Laboratory of Oral Connective Tissue Biology

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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. 1. Cell, Tissues and Animal Models of Periodontal Disease: a. Animal Models for regeneration of the periodontal apparatus: 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. Thus, we initiated studies to deliver TNAP locally in Ibsp KO mice. Results were promising (i.e., cementogenesis promoted, manuscript to submit Oct.2020), leading to a collaborative studies with Drs. Samara and Shiloach at NIH, as well as Dr. Braddock to design TNAP targeted to mineralized tissues for local delivery. We have a contract with Thermofisher to design mineral targeted TNAP and anticipate being able to test this TNAP by Jan. 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. Our data suggest different effects of osteoclast-like cells on wound healing between tissues analyzed from WT and KO mice and between tooth root and surrounding bone. We are defining the osteoclast profile in a collaboration with Dr. Ozato s lab. These collective results highlight that factors modulating PPi/Pi levels at local periodontal sites promote cementogenesis. 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 are contrasting periodontal wound healing capabilities of Ank vs Enpp1 KO mice (manuscript to submit Dec.2020). To complement in vivo studies, in vitro osteoclast assays using bone marrow stromal cells from Ank and Enpp1 single and dKO mice are being conducted to assess osteoclast differentiation and resorption with ANK and/or ENPP1 loss. 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. Project B: Defining the role of extracellular matrix proteins in periodontogenesis with a focus on SIBLINGS and collagen. BSPxOPN: In 2018 (Foster et al. Bone) we concluded that OPN has specific roles regulating mineralization of dentin and bone as well as in 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, obtained from these KO mice. (manuscript to submit Feb. 2021) 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 significantly 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 anticipate submitting our manscript by Dec.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. Collagen: In collaboration with Dr. Nan Hatch, we published a paper (Dev Dyn 2020), highlighting craniofacial and dental defects in tissues obtained from Crtap mice (inactivating mutations in the gene for cartilage-associated protein). 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 include analyses of genes/proteins associated with dentoalveolar development and potentially perturbed by BGJ398. Manuscript in progress to be submitted in December 2020.