researchers have begun investigating whether a joint of cartilage known as the spheno-occipital synchondrosis (SOS) that connects two bones at the base of the cranium could be used instead. Because the SOS is located at the base of the skull, it is thought to be a much more stable indicator compared to the neck or hand and wrist, which can be exposed to factors that could change these structures in ways unrelated to age. However, SOS assessments have had their problems, too. Mupparapu explains that earlier categorizations of the SOS were unreliable because researchers were developing and testing these measures on cadavers, which might not provide the same data as living subjects, or with 2D radiographs. To obtain much higher resolution and contrast than regular X-rays can provide, Mupparapu’s team turned to 3D cone-beam computed tomography (CBCT), a radiographic imaging method that allows accurate, three-dimensional imaging of hard tissue structures. By reviewing and analyzing CBCT scans that were already obtained as part of patients’ treatment plans, he and his team developed a new five-stage categorization system for bone maturation, which proved to be more reliable and easily reproducible. The team is currently expanding the CBCT study to validate the findings. In addition, they are testing an artificial intelligence tool in the hopes that someday, clinicians could simply feed the images into the tool to obtain an accurate skeletal age, saving valuable time.

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eriodontitis, the most severe form of gum disease, affects roughly two in five adults 30 or older in the U.S., with potentially serious consequences. Microbiologist and periodontist Flavia Teles, an Associate Professor in the Department of Basic & Translational Sciences, seeks to better understand how organisms living below the gum line contribute to the disease, generating new insights on this condition with the intent of harnessing them to improve treatment. Not only is periodontitis the most common cause of tooth loss among American adults, it also increases the risk for systemic conditions, such as diabetes, cardiovascular, and respiratory diseases. However, physicians and dentists still lack a practical means of identifying those at high risk — Teles is working to change that. Teles’ research has two major components: First, she is using metagenomic sequencing to investigate the microbial communities associated with severe periodontitis. This type of sequencing looks at all the genetic material inside a sample to analyze the biodiversity and functional capabilities of the microbial community. Then, using artificial intelligence, she and her lab are devising disease-based classifications and establishing models to predict disease progression. These models will integrate the new microbiological information with existing immunological, clinical, and demographic data.

Responsible for Gum Disease A related project, conducted in collaboration with David Issadore at Penn Engineering, focuses on detecting pathogens responsible for periodontitis and other diseases. Together, they are designing an inexpensive, modular device that rapidly isolates the vesicles released by microbes; vesicles are cargocarrying spheres formed inside a cell. These vesicles are then used to determine the presence of particular microbes and the diseases they cause.

Focusing on Fungi to Fight Childhood Tooth Decay

Confocal image showing bacterial-fungal interspecies biofilm structure: Streptococcus mutans (bacteria, green); Candida albicans (fungi, cyan); extracellular polysaccharide (red).

Physicians and dentists still lack a practical means of identifying those at high risk — Teles is working to change that. Just as the oral microbiome can cause periodontitis, it can also drive malignancy. In another line of study, the Teles lab is currently exploring this relationship through the lens of Fanconi Anemia, a rare genetic disorder that causes a predisposition to oral cancer. In this project, they are investigating the compounds made by the oral microbiome and how they damage human cells.

Early childhood caries (ECC) is the most common chronic childhood infectious disease and a significant public health problem, which can destroy teeth in young children and cause long-term, systemic health problems. Research links this potentially painful and costly dental disease with previous oral fungal infections called thrush that promote the growth of pathogenic biofilms, or plaque. A longitudinal study at Penn Dental Medicine is now further investigating this connection. Yuan Liu, an instructor in the Division of Pediatric Dentistry, and her colleagues are following young children who had oral thrush and their counterparts who did not. When complete, this study will determine if children diagnosed with the fungal infection in the first year of life are at higher risk of developing severe ECC later. Globally, there are nearly 1.8 billion cases a year of ECC, which affects children under six years of age. The severe form of ECC is particularly problematic because it progresses to the rapid and rampant destruction of primary teeth within as little as 6 months and complicated systemic infections.

