HSDM Theses and Dissertations

Periodontitis and peri-implantitis are chronic inflammatory diseases in which dysregulated host-microbial homeostasis compromises the oral mucosal barrier and leads to progressive soft-tissue destruction and bone loss. Although epithelial and stromal compartments are positioned to regulate barrier integrity and immune infiltration, how these non-immune cells differentially mediate inflammation in periodontitis versus peri-implantitis remains incompletely understood. The present study tested the hypothesis that distinct inflammatory profiles in periodontitis and peri-implantitis are associated with condition-specific changes in epithelial and stromal cellular states and their interactions with immune cells. Soft-tissue biopsies were collected from adults with periodontal health (n = 5 sites), periodontitis (n = 7 sites), and peri-implantitis (n = 8 sites). Single-nucleus RNA sequencing generated a cell-resolved atlas across epithelial, stromal, and immune compartments. Differential abundance, gene set enrichment, and pseudotime analyses assessed condition-specific shifts in cell proportions, biological pathways, and epithelial differentiation trajectories. Spatial transcriptomics and multiplexed immunofluorescence were used to validate candidate epithelial and endothelial niches and to visualize proximity-based interactions in situ. An in vitro endothelial assay tested cytokine inducibility of MHC class II under pro-inflammatory stimulation. The epithelial compartment resolved into seven differentiation states spanning a pseudotemporal continuum from basal repair-associated cells to suprabasal barrier-forming cells. Periodontitis was characterized by expansion of least differentiated, repair-skewed epithelial states and loss of suprabasal barrier-forming states. Peri-implantitis retained suprabasal differentiation programs but showed enrichment of stress keratins (KRT6A/B/C and KRT16). CXCL14 expression localized predominantly to suprabasal epithelial cells and showed the strongest spatial association with immune proximity in periodontitis. In the stromal compartment, a rare HLA-DR/MHC II-positive endothelial subset was enriched in disease and localized adjacent to T cells. This phenotype was recapitulated in cultured human endothelial cells under pro-inflammatory stimulation. These results support a model in which epithelial differentiation state and endothelial immune adaptation contribute in distinct ways to inflammatory niche organization in periodontitis and peri-implantitis. By integrating high-resolution single-cell and spatial data, this work delineates cellular niches and regulatory signatures that distinguish these diseases and identifies barrier-associated and vascular programs relevant to future host-modulatory strategies.

