Harvard School of Dental Medicine

Introduction: An increase in the size of the U.S. population is accompanied by an increasingly aging population, with adults 65 and older experiencing the highest prevalence of edentulism. Studies indicate that 25% of persons aged 35 to 74 are edentulous and require a high amount of prosthetic care. These patterns demonstrate an increasing quantity of older patients with systemic medical conditions seeking oral care. Complete edentulism is associated with several comorbid conditions, where pre-treatment and peri-treatment oral health status are critical for a favorable long-term prognosis of prosthodontic treatment. It is essential to recognize that systemic diseases may affect or limit restorative treatment options, complicating treatment planning and implementation. Purpose: This study aims to explore the oral health needs among patients identified as medically complex. In addition, to determine the most prevalent systemic conditions among medically complex patients who are concurrently seeking dental care, evaluate the major restorative and periodontal treatments needed, and explore how these systemic conditions limit their restorative options. Methods: An estimate of 80 e-records from the Medically Complex Clinic at Harvard Dental Center will be evaluated retrospectively using a statistical software package for analysis. Result: Of the 62 patients treated at the Medical Complex Clinic of HSDM that were evaluated for this study, 47 were diagnosed with cancer. Twenty-four had at least one episode of osteoradionecrosis. We could observe that the MCC of HSDM receives a substantial number of cancer patient referrals. Conclusion: Cancer is a treatment-limiting factor that severely affects a patient’s oral health. Cancer treatment can create immunosuppression, which affects patient's ability to fight infections. The recorded data allowed us to better understand the medically complex patients that are referred. With this information, existing protocols can be updated and adjusted to meet the needs of the patient pool. This may promote further brainstorming of preventive oral therapies that can be given concurrently with treating systemic conditions.

The role of bacterial plaque and its role as the primary etiology for periodontal disease is well established. The treatment of periodontitis and maintenance of periodontal health is dependent on the elimination of plaque, and calculus, and establishing a stringent dental hygiene maintenance program. The non-surgical treatment of periodontitis relies heavily on scaling and root planning (SRP) therapy to remove subgingival deposits and toxins to allow for the reattachment of periodontal and gingival fibers.

A multitude of sharpening devices are available for maintaining periodontal hand instrument sharpness. However, the impact of these devices on the cutting-edge morphology of periodontal curettes, and subsequently on root surface roughness remains unclear. . Using sharp periodontal hand curettes and scalers is pivotal in the thoroughness of calculus removal, root planing, and achieving a smooth root surface.

This study aims to investigate the effects of sharpening cards, sharpening stones of varying grits, and the Sidekick® on the lateral edge of an unused factory sharpened Gracey 11/12 curette and to evaluate the influence of these sharpened curettes on root surface roughness post-SRP.

The results show that there was a statistically significant difference between the roughness of the medium, fine, and extra-fine cards and between the method of sharpening (horizontal vs vertical) using a stationary-instrument-moving-stone technique. However, no statistically significant difference was observed in the roughness of curette edges sharpened by cards and stones of varying grit sizes, nor in the root surfaces root-planed by these sharpened curettes. This suggests that irrespective of the card or stone utilized for sharpening, the resulting edge of the curette yields relatively similar smooth surfaces. Further studies will need to be conducted to verify this finding.