If the longitudinal study confirms the connection between the two conditions, it could open the door for pediatricians and dentists to treat prior oral thrush infections as a sign of heightened risk and an indicator that a child may benefit from preventative measures. This study follows retrospective research in which Liu and her colleagues found a strong association between a thrush diagnosis in infancy and the development of ECC, especially at younger ages. Her group’s previous studies also show a synergistic interaction between the fungus Candida albicans and cavity-causing bacteria such as Streptococcus mutans that enhances the formation of caries (cavities). “Our ongoing research will provide much needed insight on whether Candida infection can alter the biofilm-forming microbiome and contribute to the aggressive onset of this disease,” Liu says, who ultimately seeks to develop practical applications for diagnosing and preventing caries in at-risk children, while also improving the understanding of the mechanisms by which biofilms promote them.

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Dental Diagnosis & Treatment

Advancing EvidenceBased Care through Cochrane Oral Health Collaborating Center Dr. Alonso CarrascoLabra (right) and Olivia Urquhart (left) of the Center for Integrative Global Oral Health.

In 2023, Penn Dental Medicine’s Center for Integrative Global Oral Health (CIGOH) partnered with Cochrane Oral Health to form the Cochrane Oral Health Collaborating Center at Penn Dental Medicine. The Center marks a significant step in helping to expand evidence-based knowledge to inform practice and oral health policy worldwide. Cochrane Oral Health, headquartered at The University of Manchester in the UK, is one of over 50 review groups within Cochrane, a global independent not-forprofit network that produces systematic reviews across all areas of medicine. Since its inception, Cochrane has defined the methodological standards for conducting systematic reviews around the world. The Collaborating Center at Penn Dental Medicine is building upon the work of Cochrane Oral Health, engaging researchers from across the globe in systematic reviews summarizing the best available evidence on oral health topics to help patients, caregivers, clinicians, and policymakers, make well-informed decisions. Center Director and Associate Professor of Preventive and Restorative Sciences Alonso Carrasco-Labra shares some of the goals and vision for the Center.

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What is the primary focus of the Center? The primary focus of the Center is to help patients, clinicians, and policymakers make decisions related to health outcomes using trustworthy evidence that can provide an objective perspective on what is available to inform a decision. A secondary goal of the Center is capacity building and the training of future researchers and clinicians who are interested in producing these types of documents with the standards that Cochrane has established. Cochrane is distinguished for the rigor of its reviews — committed to high quality methods, transparency and engagement with patients and other consumers.

How do systematic reviews impact oral healthcare delivery and policy? Who makes the case that a new intervention creates more good than harm? That is where these types of reviews can play a key role in helping to accelerate the process of translating research findings into practice and improve outcomes. In addition, the pace of the biomedical literature makes evidence-informed decision-making a daunting task. In a systematic review, investigators look at the totality of the body of evidence very closely, summarize the findings, and put it in a simple format that is outcome-centered

and trustworthy. Systematic reviews simplify the process of evidence-based decision making for clinicians — they can read this one review document rather than all the published studies.

How is our Center filling any void in this area? Currently, there is an underutilization of systematic reviews for high-level decisionmaking. One speciﬁc goal for our Center is to be able to connect these reviews with policymakers and other stakeholders. Currently, that partnership is still suboptimal and needs to be improved to truly make policies and guidelines evidence based, which is the ultimate goal.

How long does it take to do a systematic review? A novel review involves a one- to two-year process with a multidisciplinary group of statisticians, methodologists, information specialists, clinical experts, and consumers. We are gathering investigators from all over the world to produce these reviews. Currently, Cochrane Oral Health has over 1,600 collaborators and over 900 review authors from more than 40 different countries. All reviews produced through our Center will become part of the Cochrane Library (www.cochranelibrary.com).