Periodontal ligament stem cells (PDLSCs) maintain periodontal tissue homeostasis and play a critical role in tissue regeneration and the resolution of inflammation. They release Specialized Pro-resolving Lipid Mediators (SPMs) to regulate inflammation, but less is known about how SPMs stimulate PDLSCs to promote regeneration. This study aims to determine the role of Göttingen Miniature PDLSCs (mpPDLSCs) in Benzo-lipoxin A4 (BLXA4)- mediated periodontal regeneration. The study was conducted on four Göttingen female minipigs (~30 kg; 12-36 months old). This study was designed as a single experimental arm for each interventional group, where each minipig had eight surgically created two-wall bony defects in all four posterior quadrants to establish the periodontal disease model. The interventional groups were allocated as Control, BLXA4, BLXA4+PDLSC, and PDLSCs. After surgical debridement, the groups receiving the BLXA4 treatment were administered 0.05 μg/defect in 18 μl of the BLXA4 lipid nanoparticle, while those receiving PDLSCs had 1x106 cells of the cell suspension following the surgical debridement and the control group received only surgical debridement. Bone regeneration was assessed by microCT. The mpPDLSCs were isolated from freshly extracted lateral incisors from the same host pig. Stem cell markers were characterized by using flow cytometry (CD11b, CD163, CD90, CD105). The multilineage differentiation of mpPDLSCs was assessed by osteogenic, adipogenic, and fibroblastic differentiation. Alizarin Red S staining was used to evaluate osteogenic differentiation; Oil-Red O staining was used for adipogenic differentiation; and Picro Sirius Red staining was used for fibroblastic differentiation after 21 days. A colony-forming unit-fibroblast (CFU-f) assay was utilized to assess cell proliferation using 0.5% crystal violet staining. Furthermore, the in vitro impact of BLXA4 on mpPDLSCs were investigated concerning cell viability along with SPM receptor expression (FPR2 and GPR32) using immunocytochemistry. The isolated mpPDLSCs expressed mesenchymal stem cell markers and lacked hematopoietic stem cell markers. mpPDLSCs were multipotent and differentiated along osteogenic, adipogenic, and fibroblastic lineages with appropriate stimulation. Seeding 100 cells per well in a 6-well plate resulted in colony formation after 10 days. Additionally, mpPDLSCs exhibited enhanced proliferation when stimulated with BLXA4, with or without E. coli LPS stimulation. Moreover, BLXA4-stimulated mpPDLSCs expressed FPR2 and GPR32. The lipidomic results indicated that BLXA4 and PDLSCs exhibited distinct effects on the lipid mediator profiles, leading to differential modulation of pro-inflammatory and pro-resolving pathways. It is noteworthy that their combination did not invariably result in additive effects, suggesting intricate and potentially synergistic interactions between BLXA4 signaling and PDLSC-mediated mechanism. The microCT data, in conjunction with the qualitative histological results, demonstrated that all interventional groups exhibited a significantly improved periodontal regeneration (P.05) in comparison to debridement alone. In summary, this study demonstrates the potential of mpPDLSCs in enhancing periodontal regeneration, alone or when stimulated with BLXA4. The findings indicate that BLXA4 promotes cell proliferation and stimulates the expression of receptors associated with inflammatory resolution (FPR2 and GPR32) in mpPDLSCs. The significant improvements in bone regeneration observed across all interventional groups underscore the therapeutic potential of combining SPMs with stem cell therapy. These results provide a strong foundation for a larger randomized clinical trial designed to further evaluate and translate SPM-enhanced stem cell therapy for periodontal regeneration in humans, potentially offering a novel approach to managing periodontal diseases.

Bone regeneration remains a significant challenge in orthopedic and dental medicine, particularly in cases involving complex defects, persistent inflammation, or underlying pathology. Traditional bone graft materials, while widely used, are limited by factors such as donor site morbidity, immune rejection, and insufficient integration. To address these limitations, bioactive materials with osteoinductive and osteoconductive properties have emerged as promising alternatives. In parallel, specialized pro-resolving mediators (SPMs) have gained attention for their ability to modulate inflammation and promote tissue repair. Among them, resolvin E1 (RvE1)—a lipid mediator biosynthesized from eicosapentaenoic acid (EPA)—has demonstrated strong potential in accelerating inflammation resolution and supporting bone regeneration. This study evaluated the regenerative effects of a novel bone graft system composed of highly porous dicalcium phosphate dihydrate (DCPD) granules functionalized with RvE1 in a preclinical model of alveolar ridge preservation. Thirty male Sprague–Dawley rats underwent maxillary first molar extraction with standardized socket preparation and were randomly assigned to one of three treatment groups: negative control (unfilled socket), DCPD granules alone, or DCPD granules coated with RvE1. Specimens were collected at 3- and 6 weeks post-extraction for comprehensive analysis. Bone regeneration was quantified using micro-computed tomography (µCT) to assess bone volume/total volume (BV/TV) and bone mineral density (BMD). Soft tissue healing was evaluated using a composite scoring system based on occlusal photographs, and histological staining provided tissue-level validation. At 3 weeks, the DCPD+RvE1 group demonstrated significantly higher BV/TV compared to both the control and DCPD-only groups (p 0.01). Improved soft tissue healing was also observed in the RvE1-treated group (p 0.05), characterized by reduced erythema and smoother mucosal surfaces. These early enhancements were attributed to the localized burst release of RvE1 during the acute inflammatory phase, promoting a pro-resolving microenvironment conducive to regeneration. By 6 weeks, all groups showed progressive bone fill and soft tissue maturation, and differences in BV/TV, BMD, and healing scores were no longer statistically significant. Histological analysis corroborated the imaging results, showing more organized connective tissue and early woven bone in RvE1-treated sockets at 3 weeks. The DCPD granules provided a biocompatible and resorbable scaffold capable of one-time local drug delivery, aligning with clinical needs for minimally invasive and scalable treatments. In conclusion, RvE1-functionalized DCPD granules significantly enhance early bone and soft tissue healing following tooth extraction. This single-step, bioactive bone graft system presents a promising strategy for socket preservation and regenerative therapies by coupling osteoconductive support with targeted immunomodulation.

