Jill A. Helms

The aims of this study were 1) to determine how ligature-induced periodontitis (LIP) disrupts barrier functions of the junctional epithelium (JE); 2) to determine, using a genetic approach, the necessity of Wnt signaling for JE barrier functions; and 3) to test, using a biochemical strategy, whether a WNT therapeutic is sufficient to improve barrier functions of a pocket epithelium. In a murine model of LIP, quantitative analyses performed at multiple time points assessed epithelial apoptosis; expression of attachment proteins laminin 5 and β4 integrin, inflammation, and bone resorption. Axin2 ^CreERT2/+ ; R26R ^mTmG/+ mice were used to evaluate how LIP impacted Wnt-responsive cells and their progeny, and K14 ^CreERT2/+ ;Wls ^fl/fl mice were used to determine whether Wnt signaling was required for JE barrier functions. In some cases, LIP was followed by a recovery period to assess molecular changes in pocket epithelium, and in a subset of these mice, a liposomal formulation of human WNT3A protein (L-WNT3A) was tested for its effects on early repair dynamics in pocket epithelium. LIP triggered apoptosis, significantly reduced expression of laminin 5 and β4 integrin in the JE, and disrupted the Wnt-responsive compartment; these epithelial changes were accompanied by inflammation and alveolar bone resorption. Reepithelialization occurred even with a ligature present, but this pocket epithelium had compromised barrier functions. Wntless (Wls) deletion was sufficient to convert a JE into pocket epithelium, while topical L-WNT3A treatment was sufficient to increase hemidesmosomal protein expression in pocket epithelium and reduce inflammation at early time points. LIP destroys barrier functions and thus converts a JE into pocket epithelium. Deletion of epithelial Wls demonstrates that this conversion is a Wnt-dependent event. L-WNT3A restores some early barrier features to pocket epithelium; future studies will focus on the durability of these effects.

Epithelial rests of Malassez (ERMs) constitute a 3-dimensional epithelial meshwork extending from the tooth apex to the junctional epithelium (JE). This position, coupled with observations from a variety of injury models, suggests that ERMs may be involved in sensing disruptions to the mechanical integrity of the periodontium. To test this hypothesis, finite element modeling was employed to predict strain states in tissues surrounding ERMs (e.g., the periodontal ligament [PDL], supracrestal connective tissues, and the JE), which, by virtue of their attachment to the tooth surface, experience distortional strains during mastication. An in vivo hyper-loading mouse model was employed to map these distortional strains to location-specific and time-dependent morphological changes in ERMs. Quantitative immunohistochemistry was used to interrogate ERMs for changes in the expression of Piezo1, β4 integrin, plectin, YAP, TRPV4, and phosphorylated FAK. Collagen and oxytalan fiber organization around ERMs was compared between normal- and hyper-loading conditions. Axin2Cre ^ERT2 ; R26R ^mTmG/+ mice were used to lineage-trace Wnt-responsive ERMs under normal and hyper-loading conditions, and Wnt signaling from ERMs and the JE was disrupted using K14Cre ^ERT2 ; Wls ^fl/fl mice. Collectively, these analyses predicted that hyper-loading produced high distortional strains in the JE and supracrestal connective tissues, which correlated with sites of ERM enlargement. ERMs expressed mechanosensory proteins, whose levels were significantly elevated by hyper-loading. Load-activated ERMs were Wnt-responsive, and if Wnt secretion was blocked, then ERMs no longer expressed mechanosensory proteins and no longer enlarged in response to hyper-loading. A deterioration in the JE attachment and the PDL followed. Thus, the ERM network is positioned to sense distortional strains produced by mastication; they possess the molecular machinery to sense and respond to masticatory forces and, via a Wnt-dependent mechanism, coordinate tissue-level responses to mechanical loading in the PDL, supracrestal connective tissues, and the JE.