Addressing the Opioid Epidemic with a Personal Approach New Guideline Details Acute Dental Pain Management Strategies

elping to advance evidence-informed clinical and population-level decision-making is a primary focus of the School’s Center for Integrative Global Oral Health (CIGOH), with faculty involved in producing systematic reviews and clinical practice guidelines across the varied areas of dentistry. Among recent projects, Dr. Alonso Carrasco-Labra, Associate Professor and Director of the Cochrane Oral Health Collaborating Center at Penn Dental Medicine, and Olivia Urquhart, a data analyst and instructor with CIGOH, co-led the development of a new clinical practice guideline for managing acute pain in children, adolescent, and adult dental patients. According to the new guideline developed by CIGOH, the American Dental Association (ADA), and the University of Pittsburgh School of Dental Medicine, nonsteroidal anti-inflammatory drugs (NSAIDs) taken alone or along with acetaminophen are recommended as first-line treatments for managing short-term dental pain in adults and adolescents aged 12 or older. The guideline has been endorsed by the ADA. Based on a review of the available evidence, a guideline panel concluded that, when used as directed, NSAIDs (like ibuprofen and naproxen) alone or in combination with acetaminophen can effectively manage pain after having a tooth removed or when experiencing a toothache when dental care is not immediately available. The guideline also offers clinicians recommendations for prescribing opioid medications in the limited circumstances in which they may be appropriate. These include avoiding “just in case” prescriptions, engaging patients in shared decision-making, and exerting extreme caution when prescribing opioids to adolescents and young adults. When prescribing opioids, the guideline suggests advising patients on proper storage and disposal and considering any risk factors for opioid misuse and serious adverse events. This is the second of two guidelines on acute dental pain management from this research team. A previous set of recommendations for pediatric patients was published in 2023. Both guidelines are published in the Journal of the American Dental Association (JADA) and can be found at ada.org/painmanagement. Carrasco-Labra also participated as a guideline panel member in developing a clinical practice guideline for managing chronic pain associated with temporomandibular disorders, published in The British Medical Journal in December 2023.

Although opioids can provide much-needed pain relief to patients, especially after undergoing surgical procedures, these drugs are highly addictive. Abuse or overuse of prescription or illegal opioids was responsible for nearly 645,000 overdose deaths from 1999 to 2021, fueling a national crisis. But, not every patient given a prescription actually needs it. “Most people do very well with just over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs), like ibuprofen, as their primary method for pain relief after wisdom tooth extraction surgery, but others need something extra,” says Katherine Theken, Assistant Professor in the Department of Oral and Maxillofacial Surgery and Pharmacology, who is studying the possibility of using precision medicine approaches to predict the best pain management strategy. The key, notes Theken, is to figure out ahead of time which patients will need that something extra — an opioid. Understanding the molecular mechanisms that enable individuals to achieve relief with NSAIDS may identify biomarkers that are predictive of response, so only those patients requiring opioids get them. Underlying this variability is the fact that many different molecular processes can contribute to the sensation of pain. One way that pain is felt is through the production of prostaglandins, hormone-like fats that affect processes involved in several bodily functions, including pain. NSAIDs work by putting the brakes on prostaglandin production.

“Most people do very well with just overthe-counter drugs, like ibuprofen, as their primary method for pain relief after wisdom tooth extraction surgery, but others need something extra.” — Katherine Theken

In a small initial study of patients who had surgery to remove their wisdom teeth, Theken’s team found that, indeed, those who had higher levels of prostaglandins after the procedure responded better to NSAIDs. Theken observed that other factors were also at play, so her lab is now conducting a larger study to take the findings further. Expanding on this work, the current clinical study will determine whether substances in patients’ blood or urine can predict how they will respond to ibuprofen after wisdom tooth extraction. Although it’s too early to draw conclusions, Theken has gleaned a few interesting insights. For example, women and people who have a higher body mass index are more likely to require opioids in addition to an NSAID for pain management. “We’re planning to look more into what’s going on at a molecular level with regard to inflammation and the response after surgery,” says Theken, who, in a future study, will also be looking at whether gut microbes play a role in the variable pain response. 2 024 | RESEARCH AT PENN DENTAL MEDICINE

Dental Diagnosis & Treatment

Diagnosing Oral Lesions with an Eye to Systemic Connections

“It is often the dentist who makes the connection between oral and systemic conditions, which starts the patient down the path toward effective management.” — Eric T. Stoopler