PTH signaling is of primordial clinical importance in the regulation of skeletal development and homeostasis, as well as in tooth formation and eruption. Both skeletal and dental mesenchymal cells express PTH1r and are target of PTH signaling. Our lab has been investigating the function of a recently identified periosteal stem cell (PSC) population labeled by Cathepsin K (Ctsk) in the regulation of cortical bone homeostasis. Ctsk+ lineage PSCs, which fulfil stemness criteria, express high levels of the PTH1r and respond to iPTH treatment. Ctsk is also expressed in dental pulp cells, dental follicle cells, and the periodontal ligament which are known to play a key role in tooth development and eruption. Whether PTH signaling in Ctsk+ lineage cells is required for proper periosteal bone formation and tooth development and eruption is not known. We, therefore, undertook a study to investigate whether PTH signaling in the Ctsk expressing PSCs and dental mesenchymal cells regulates these processes. To this end, we generated mice lacking PTH1r, specifically in Ctsk+ cells, using the CtskCre mice and analysed their cortical bone and teeth.

Eight-week-old CtskCre;PTH1rfl/fl (CtskPTH1r) male and female mice are significantly smaller than their control littermates (PTH1rfl/fl). microCT and bone histomorphometry analyses revealed a significant decrease in cortical bone volume (%), cortical thickness, and periosteal MAR in CtskPTH1r mice compared to PTH1rfl/fl littermates. Notably, CtskPTH1r male and female mice present with failure of molar eruption and impaired incisor eruption. microCT analyses and histological examination revealed several abnormalities in 8-week-old CtskPTH1r mice, including truncated molar roots, loss of the periodontal ligament, root ankylosis, reduced cementoblasts, and markedly decreased alveolar bone. Severe dental anomalies werealso seen in CtskPTH1rmice at P12 and P19. Confirming that both the skeletal and the tooth phenotype are a consequence of deletion of the PTH1r in the mesenchymal cell lineage, Lys2Cre mice, widely used to deleted genes of interest in osteoclasts, do not present with any skeletal and dental phenotype.

While our findings confirm the significance of PTH signaling in both periosteal bone formation and tooth development and eruption, they reveal for the first time a crucial role for Ctsk+ lineage cell- dependent PTH signaling within the periosteum and dental mesenchyme. The periosteum is a significant source of stem cells and progenitors contributing to bone growth and homeostasis, regeneration and response to anabolic drugs. Investigating the signaling molecules and pathways regulating periosteal stem cells offers an opportunity to advance our understanding of the mechanisms involved in these processes and may open novel and targeted therapeutic approaches for human diseases associated with bone fragility and impaired bone regeneration. Similarly, a comprehensive understanding of distinct subsets of dental mesenchymal cell populations and unravelling their regulation is of significance for the effective pursuit of novel dental regenerative strategies.

Systemic administration of ABL significantly reduces the risk of fragility fractures in the femur, hip, and vertebrae, but no data exists on the likely positive effects that could be exerted on alveolar bone of the jaws. Considering the mechanism of action in which ABL directly influences osteoblastic cell lines and the previous clinical data reporting a substantial treatment effect of TPD in the regeneration of periodontal defects, the osteoanabolic effect of ABL could be leveraged to enhance procedures that aim to regenerate alveolar bone.

The preclinical investigations in the present report evaluate the potential of systemic ABL therapy to increase alveolar bone formation and bone mineral density (BMD), in applications of tooth extraction socket healing, implant osseointegration, and peri-implant regeneration. A series of two experiments were performed in female, Sprague-Dawley rats to assess the effects of ABL under conditions simulating: 1) osteoporosis, in animals that received ovariectomy surgery to induce bone loss via estrogen-deficiency and; 2) in healthy, young animals with normal bone metabolism. These experiments assess the biologic possibility that ABL may increase alveolar bone quality prior to implant placement, enhance primary dental implant fixation, and accelerate healing in dental implant therapy.

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.