Parathyroid hormone (PTH), an 84-amino acid peptide synthesized and secreted by the parathyroid glands, plays a crucial role in calcium and phosphorus metabolism, acting on bone and the kidney. Although PTH can induce both bone catabolic and anabolic effects, depending on the duration and periodicity of exposure, daily injections increase bone density in both human and animal studies. PTH acts directly on osteoblasts and osteocytes, indirectly influencing osteoclasts through interactions with osteoblasts and osteocytes. Osteocytes constitute 90%-95% of all bone cells in normal adult bone. They play an essential role in bone homeostasis by secreting various regulators that control the activity of both osteoblasts and osteoclasts during bone modeling and remodeling processes. Additionally, osteocytes release an endocrine factor, FGF23, to regulate other organs, such as the kidneys, playing a crucial role in maintaining phosphate and vitamin D homeostasis. The Baron-Gori Lab's bulk-RNA seq database from osteocyte-enriched cell populations in response to in vivo PTH treatment in mice reported an increase in several gene expressions, with Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor (Bambi) being one of the most significantly upregulated genes. Furthermore, analysis of pooled scRNA-seq public databases of bone cells revealed that BAMBI is expressed in a relatively restricted manner in osteocytes. BAMBI is a transmembrane protein pseudo-receptor that exhibits structural homology to the transforming growth factor β type I receptor (TGF-βRI) but lacks an intracellular kinase domain. It has been identified as a common transcriptional target and mediator between the Wnt and TGF-β signaling pathways, ultimately enhancing cellular growth by promoting Wnt signaling and inhibiting the TGF-β signaling pathway. Although an increase in BAMBI expression has been associated with the pathogenesis of various human pathologies, there is a limited body of literature on BAMBI's role in bone-related events, and its impact on osteocyte biology remains unexplored. Here, we demonstrated that during the differentiation of the OmGFP66 osteocyte cell line, Bambi was expressed at basal levels and continued to increase during differentiation. Importantly, Bambi expression was elevated with PTH treatment, both in a time-dependent manner—most prominently at the 1-hour time point with PTH 50 nM—and during long intermittent treatment with PTH 50 nM for 1 hour per day over 5 days. Subsequently, we knocked down Bambi expression using shRNA. Following the knockdown, we observed an upregulation of Sost and a significant increase in Tnfsf11 (RANKL) expression. Additionally, there was a decrease in non-phosphorylated β-catenin and an increase in pSMAD2, the signaling molecules of the Wnt and TGF-β pathways, respectively. Bambi knockdown (Bambi-KD) cells exhibited impaired osteocyte mineral deposition, as assessed by Alizarin Red staining, and decreased levels of the endocrine factor FGF23, as evidenced by reduced RNA and protein levels. Furthermore, the knockdown affected osteocyte morphology, with a reduction in phalloidin-stained osteocytic dendrites and decreased expression of dendritic markers Sp7 (Osterix) and Ostn (Osteocrin). Importantly, with PTH treatment, Bambi-KD cells demonstrated a significant increase in Sost expression, mirroring the inhibition of HDAC4/5 by phosphorylation, which facilitates MEF2C-driven Sost expression. Interestingly, despite reversing PTH's typical repression of Sost, Bambi-KD had no reversed effect on Tnfsf11 (RANKL) expression. Furthermore, in contrast to the usual upregulation of Fgf23 by PTH, we observed a significant downregulation, likely attributed to PTH's inability to suppress Sost in the knockdown cells. In summary, our findings reveal that BAMBI is implicated in osteocyte biology, potentially playing pivotal roles in osteocyte signaling, functions, and dendritic morphology. Notably, BAMBI may be essential for PTH, an agent routinely used to increase bone density in osteoporotic patients, to mediate the suppression of Sost and the increase in Fgf23 in osteocytes.