Implant dentistry has historically centered on osseointegration, yet it is now evident that soft‐tissue integration is equally critical for long‐term success. The stability of bone‐anchored dental implants hinges on the establishment and maintenance of a stable attachment that mirrors, at least functionally, the supracrestal attachment around teeth. This review synthesizes current understanding of how the soft tissue–implant interface forms and how it is maintained over time. This interface comprises (i) a rapidly developing peri‐implant epithelium and (ii) a connective‐tissue component that gradually rebuilds its collagen architecture after implant placement. Although current knowledge is surprisingly limited, evidence indicates that neither the epithelium nor the connective tissues form a true attachment to the implant surface or prosthetic components. Soft‐tissue dimensions, the role of keratinization of the peri‐implant epithelium, the characteristic presence of inflammation within peri‐implant connective tissue, and the biological and clinical determinants shaping the formation and maintenance of this interface are discussed, highlighting major knowledge gaps for future research. Finally, we examine the contribution of soft‐tissue phenotype to peri‐implant diseases and soft‐tissue dehiscence. A better understanding of the principles governing the development and homeostasis of soft tissue–implant interfaces may inform strategies to improve the barrier function of peri‐implant soft tissues around all bone‐anchored implants, ultimately guiding future translational research to enhance implant longevity.

Once considered vestigial structures, epithelial rests of Malassez (ERMs) have recently been implicated in periodontal repair, largely based on their location in the periodontal ligament space, adjacent to damaged tissues. This study decodes what ERM activation entails and then tests in a variety of periodontal injury models the consequences of injury activation on ERM behavior and function. A Wnt lineage tracer strain Axin2Cre ^ERT2/+ ; R26R ^mTmG/+ was employed to map the distribution of Wnt-responsive cells and their descendants during root development and in response to injuries to the periodontium. Injury-activated murine ERMs were compared against developmental ERMs, and both were analyzed by histology and immunohistochemistry (IHC). Additionally, ERMs isolated from human tissues were analyzed by single-cell RNA sequencing and IHC. During root development, ERMs were surrounded by Wnt-responsive cells and their progeny. In response to injury, both the number and size of ERMs significantly increased, and this injury-induced enlargement did not involve cell proliferation. The injury-activated state of ERMs was accompanied by expression of Wnt pathway components. Compared to ERMs in uninjured tissues, injury-activated ERMs exhibited a shift toward expression of molecular markers associated with the epithelial-to-mesenchymal transition (EMT). When ERMs were juxtaposed to an injured junctional epithelium (JE) or damaged cementum, some ERM cells adopted a stellate morphology, exhibited evidence of matrix remodeling, and showed a loss of cell–cell adhesion, contributing to the repair of cementum and the JE. A dynamic state of Wnt responsiveness exists in injury-activated ERMs, and a subset of ERM cells undergo EMT. Together, these findings raise the possibility that cells in and around these activated ERMs may adopt an active role in periodontal repair.

As a first line of defense against periodontal diseases, the junctional epithelium must establish a firm attachment to the tooth surface while allowing new cells arising from the stem cell niche to migrate through the tissue. How these static and dynamic cell behaviors are coordinated in the junctional epithelium is not clear; we hypothesize it involves the epithelial-to-mesenchymal transition (EMT). An intact junctional epithelium was interrogated via quantitative immunohistochemistry for molecular markers of EMT; since EMT is also critical for tissue repair, the junctional epithelium was also interrogated at multiple time points after ligature-induced periodontitis. These analyses revealed that junctional epithelial cells transition from a polarized, cell-adherent phenotype to one in which cytoskeletal remodeling and increased motility occur. To gain insights into molecular mechanisms regulating EMT, we used a genetic system to delete the BMP type I receptor (BMPR1a) in postnatal mice. Within 7 d of BMPR1a inactivation, multiple EMT signaling pathways including Wnt/β-catenin, transforming growth factor–β, and Notch signaling were upregulated in the junctional epithelium. The mutant junctional epithelium exhibited extensive EMT accompanied by downregulation of the hemidesmosomal proteins Laminin5, plectin, and β4 integrin. Together, these findings demonstrate that normal junctional epithelium turnover used EMT and that this physiological process is controlled in part by WNT/BMP signaling.