Background: Peri-implant mucositis and peri-implantitis are pathological conditions occurring in tissues around dental implants. Both diseases are characterized by inflammation in the peri-implant connective tissue, but peri-implantitis additionally involves progressive loss of supporting bone. Previous studies have utilized 16sRNA sequencing which have revealed the severity of human peri-implant disease lesions correlated with the level of submucosal microbial dysbiosis. However, the spatial arrangement of these microbial communities remains largely unknown. Hypothesis & Specific Aims: The aim of this study is to elucidate the composition and spatial organization of oral microbial communities in periimplant health (PIH) and peri-implant disease (PID) at the single-cell resolution. Our central hypothesis is that peri-implant microbiomes in PIH and PID exhibit different compositions and spatial organizations indicative of their ecological interactions. Consequently, our specific aims are: 1) 1) To identify structural differences in microscale spatial arrangement of peri-implant microbial communities in PIH and PID. 2) To identify characteristic motifs in the microscale spatial organization of taxa in PIH and PID 3) To explore the relationship between the severity of PID and microbial spatial organization. We hypothesize that PIH and PID have different spatial arrangement and distinct characteristic motifs. In addition, microbial spatial organization may differ depending on diseases severity. Material & Methods: Subgingival microbial plaque samples from health peri-implant sulcus (n = 26), peri-implant mucositis sulcus (n = 15), untreated peri-implantitis sulcus (n = 19) were obtained from 60 non-smoking, systemically healthy patients during standard of care clinical procedures (IRB21-0662). All patients completed baseline clinical measurements (probing depth, recession, bleeding on probing, plaque index), and full-mouth radiographs (periapical and/or bitewing) to assess peri-implant disease severity and establish a diagnosis. For measuring microbial composition, we used third generation long read sequencing of the full 16sRNA gene. For surveying microbial spatial organization, we utilized highphylogenetic-resolution microbiome mapping by fluorescence in situ hybridization (HiPR-FISH), which is a novel and versatile technology that uses binary encoding, spectral imaging, and machine learning decoding to create micrometer-scale maps of the locations and identities of microbial species in complex communities. Key Innovation: High-phylogenetic-resolution microbiome mapping by fluorescence in situ hybridization (HiPR-FISH) provides an advanced and contemporary framework for analyzing the microscale spatial ecology of environmental microbial communities at the single-cell resolution. Thus, the novel technology can be used to make significant progress in our understanding of peri-implant microbiome ecology

Sfrp4 (Secreted Frizzled Receptor Protein 4) serves as a decoy receptor for Wnts and differently from sclerostin (Sost) that blocks canonical (c)Wnt signaling, it suppresses cWnt and non-cWnt cascades. Loss of function mutations in Sfrp4 cause Pyle disease, a skeletal disease-causing cortical thinning and fractures. Using Sfrp4-/- mice, we demonstrated cortical thinning is by decreased periosteal bone formation and increased endosteal remodeling through non-cWnt/Ror2/Jnk cascade activation. Given that periosteum contains stem cells which forms new bone to injury, we investigated role of activation of cWnt and non-cWnt signaling (Sfrp4-/- mice) in bone regeneration. We created calvarial critical defects (which don’t heal spontaneously) or subcritical defects (which heal spontaneously). We used Sost-/- mice for cWnt signaling activation. We confirmed cWnt activation in Sost-/- and Sfrp4-/- calvariae, and non-cWnt/Jnk activation only in Sfrp4-/- calvariae. MicroCT analyses indicate while cWnt activation (Sost deletion) favors bone regeneration (BV/TV(%)) within initial critical defect (p.0001) 6-wk after surgery, Sfrp4 deletion did not. In subcritical defects, cWnt activation (Sost-/- mice) led to accelerated bone regeneration (p.01), while Sfrp4-/- mice did not. Sfrp4 deletion leads to significant decrease in percentage and function of Cathepsin K (Ctsk+) labelled calvarial periosteal stem cells (PSCs). We investigated effect of Sfrp4 deletion in response to injury by using CtskCre;mTmG;wt and CtskCre;mTmG;Sfrp4-/- mice. Preliminary confocal analyses indicate Sfrp4 deletion impairs response of Ctsk+ PSCs. We explored whether activation of Ror2 cascade in Sfrp4 null mice might improve bone formation in subcritical defects. We generated mice lacking Ror2 receptor in Ctsk+ cells in Sfrp4 null background. Preliminary findings suggest Ror2 signaling does not impact response to injury found in Sfrp4-/- mice. Our findings demonstrate Wnt signaling fine-tuning is critical for bone responses and activation of non-cWnt signaling in Sfrp4-/- mice might be responsible for improper function of stem cells within sutures and periosteum.