Alveolar bone serves to support and anchor teeth. It differs from most other skeletal tissue due to its unique origin from neural crest-derived mesenchymal cells and its formation through intramembranous ossification. The development of alveolar bone occurs coupled with tooth eruption through various signaling networks, including the parathyroid-related protein (PTHrP) pathway. Salt inducible kinases (SIKs) are important downstream regulators of PTH/PTHrP signaling in long bones, growth plate chondrocytes, and kidneys. Previous studies reported that the deletion of SIKs increases bone turnover and trabecular bone mass in long bones. However, the role of SIKs in alveolar bone remains unknown. To address this question, we used Ubq-CreERt2; Sik2f/f; Sik3f/f mice to perform tamoxifen-inducible global deletion in 3 different mouse models: development, adult onset, and following molar extraction. Tamoxifen-induced global deletion of SIK2/SIK3 was performed at postnatal day 3 in the developmental model, at postnatal week 12 in adult mice, and in some adult mice tooth extraction was performed 10 weeks post-tamoxifen injection. Surprisingly, compared to wild-type controls, SIK2/SIK3 mutant mice showed reduced alveolar bone mass in all models. Micro-CT demonstrated less bone volume and mineralization in the mutant alveolar bone lacking SIK2 and SIK3. Reduced alveolar bone in SIK2/SIK3 mutants was associated with reduced bone formation as assessed by calcein labeling. Furthermore, TRAP staining failed to show increased osteoclast activity, indicating that the alveolar bone loss is primarily caused by reduced bone formation rather than increased bone resorption. In regions of absent alveolar bone, increased alkaline phosphatase with reduced osteocalcin expression was noted, suggesting increased immature osteoblasts in the mutant mice lacking SIK2/SIK3. At 4 weeks post-tooth extraction, control maxillary first molar sockets displayed complete bone healing, while SIK2/SIK3 mutant mice exhibited absent bone healing in the tooth socket. The mutant extraction socket was filled with fibrous-like tissue showing increased alkaline phosphatase staining with decreased osteocalcin expression, consistent with the developmental model result. Our results demonstrate that the absence of SIK2/SIK3 impairs terminal osteoblast maturation in alveolar bone. Taken together, these findings emphasize the importance of SIKs in alveolar bone osteoblast differentiation, maturation, and alveolar bone formation.

Contemporary orthodontic practices slowly adopt modern technologies, new treatment tools, and soft-tissue-based diagnostic approaches. Classic diagnostic tools, especially those concerning the cephalometric positioning of the incisors, are gradually being discarded from routine use. However, it is important to understand whether any of the classic treatment planning tools have a direct relation to perceived success in facial harmony. In this study, we aimed to evaluate the perceived success in posttreatment facial outcomes in a randomized orthodontic population using several components of the Steiner analysis. Inclusion criteria included an ANB angle ranging from -1 to 6 degrees and an SN-GoGn angle ranging between 26 and 39 degrees at the beginning of the treatment. In addition, to be enrolled in the study, the selected patients were required to have a Class I canine relationship, normal overjet and overbite, standard curves for Wilson and Spee, and absence of visible spacing at the end of the treatment. The records of 119 qualifying orthodontic patients were included in the study. Ten orthodontists were presented with pre-and posttreatment cephalometric radiographs of the sample and asked to review the facial outcomes of the treatment using a ten-point visualized analog scale (VAS). Based on the evaluations of the panel, three groups were formed: ideal (n=20), average (n=82), and below average (n=17). Cephalometric variables from the Steiner analysis were compared between the three groups using a one-way analysis of variance (ANOVA). In addition, Pearson Correlation coefficients were calculated between the evaluations of the judges and the cephalometric variables. The level of significance was set at p.01. Of the cephalometric variables used in this study, the measurements related to the position and inclination of the mandibular incisors and the position of the lower lip were significantly different between the three groups (p.01). Bonferroni post hoc comparisons showed that when the below average group was compared to the other two groups (p.01), significant differences occurred for L1-NB (mm and degrees) and the lower Lip to E line measurements. In addition, there was a significant difference in IMPA measurement between the below average and average groups (p.01). Significant negative correlations occurred between the judges' esthetic scores and the cephalometric variables, including all the mandibular incisor measurements and the posttreatment lower-lip position. Increased lower incisor proclination and protrusion both before and after treatment as well as posttreatment lower lip protrusion had strong affiliations with poor facial outcomes.