Background: We undertook a comparative analysis of bone healing in the craniomaxillofacial (CMF) skeleton, focusing on the extent to which the physiological processes of bone repair and remodeling differed between different species. Understanding these differences can enhance the value of research outputs and thus improve treatment strategies. Methods: An osteotomy was produced in an equivalent anatomical location in mice, rats, minipigs, and human patients. Quantitative analyses were performed using micro‐computed tomographic (µCT) imaging, histology, dynamic histomorphometry, immunohistochemistry, and cellular activity assays. Results: Non‐invasive imaging analyses, histomorphometry, and molecular and cellular analyses together demonstrated that the overall bone repair program was highly conserved between rodents and minipigs, correlating with limited human data. Comparing the rate of bone repair across species was confounded by limited time points for analysis in human tissues. However, at later time points, CMF bone repair in minipigs was equivalent to CMF bone repair in rodents. Conclusions: The molecular and cellular features of bone repair are shared among large and small animals, suggesting a common bone healing mechanism among species. Combined with the availability of transgenic lines and an extensive array of molecular and cellular analyses, this makes rodents an appealing choice for preclinical testing. Plain language summary: We conducted this study to understand how bone heals in the head skeleton. We compared this repair process in animal models, including mice, rats, and minipigs, against a limited set of data from humans. Our goal was to determine whether bone repair and remodeling were conserved between species because this would have implications for preclinical studies to improve treatment strategies. We created a controlled injury in similar bone areas in each species, then examined the healing process using various research techniques, analyzing bone architecture and cellular activity. Our findings showed that the overall process of bone healing was similar between species. Although we had limited data from humans at later stages of healing, the healing patterns in minipigs closely matched those in rodents at the later stages. These similarities suggest that the way bones heal is equivalent among mammals. Therefore, with the added advantage of advanced research techniques available, rodents are an excellent choice for testing new treatments before advancing to studies in large animals and testing in humans.

The molecular mechanisms mediating barrier functions of the junctional epithelium (JE) are incompletely understood. The aim of this study was to gain mechanistic insights into how reduced Wnt/β-catenin signaling affects the metabolism, turnover, and attachment of JE cells to the tooth surface. A membrane-permeable selective inhibitor of the Wntless protein, C59, was topically delivered to the JE. Wnt pathway suppression was verified by using Axin2 ^LacZ/+ and Axin2Cre ^ERT2/+ ; R26R ^mTmG/+ strains of mice. Quantitative analyses were carried out at multiple time points to assess mitotic activity, apoptosis, expression of hemidesmosomal attachment proteins, distribution of immune cells, collagen remodeling, and alveolar bone resorption. To complement these studies, Wntless was genetically deleted in osteocalcin-expressing cells, including those in the JE, after which the same quantitative analyses were performed. C59 caused a dose-dependent inhibition in Wnt signaling, which led to reduced mitotic activity and increased apoptosis in the JE. Continued dosing of C59 was accompanied by downregulation of the hemidesmosome attachment proteins laminin 5, plectin, and integrin β4 and a disruption in collagen orientation. A genetic approach in which Wntless function was inhibited in osteocalcin-expressing JE cells yielded similar inhibitory effects on Wnt signaling, mitotic activity, the JE’s attachment to the tooth surface, and an increase in immune cells within the connective tissue. Wnt/β-catenin signaling is required for JE homeostasis, and disruptions to the pathway are sufficient to cause JE breakdown and attachment loss. Methods to modulate Wnt/β-catenin signaling may prove beneficial in restoring JE homeostasis after injury or disease.

