Laboratory of Oral Connective Tissue Biology

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

Linked publications, trials & patents

Abstract

Project A: Establish the role of factors regulating PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, and TNAP, in root formation and cementogenesis and apply this knowledge to deliver factors locally/systemically to regenerate periodontal tissues, using rodent models of periodontal disease. Results demonstrate the importance of Pi/PPi and SIBLING family genes/proteins during mineralization, highlighting the need for both physiochemical and cellular molecular factors to achieve homeostatic balance required for formation/regeneration of periodontal tissues. a. Animal Models for regeneration of the periodontium: We reported factors controlling PPi/Pi levels have significant roles during formation of the periodontium. In a proof of principle experiment using Ibsp KO mice, TNAP lentivirus via intramuscular delivery, rescued the Ibsp KO periodontal disease phenotype, i.e., insertion of PDL into newly formed cementum and improved alveolar bone volume and mineral density. Results of these studies show TNAP is a promising candidate for local delivery for treating periodontal diseases, including perio-implantitis and socket preservation. Results published in JDR,2021 demonstrated substantial regeneration of cementum in a mouse model, with a patent pending. These studies are being continued with my fellow, Atsuhiro Nagasaki, now an Assistant Professor at Tohoku University (started April 2021). Further, periodontal fenestration defects were created in ANK and ENPP1 KO mice. At 15 and 30 days post-surgery we noted increased cementum regeneration in tissues obtained from KO mice. These studies have been published in JDR, 2021. Our data suggest different effects of osteoclast-like cells on wound healing between tissues analyzed from WT and KO mice. We are defining the osteoclast profile in a collaboration with Dr. Ozato's group, an ongoing project with NIH and Tohoku U. b. Ank, Enpp1, dKO and Ank, Alpl dKO mice and cells, in vitro: To determine whether effects of ANK and ENPP1 are additive/synergistic and if distinct mechanistic roles exist in cementogenesis, we generated Ank, Enpp1, dKO and Ank, Alpl dKO mice. Histological and microCT results indicate that acellular cementogenesis of Ank, Enpp1 dKOs was comparable to single KOs. Ank, Alpl dKO mice exhibit improved cementogenesis compared to Alpl KOs, further supporting PPi as a key regulator in cementogenesis. Bone defects appeared more severe in Ank KOs and dKO mice compared to Enpp1 KOs, suggesting that ANK and ENPP1 have non-redundant roles in upregulating extracellular PPi. Additionally, alveolar bone volume and mineral density were not improved compared to Alpl KO mice (publication, Bone 2020). Ongoing studies, using proteomics, qPCR microarray and RNA seq, are focused toward defining protein and gene expression profiles of PDL tissues obtained from Ank and Enpp1 single and dKO mice at various stages of tooth root development. Further, as mentioned in 1a, we contrasted periodontal wound healing capabilities of Ank vs Enpp1 KO mice (Bone, 2021). These projects are continuing with Dr. Emily Chu who has transitioned to University of Maryland School of Dentistry as an Assistant Professor (K99/R01 recipient). Further, we have a patent pending with Yale University for use of Enpp1 inhibitors, local delivery, to promote periodontal regeneration. c. Orthodontic tooth movement: In continuing studies with Dr. Wolf investigating the role of Pi/ PPi in regulating osteoclast/odontoclast distribution, Ank-/- mice were subjected to orthodontic loading. Also, to characterize the effect of a compromised periodontia on tooth movement, we exposed Ibsp-KAE (BSP RGD replaced by KAE), Ibsp KO, and WT mice to orthodontic loading. We are performing microCT and histological analyses in tissues obtained from these orthodontically treated mice and data are being analyzed in Germany. This project is continuing with Germany, Tohoku University and Ohio State U. Project B: Defining the role of extracellular matrix proteins in perio-odontogenesis a.BSPxOPN: In 2018 (Foster et al.Bone) we reported OPN has specific roles in regulation of dentin, bone, PDL and pulp, but not acellular cementogenesis. As a next step, we generated and are characterizing periodontal tissues obtained from Spp1(OPN) x Ibsp (BSP) dKO mice to determine if OPN loss rescues the Ibsp KO periodontal phenotype. Preliminary micro CT analysis indicate decreased alveolar bone proper volume in dKO vs. WT, yet greater than that of Ibsp KO mice. Histological analyses revealed defective periodontal complex and increased number of osteoclasts along the alveolar bone in dKO similar to Ibsp KO. Thickened alveolar crest was noted as a unique phenotype in dKO mice further suggesting complex interactions between OPN and BSP in maintaining periodontal homeostasis. Additionally, huge osteoclasts were noted in cells from femurs of Spp1 KO mice but not Ibsp KO or dKO cells. In collaboration with Dr. Hanson Fong, TEM imaging will be used to define further the morphology and function of osteoclasts in vivo and in vitro. This project has moved to Tohoku University (Dr. Karin Nagasaki) and Ohio State University. b.Ibsp-KAE: Toward defining BSP mechanistic functions in cementogenesis, we generated mice where the BSP RGD domain (integrin binding domain) was replaced by a non-functional KAE sequence (Ibsp-KAE). In collaboration with Dr. Ralston, results from histology and second harmonic generation microscopy reveal that in contrast to Ibsp KO mice, Ibsp-KAE mice display normal cementogenesis, but disorganized PDL with increased osteoclasts along the alveolar bone, like Ibsp KO mice. Micro CT analysis revealed a higher alveolar bone volume in older age Ibsp-KAE mice. In vitro data demonstrated comparable osteoclastogenesis potential between Ibsp-KAE, Ibsp KO, and WT mice. Based on these results we hypothesize that the weakened PDL insertion in Ibsp-KAE mice triggers inflammatory cytokines production, which in turn promotes expression of RANKL, resulting in increased local osteoclast activity. Ibsp-KAE cells migrated slower and at the same rate as Ibsp KO cells, when compared to WT cells providing evidence for loss of the RGD region as the factor mediating weakened PDL properties we noted in vivo. We will submit a manuscript by end of Sept. 2021. Another unanticipated finding was that Ibsp-KAE mice exhibit an increase in body weight over time vs WT and Ibsp KO mice. We have initiated studies to determine if these mice (using male mice only at this time) exhibit alterations in specific blood markers to include insulin, glucose and triglycerides and in specific tissues associated with fat metabolism, e.g., liver, pancreas, kidney and epididymis. This is a collaboration with Dr. Oksana Gavrilova, NIDDK/NIH. These studies continue with Karin Nagasaki, NIDDK and Somerman (advisor). c.Pan Fibroblast Growth Factor Receptor Inhibitor Infigratinib (BGJ398): In collaboration with QED Therapeutics, we are examining effects of BGJ398 on dentoalveolar development. Male and female Wistar rats were dosed daily (starting on 7 days postnatal) with vehicle, 0.1mg/kg BGJ398 (low-dose), or 1.0mg/kg BGJ398 (high-dose) until euthanasia on 37dpn. Third molars exhibited aberrant crown and root morphology in high-dose groups (100% females, 80% males), demonstrating sensitivity of early tooth morphogenesis to BGJ398. Clinically, the low-dose data implies a potential therapeutic treatment with minimal effect on dental structures. Future studies under Emily Chu at UMD include analyses of genes/proteins associated with dentoalveolar development and potentially perturbed by BGJ398. Manuscript in progress to be submitted by Dec. 2021.