The study of peri-implant diseases is constantly evolving, from the best practices for detecting mucositis to treating mucositis to return the patient to peri-implant health and the interventions that can be used to treat peri-implantitis. Probing dental implants is the current standard of care for early diagnosis, prevention, and intervention. Ultrasonography in dentistry is an innovative and non-invasive tool that can be useful in the early detection of peri-implant disease to measure tissue perfusion. The pilot case-control study aimed to assess ultrasonographic features and doppler tissue perfusion at healthy implants (HE) and sites diagnosed with peri-implant mucositis (PM); changes within clinical, ultrasonographic, and patient-reported outcomes were assessed after non-surgical therapy. Twenty (20) patients receiving treatment at Harvard Dental Center with two non-adjacent implants diagnosed as HE and PM were included in the present study. The peri-implant region of interest was scanned using the ultrasound before non-surgical therapy at T0 and four weeks after non-surgical treatment at T1. Clinical measurements such as probing depth (PD), keratinized mucosa width (KMW), and peri-implant soft tissue dehiscence (PSTD) of the healthy and diseased implants were assessed. Tissue perfusion, mucosal thickness (MT), buccal bone distance (BBD), buccal bone thickness (BBT), supracrestal tissue height (STH), and mesial and distal papilla height (PH) at the healthy and diseased implants at the midfacial and interproximal was measured at both time points. Non-surgical therapy was performed during the same visit at the diseased implant site, and oral hygiene techniques were reviewed. Thirteen of the twenty diseased implant sites showed complete resolution of the inflammatory condition at the re-evaluation visit. A reduction of mucosal thickness was observed in its coronal portion, while no changes were observed in the most apical areas. PM sites displayed an ultrasonographic feature of an isolated darker area, analogous to a radiolucent area on an x-ray, adjacent to the junctional epithelial surface of the peri-implant mucosa, termed a hypoechoic supracrestal area/lesion (HSA). Without BOP on the midfacial aspect, 19 out of 20 (95%) ultrasound scans did not exhibit HSA. The vertical extension of the HSA (in a corona-apical dimension) was 5.75 mm at baseline and 3.2 mm at the re-evaluation. At the same time, the area of the HSA was 4.94 mm2 at baseline and 2.61 mm2 at the re-evaluation visit. Color Doppler Velocity and Power Doppler were significantly increased in PM sites compared to the HE group (p.01), and their magnitude was correlated to the area of the HSA (p.05). Echo-Doppler ultrasonography is a valuable tool for the characterization of healthy and diseased implant sites in a non-invasive manner. Ultrasonographic outcomes, such as the presence/absence of a “lesion” (HSA) and Doppler tissue perfusion, correlated with the clinical diagnosis of health vs. disease while providing additional information that may be associated with the severity of the condition, the likelihood of resolution, and diseased progression over time. Future studies with longer follow-ups are needed to validate these preliminary findings.

Introduction: Secreted Semaphorin-3F (SEMA3F) is a chemorepulsive protein in Neuropilin-2 (NRP2)-expressing cells. It guides nervous and circulatory system patterning during embryogenesis. SEMA3F is frequently deleted in human small cell lung cancer and other cancer types including head and neck squamous cell carcinoma, as such it is a postulated as a potential tumor suppressor gene. Recent data from our group has identified NRP2 in CD4+ T cells. Aims: Our goal is to investigate the role of endogenous Sema3F and NRP2 in oral carcinogenesis, tumor progression, and tumor immunity. Methods: Different stages of oral carcinogenesis were evaluated for NRP2 expression. Sema3F expression was evaluated in OSCC cell lines and compared to normal mucosa. Transgenic mice with Sema3F deletion in adult keratinocytes were used to analyze cancer initiation and progression before or after exposure to carcinogen compared to controls. Tongues of Nrp2-global knockout (KO) or wildtype mice were injected with syngeneic oral cancer xenografts and tumors were evaluated for CD4+ and CD8+ T cell infiltration. Antigen-induced hypersensitivity reactions in Sema3F-KO, K14-Sema3F-iKO, Nrp2-KO, and CD4-Nrp2-KO mice were used to evaluate for ear swelling and CD4+ T cell infiltration in experimental mice compared to controls. Results: Nrp2 expression was absent in normal oral and skin epithelium but was upregulated during the late stages of dysplasia in both human and mouse carcinogenesis. Conversely, Sema3F was present in normal mouse tongue epithelium but decreased in OSCC cell lines. When Sema3F was deleted prior to exposure to carcinogen, the majority of control mice (Sema3F-intact) (25 out of 30) developed carcinoma in situ (CIS) or invasive oral squamous cell carcinoma (OSCC), while only 17% of K14-Sema3F-KO mice (4 out of 23) developed CIS, with none progressing to OSCC. When Sema3F knockout was induced post-carcinogen exposure, mice that lacked Sema3F in their epithelium exhibited more infiltrative tumors compared to controls. Oral cancer grafts displayed increased infiltration of CD4+ and CD8+ lymphocytes in Nrp2-/- mice compared to Nrp2+/+ mice. Mice lacking epithelial Sema3F or Nrp2-expressing T cells exhibited prolonged inflammation, tissue swelling, and CD4+ T cell infiltration, whereas control mice resolved quickly. Conclusion: Sema3F is not a tumor suppressor in OSCC, primarily because normal oral epithelium lacks Nrp2 expression and Sema3F cannot function in an autocrine fashion to inhibit carcinogenesis. Furthermore, mice that lacked Sema3F in their epithelium exhibited reduced carcinogenesis. Cancerous and inflamed tissues lacking Sema3F in the epithelium or Nrp2 on CD4+ T cells exhibited increased immune surveillance and T lymphocyte recruitment abrogating cancer initiation and preventing oral cancer progression. We conclude that the SEMA3F/NRP2 pathway suppresses host immunity and provides new potential targets for immunotherapy in oral cancer.