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ost often, an ulcer or sore in the mouth is little more than an annoy annoyance. But sometimes such an abnormality can herald a bigger problem. “For some serious, systemic diseases, we see the first sign within the mouth,” says Thomas P. Sollecito, Professor and Chair of the Department of Oral Medicine. The list of such conditions is long: cancer, digestive, autoimmune and blood diseases can all first manifest through oral lesions. “Because the presentation of diseases in the mouth can be harbingers of underlying issues, our responsibility is to the patient’s health as a whole and to approach oral care from a broader perspective,” says SSollecito. He and his frequent collaborator Eric T. Stoopler, also a Professor in the Department of Oral Medicine, often encounter patients with these tell-tale abnormalities. These patients may see multiple physicians in their unsuc unsuccessful attempts to get help for sores, masses, or other changes that have often gone undiagnosed for years, according to Stoopler. “It is often the dentist who makes the connection between oral and systemic conditions, which starts the patient down the path toward effective manage management,” Stoopler says. In one case, Sollecito determined that ulcers in a patient’s mouth resulted from an immune-related condition, mucous membrane pemphigoid, which also attacks the eyes. This diagnosis led the patient to get treatment for diseaserelated eye problems that could have had the potential to blind him. In another, a middle-aged patient presented with ulcers unusual for his age. Blood tests revealed anemia and follow-up gastrointestinal testing lead to a cancerous polyp — a discovery that caught the cancer at its early stage before pro progression. Through professional publications, Sollecito and Stoopler educate other physicians to recognize the causes of oral lesions. Meanwhile, they also study the conditions responsible. One area of study is oral lichen planus, which commonly manifests as white lacey lines and/or sores. Often, lichen planus clears on its own, but has the potential to develop into oral squamous cell cancer. Currently, there is no way to stop this transition, however, Sollecito and Stoopler are studying what may impact it. In one study, they found that treatment with topical steroids delayed the development of malignancy by about four years, but with an important caveat. Steroids can promote the growth of the fungus Candida, and their studies also showed that growth of this fungi accelerates the development of oral cancer, making measures to inhibit fungal growth an important part of care for patients with the condition. Lichen planus was included in an assessment Sollecito and other colleagues conducted of the likelihood of oral lesions of varied origin becoming malignant. Through it, they uncovered an ever-present risk of transformation to cancer, requiring patients with lesions to be closely monitored. Together, Sollecito and Stoopler are now examining tissue samples taken from oral lesions that ultimately became cancerous, including those that started out as lichen planus and other white lesions. By looking at the architecture of the skin and the cells within it, they are identifying patterns they can link to a more rapid versus a slower transformation to cancer. In the future, they hope to identify molecular markers that indicate how quickly cancer will develop, a less invasive way to help physicians identify more aggressive cases and tailor treatment to the patient’s needs.

SCHOOL LEADERSHIP Mark S. Wolff, DDS, PhD Morton Amsterdam Dean Faizan Alawi, DDS Associate Dean, Academic Affairs Hydar Ali, PhD Associate Dean, Faculty Diversity & Mentorship Markus Blatz, DMD, PhD, Dr med dent habil Assistant Dean, Digital Innovation & Professional Development Kathleen Boesze-Battaglia, PhD Assistant Dean, Academic Initiatives Bruce Brandolin Assistant Dean, Intramural Practice and External Partnerships Maren Gaughan Associate Dean, Leadership Giving Joan Gluch, RDH, PhD Associate Dean, Academic Policies Marco Georeno Associate Dean, Finance and CFO Dana Graves, DMD, DMSc Vice Dean, Scholarship & Research

Kelly Jordan-Scuitto Associate Dean, Organizational Effectiveness Elizabeth Ketterlinus Vice Dean, Institutional Advancement Syngcuk Kim, DDS, PhD, MD (Hon) Associate Dean, Global Affairs