Background and objectives: Parathyroid hormone (PTH) is essential for skeletal homeostasis and PTH[1-34] (teriparatide) is used to treat severe osteoporosis, medication-related osteonecrosis of the jaws, and enhance implant osseointegration. PTH exerts its anabolic actions by acting on osteoblasts, bone lining cells, and osteocytes. Recently, marrow adipose lineage precursors (MALPs) have been reported to suppress osteogenesis and enhance osteoclastogenesis. We hypothesized that, since MALPs express the PTH receptor 1 (Pth1r), they may contribute to the skeletal response to PTH. Methods: We deleted Pth1r specifically in MALPs and their lineage (adipocytes) using Adiponectin Cre (AdipoqCre) and tdTomato was used as a reporter. AdipoqCre;Pth1rfl/fl,tdTomatofl/fl mice (Pth1rMALPs) were used as experimental mice and age- and sex-matched AdipoqCre;Pth1rfl/fl,tdTomatofl/fl and Pth1rfl/fl,tdTomatofl/fl were used as controls. The skeletal phenotype was characterized by microscopic computed tomography (CT) and dynamic histomorphometry at 4, 7, and 12 weeks of age. Bone marrow adipose tissue (BMAT) was assessed by osmium tetroxide staining and CT analysis. The proximal one-third of the tibiae was designated as regulated BMAT (rBMAT) and the distal one-third was designated as constitutive BMAT (cBMAT). Colony forming unit (CFU) assays, flow cytometry, and fluorescence-activated cell sorting (FACS) were performed on flushed bone marrow stromal cells (BMSC). Two-way ANOVA and Student’s t-test were used for statistical analysis ( = 0.05). Results: Pth1rMALPs female mice exhibited a 54.2%, 15.8%, and 42.7% increase in trabecular bone volume at 4, 7, and 12 weeks of age, respectively (p = 0.001). The increase in bone volume was associated with a significant increase in labeled surfaces (MS/BS and dLS/BS, p = 0.0066 and p = 0.0429, respectively by two-way ANOVA) but an increase in bone formation rate (BFR/BS) was significant only at 4 weeks of age (p = 0.03). Flow cytometry analysis revealed that osteochondrogenic progenitor cells were decreased by half in Pth1rMALPs mice (p = 0.01). Since mineralizing surfaces were increased, these results suggest a shift of the progenitor pool towards differentiated and functional osteoblasts. Additionally, the number of CFU-F colonies was significantly decreased in Pth1rMALPs cells, further confirming the decrease in the progenitors. In contrast, the CFU-Ob remained similar between the two groups, suggesting an increase in the osteogenic potential of cells from Pth1rMALPs mice despite their decreased number. Only 6.4% ± 4.6% of CFU-Ob cells represented a distinct population of tdTomato+ cells, and 5.7% ± 2.7% had a dim tdTomato+ signal (herein, tdTomato+ dim). Interestingly, cells from Pth1rMALPs mice exhibited an 8-fold increase in tdTomato+ dim cells, and unfractionated CFU-Ob cells exhibited a 90.3% decrease in Pth1r mRNA gene expression (p = 0.001). Additionally, Pth1rMALPs mice showed a significantly higher rBMAT density, confirming that PTH suppresses adipogenesis. Importantly, sorted MALPs expressed Rankl, and its expression is increased in MALPs lacking Pth1r. The endosteal osteoclast number and surface was increased in Pth1rMALPs mice, indicating that PTH signaling in MALPs suppress osteoclastogenesis. Conclusions: Deletion of the Pth1r in MALPs partially prevents their suppression of osteogenesis through driving the progenitor pool to a differentiated state. Moreover, PTH signaling in MALPs suppresses adipogenesis and osteoclastogenesis.