Introduction: Presently, patients’ aligner change frequency and wear time are primarily determined by the clinician’s judgment rather than by literature comparing distinct protocols. The aims of this study are to evaluate DentalMonitoring (DM; Paris, France)’s ability to personalize patients’ treatment schedules and to determine the effect of full-time versus half-time wear as well as usage versus non-usage of VPro+ (Propel Orthodontics; Ossining, NY) on treatment duration. Methods: Clear aligner patients at the Harvard School of Dental Medicine Orthodontics Clinics were randomized into 4 groups: Group 1 (full-time wear, no VPro+), Group 2 (full-time wear, VPro+), Group 3 (half-time wear, no VPro+), and Group 4 (half-time wear, VPro+). All groups submitted intraoral scans to DM every 4 days and followed DM’s aligner change instructions for 10 trays. Treatment durations for each group were compared with standard protocols of 7-14 days/tray and between groups by a statistician blinded to the group descriptions. Results: Of the 76 patients enrolled, 48 patients (12 per group) completed the study. All groups progressed through aligners more efficiently (4.315-5.370 days/tray) than standard protocols of 7-14 days/tray (p.01). Full-time groups (4.407 days/tray) progressed through aligners more efficiently (p=0.0007) than half-time groups (5.310 days/tray). No effect (p=0.6931) was observed between VPro+ (4.782 days/tray) and no VPro+ groups (4.935 days/tray). Conclusions: Clear aligner protocols should be individualized for each patient and each set of aligners. Patients scanning every 4 days with DM progressed through aligners more quickly than a standardized change frequency of 7-14 days/tray. Treatment efficiency was improved with increased daily wear time but not affected by VPro+ usage. DentalMonitoring can serve as a valuable treatment adjunct and future research tool by improving the efficiency and individualization of clear aligner therapy.

Registration: This randomized clinical trial was registered and reported at ClinicalTrials.gov (NCT04260633). Protocol: The protocol was not published before trial commencement. Funding: This study was funded by an AAO Foundation Biomedical Research Award.

Abstract

Effect of endodontic treatment on local inflammatory biomarkers in teeth with apical periodontitis

Sam Yun DDS, Joshua Hong DMD MMSc, Pallavi Suhag DDS MMSc, Summer Tan, Ozge Erdogan DDS DMSc, Jennifer Gibbs DDS PhD

Introduction: The purpose of this study was to measure biomarkers from teeth with apical periodontitis through gingival crevicular fluid (GCF) and periapical fluid( PF) to capture the host response after root canal therapy.