DEPARTMENT CHAIRS

CENTER DIRECTORS

BASIC & TRANSLATIONAL SCIENCES

CARE CENTER FOR PERSONS WITH DISABILITIES

Robert P. Ricciardi, MA, PhD Acting Chair Henry Daniell, PhD Vice Chair ENDODONTICS

Miriam Robbins, DDS, MS Director Alicia Risner-Bauman, DDS, FADPD, DABSCD Associate Director

Sean C. Meehan, DMD Assistant Dean, Office of Clinical Affairs, Chief Dental Officer

Bekir Karabucak, DMD, MS Chair ORAL MEDICINE

Temitope Omolehinwa, BDS, DMD, DScD Associate Director

Mark Mitchell Assistant Dean, Admissions

Thomas Sollecito, DMD, FDS, RCSEd Chair

CENTER FOR CLINICAL & TRANSLATIONAL RESEARCH

Sinem Esra Sahingur, DDS, MS, PhD Associate Dean, Graduate Studies and Student Research

ORAL & MAXILLOFACIAL SURGERY & PHARMACOLOGY

Eugene Ko, DDS Deputy Director

Anh D. Le, DDS, PhD Chair

CENTER FOR INNOVATION & PRECISION DENTISTRY (CiPD)

Thomas P. Sollecito, DMD Associate Dean, Hospital and Extramural Affairs

ORTHODONTICS

Michel Koo, DDS, PhD Co-Founding Director

Kim Wolcott Chief of Staff, Senior Director, Faculty Affairs

Nipul Tanna, DMD, MS Associate Chair

Najeed Saleh, DMD Associate Dean, Clinical Affairs

Uri Hangorsky, DDS, MS Associate Dean, Student Life

Chun-Hsi Chung, DMD, MS Chair

PERIODONTICS

Rodrigo Neiva, DDS, MS Chair PREVENTIVE & RESTORATIVE SCIENCES

PAGE 2: top, Min Jun Oh (Koo/Steager Lab); left, Altmeyers Encyclopedia;

Markus B. Blatz, DMD, PhD, Prof Dr med dent Chair

PAGE 11: Illustration by Katie Vicari

DIVISION OF COMMUNITY ORAL HEALTH

PHOTO CREDITS right, AdobeStock/JosLuis

PAGE 15: top, AdobeStock/Ilia; bottom, Campagno KE, Lu W, Jassim AH,

Albalawi F, Cenaj A, Tso HY, Clark SP, Sripinun P, Gómez NM, Mitchell CH. Rapid morphologic changes to microglial cells and upregulation of mixed microglial activation state markers induced by P2X7 receptor stimulation and increased intraocular pressure. J Neuroinflammation.18:217, 2021.

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Joan Gluch, PhD, RDH, PHSHP Division Chief DIVISION OF PEDIATRIC DENTISTRY

CENTER FOR INTEGRATIVE GLOBAL ORAL HEALTH (CIGOH)

Michael Glick, DMD Executive Director COCHRANE ORAL HEALTH COLLABORATING CENTER AT PENN DENTAL MEDICINE

Alonso Carrasco-Labra, DDS, MSc, PhD Director DIGITAL DESIGN & MILLING CENTER

Julian Conejo, DDS, MSc Director of Chairside Digital Dentistry CENTER FOR VIRTUAL TREATMENT PLANNING

Michael Bergler, MDT Director

Betty Hajishengallis, DMD, DDS, PhD, MSc Division Chief DIVISION OF RESTORATIVE DENTISTRY

David Hershkowitz, DDS, AAS Division Chief

PAGE 36: Volume 38.4 of Dermatologic Clinics, October 2020, Elsevier PHOTOGRAPHERS

Mark Garvin: Page 23 (top), 24 (top), 25 (left) Kevin Monko: Page 3 (bottom), 5 (top), 14 (top), 27 (top), 34 Peter Olson: Page 2, 5 (bottom), 8, 12, 30

ON THE BACK COVER: Co-culturing human-induced pluripotent stem cell derived astrocytes,

microglia, and neurons in one dish provides an effective model to study intercellular impact of HIV and other diseases of the central nervous system. (From the lab of Dr. Kelly Jordan-Sciutto, see story p. 9)

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