The development of craniofacial bones and teeth relies heavily on the Wnt signaling pathway, yet the specific mechanisms and Wnt variants involved remain under continual investigation. Using publicly available single-cell sequencing data from the mouse incisor, we reveal Wnt1 expression across dental structures and investigate its role using a Col1a1 -dependent Wnt1 transgenic mouse model. Inducing Wnt1 early on affects craniofacial bone without disturbing tooth development, but prolonged embryonic induction leads to postnatal mortality with osteopetrosis-like bone overgrowth and malformed teeth. While tooth formation was initially unaffected by postnatal Wnt1 induction, prolonged activation impaired tooth root formation and odontoblast differentiation, resulting in shortened roots and thinner dentin. Three-dimensional micro–computed tomography quantification reveal that both embryonic and postnatal activation of Wnt1 significantly increase neural crest–derived craniofacial bone volume, whereas mesenchymal-derived craniofacial bones are unaffected. Importantly, osteoclastogenesis is suppressed by Wnt1 in a dose-dependent manner, revealed through bulk RNA sequencing and in vitro experiments. These findings emphasize the differential effects of Wnt1 on bone development based on origin and highlight its role in modulating osteoclast activity, indicating broader implications for craniofacial development and potential therapeutic avenues.

Aim: To investigate additional factors contributing to the pathophysiology of chemotherapy‐induced oral mucositis and periodontitis beyond the systemic immune suppression caused by the chemotherapeutic agent 5‐Fluorouracil (5‐FU). Methods: 5‐Fluorouracil was topically delivered to the non‐keratinized, rapidly proliferating junctional epithelium (JE) surrounding the dentition, and acts as an immunologic and functional barrier to bacterial ingression. Various techniques, including EdU incorporation, quantitative immunohistochemistry (qIHC), histology, enzymatic activity assays, and micro‐computed tomographic (μCT) imaging, were employed to analyze the JE at multiple time points following topical 5‐FU treatment. Systemic 5‐FU delivery was used for comparison, and all 5‐FU treated tissues were compared to vehicle‐treated controls. Results: We first showed that systemic 5‐FU blocked mitotic activity that rapidly led to JE atrophy. This atrophy was accompanied by suppression of the immune system. We then demonstrated that topical 5‐FU delivery effectively inhibited cell proliferation in the JE. Quantitative immunohistochemical (qIHC) analyses further demonstrated a progressive breakdown in JE barrier functions following topical 5‐FU. CBC analyses confirmed that topical 5‐FU did not alter the innate immune system but did suppress the local immune response of the JE. The longer‐term consequences of this disruption in JE barrier functions were significant alveolar bone loss and an increase in porosity. Together, these results document the essential requirement for rapid JE cell proliferation to maintain homeostasis of the periodontium. Conclusions: The reduction of cell division in the JE due to 5‐FU treatment directly compromises both its structural integrity and immune surveillance capabilities, contributing to the destruction of periodontal hard tissues.

The long-term success of dental implants depends on the ability of soft tissues to form a protective barrier, limiting pathogen infiltration into peri-implant tissues. Here, we investigated the impact of an anodized surface modification on mucosal integration. Scanning electron microscopy and surface chemistry characterization were carried out on miniaturized implants. Following placement in fresh extraction sockets of mice, peri-implant tissues were examined at 4 time points. Histology along with quantitative immunohistochemistry for Keratin14, Vimentin, Laminin5, and CD68 were carried out on postimplant day (PID) 3 to assess early events in soft-tissue repair; on PID7, when peri-implant epithelialization was complete; at PID14, when osseointegration was complete; and at PID28, when soft-tissue maturation was nearing completion. In all cases, an intact junctional epithelium served as a reference. These analyses supported 3 conclusions: first, maturation of the peri-implant epithelium (PIE) is a protracted process, consistent with clinical observations. Second, maturation of the soft tissue–implant interface is slower than maturation of the bone-implant interface. Third, there is a benefit, albeit transient, to soft-tissue maturation around an anodized implant surface. Given its prolonged time course, strategies to improve and/or accelerate PIE maturation are likely to have significant clinical benefit.