Methodology: Participants with pulpal necrosis with radiographic signs of periapical pathology, requiring primary root canal treatment (RCT) were recruited for the study. Clinical/radiographic data were collected at each visit. GCF was collected from a healthy control tooth and a diseased tooth pre-operatively (1st visit), at the 2nd treatment visit, and at 1-month and 6-month follow up visits. PF samples were collected at the 1st visit after cleaning and shaping and at the 2nd visit before obturation. Samples were analyzed using multiplex immuno-assays (Meso Scale Discovery), for multiple targets.

Results: For this analysis, samples from 11 participants were analyzed. When comparing GCF biomarkers between study visit 1 and 2: In the success group, there was a decrease or no change in expression for IL-1B, IL-4, IL-13, MMP-2, and MMP-9 and an increase in expression for IL-2. In the failure group, there was an increase in expression for all biomarkers. When comparing PF biomarkers between study visit 1 and 2: In the success group, there was a decrease in expression in IL-1b, IL-4, MMP-2, and MMP-9 and an increase in expression in IL-13. In the failure group, there was an increase in expression for all biomarkers except IL-4. In both groups, IL-2 was not detected. When analyzing the correlation between GCF/PF in study visit 1 and 2: The first study visit had a moderate positive correlation with the coefficient of determination being 0.3169 and the second study visit had a stronger positive correlation with the coefficient of determination being 0.7884. Lastly, when comparing GCF biomarkers between study visit 1 and 4: In the success group, there was a decrease in expression in all cytokines except IL-1b. In the failure group, there was an increase in expression in IL-1b, IL-2, MMP-2, no change in expression, in MMP-2, and decrease in expression in IL-4 and IL-13.

Conclusion: IL-1b and MMP-9 showed decrease in expression in the GCF and PF samples in the success group and increase in expression in the failure group at the early time points. MMP-2 showed increase in expression in the GCF and PF samples in the failure group at the early time points, and decrease in expression in the success group at the late time point. IL-4 showed decrease in expression in the GCF and PF samples in the success group at late time point. IL-2 showed increase in expression in the GCF and PF samples in the failure group at both the early and late time points. For study visits 1 and 2, GCF and PF showed similar biomarker patterns, and therefore GCF can be a good reflection of what is going on in the periapical region. Potential biomarkers that may merit further investigation can include IL-2, MMP-2, and MMP-9.

Introduction: Dental pulp tissue is densely innervated and it has unique immunological characteristics. Upon injury such as deep caries, exposing the pulp, a strong neuronal and immune response occurs which modulates pain and the tissue damage. Our knowledge on the dynamics of initial innate immune response, whether this response is modulated by sensory afferents of the pulp, and how this innate immune response alters tissue damage and/or pain outcome is limited. Materials and Methods: Dental pulp exposure in mice was used as the pulpitis model. To investigate the innate immune response, pulp tissue was collected from the permanent molars at different time points and flow cytometry was performed. To investigate tissue damage, H&E staining was performed. Immunohistochemistry and in-situ hybridization were performed to capture spatial changes of innate immune cells, sensory afferents, and bacteria invasion, respectively. Finally, mechanical pain sensitivity was captured by facial Von Frey stimulation and spontaneous pain was captured using the Mouse Grimace Scale. Data were analyzed using two-way ANOVA, t-tests, or repeated measure of ANOVA with appropriate multiple comparison tests. Results: We found that neutrophils constituted 70-90% of immune cell populations up to day 7. We also found that the neuropeptide CGRP, released from sensory afferents, contributed to the recruitment of myeloid cells (p=0.017) while also increasing spontaneous pain at day 1 (p=0.02). Moreover, when we depleted neutrophils and monocytes, we found that there was more tissue necrosis at day 6 (p=0.002) and bacteria had penetrated deeper parts of the tissue, whereas mechanical pain was lower compared to control group (p=0.03). Conclusions: Neutrophils and monocytes are crucial to protect dental pulp tissue from further bacterial penetration and tissue damage while also contributing to mechanical pain sensitivity. We also found that CGRP modulates innate immune responses either by causing vasodilation or by directly mediating neutrophil recruitment while contributing to spontaneous pain behavior during pulpitis.