Martha J. Somerman - US grants

Periodontal diseases are the most common cause of adult tooth loss in the western world today. Furthermore, their effects have proven, thus far, to be irreversible as attempts to regenerate lost periodontal supporting structure have been of limited success. Regeneration requires the formation of both mineralized and connective tissues. These tissues, according to current evidence, are formed by cells which are derived from the remaining healthy portions of the periodontal connective tissues. To complete the regeneration process, such cells must undergo mitosis, migrate to the healing site and become synthetically active. The agents triggering these events are not known, but it is probable that their source is the periodontium (cementum, bone, connective tissue). The major objective of the proposed research is to identify the conditions necessary for eliciting the cells of the periodontium to function in a regenerative capacity. To do this specific characteristics of cultured fibroblast-like cells derived from two sources, healthy human periodontal ligament and gingiva, will be evaluated and compared. The specific characteristics to be investigated include the distribution of collagenous and noncollagenous proteins, types of collagen produced, proliferation rates, and attachment and chemotactic properties. Next, selected agents present in the periodontium, (e.g., demineralized extracts of cementum, dentin and alveolar bone, attachment proteins and bone associated agents) which are known to trigger alterations in other cells, will be added to determine if they alter the specific characteristics of these cells. At the same time, a second approach to this study will be implemented. Single-cell clones obtained from mass cultures of PDL cells will be evaluated for their effects on demineralized extracts of cementum, dentin and alveolar bone. This could be important since cloned cells may provide a more sensitive method to evaluate the potential of agents to alter cell function. Results from these in vitro experiments will provide the basic knowledge essential for the study of periodontal regeneration in animal models and in humans. This is a necessary step in the ultimate goal of achieving an effective clinical method to regenerate the periodontium.

spectrometry; biomedical equipment resource;

Periodontal diseases are the most common cause of adult tooth loss in the western world today. Furthermore, their effects have proven, thus far, to be irreversible as attempts to regenerate lost periodontal supporting structure have been of limited success. Regeneration requires the formation of both mineralized and connective tissues. These tissues, according to current evidence, are formed by cells which are derived from the remaining healthy portions of the periodontal connective tissues. To complete the regeneration process, such cells must undergo mitosis, migrate to the healing site and become synthetically active. The agents triggering these events are not known, but it is probable that their source is the periodontium (cementum, bone, connective tissue). The major objective of the proposed research is to identify the conditions necessary for eliciting the cells of the periodontium to function in a regenerative capacity. To do this specific characteristics of cultured fibroblast-like cells derived from two sources, healthy human periodontal ligament and gingiva, will be evaluated and compared. The specific characteristics to be investigated include the distribution of collagenous and noncollagenous proteins, types of collagen produced, proliferation rates, and attachment and chemotactic properties. Next, selected agents present in the periodontium, (e.g., demineralized extracts of cementum, dentin and alveolar bone, attachment proteins and bone associated agents) which are known to trigger alterations in other cells, will be added to determine if they alter the specific characteristics of these cells. At the same time, a second approach to this study will be implemented. Single-cell clones obtained from mass cultures of PDL cells will be evaluated for their effects on demineralized extracts of cementum, dentin and alveolar bone. This could be important since cloned cells may provide a more sensitive method to evaluate the potential of agents to alter cell function. Results from these in vitro experiments will provide the basic knowledge essential for the study of periodontal regeneration in animal models and in humans. This is a necessary step in the ultimate goal of achieving an effective clinical method to regenerate the periodontium.

biomedical equipment resource; biomedical equipment purchase;

Bone sialoprotein (BSP) is a phosphorylated glycoprotein, expressed specifically at sites of new bone formation. BSP, which contains an arginine-glycine-aspartic acid cell adhesion sequence, promotes attachment of a variety of cell types. Presently, the cell surface integrin, alpha/v/beta3, is the only cell surface receptor known to bind to BSP. Specificity of BSP to bone, and its expression at sites of new mineralization have stimulated interest in it, as a likely nucleator of mineralization. However, this has not been confirmed and the specific role for BSP in mineralized tissues remains to be determined. The hypotheses upon which this proposal is based are that; 1) follicle cells are responsible for secreting BSP onto the tooth root matrix; 2) BSP is critical for the formation of cementum on this matrix; and 3) that cell surface receptors, notably Alpha v Beta 3, is increasingly expressed by cells in the local environment, concomitant with increased secretion of BSP onto the root surface. Our studies to date demonstrate that BSP mRNA is expressed in follicle cells during cementogenesis, and BSP is localized to the root surface, consistent with the spatial and temporal initiation of cementum formation. We predict that BSP secretion onto the root surface by follicle cells is necessary for cementum formation. Moreover, the expression of specific surface receptors on cell during development is required for controlling the migration and attachment of cells onto the root surface matrix. Specifically, the studies detailed in this proposal are directed at establishing that BSP and follicle cells are responsible for inducing cementum formation and that adhesion receptors are essential in this process. The major objectives of these studies are to 1) establish the time during tooth development at which follicle cells express characteristics of cementoblast-osteoblast-like properties, in situ, including BSP mRNA, 2) prove that BSP, synthesized by follicle cells, is essential for mineralization of cementum; and 3) determine the spatial and temporal expression of alpha/v/Beta3 during cementum formation and correlate its expression with that of BSP. These studies will fill major gaps in our understanding of factors/mechanisms and cell controlling cementogenesis. Furthermore, they will clarify the role of BSP in the mineralization process. This information is critical for the development of therapies that can predictably regenerate periodontal tissues, as well as other mineralized tissues.

Cementum, a mineralized tissue localized on tooth root surfaces, is considered critical for development and regeneration of a functional periodontal apparatus. Studies to date have been limited to examining the properties of cementum in situ. Advanced technologies now enable us to ask critical questions as to the cellular and molecular mechanisms regulating cementoblast activity which will ultimately result in improved strategies for regeneration of periodontal tissues. Based on studies to date using a murine tooth root model system we hypothesize that 1) cells lining the root surface, cementoblasts, secrete a matrix for cementum formation; 2) bone sialoprotein (BSP) secreted by these root surface cells, is a critical protein, required for cementum formation 3) expression of BSP by cementoblasts is regulated by specific promoter sequences which bind cementum-specific transcription activators; and 4) cementoblasts exhibit a protein profile different from that of cells associated with bone, dentin, and/or enamel. These hypotheses are supported by results to date which include the finding that timed and spatial expression of BSP, a protein implicated in mineral formation, by root surface cells coincides with the initiation of cementogenesis. These hypotheses will be tested under three specific aims. 1) To prove that cementoblasts induce mineral matrix formation, in vitro and regeneration of cementum, in vivo and furthermore, that expression of BSP is required for biomineralization to occur; 2) To delineate BSP promoter sequences and related transcription factors controlling cementum-specific expression of BSP; and 3) To determine the molecular profile of cementoblasts. The information obtained from these studies will fill major gaps in our knowledge as to cellular and molecular mechanisms regulating development of the periodontium. This foundation is required for understanding various pathologies, e.g., hypophosphatasia, cementomas, and Paget's associated hypercementosis, and for designing effective clinical procedures for regeneration of periodontal tissues, as well as other mineralized tissues.

DESCRIPTION (Adapted from the applicant's abstract) Cementum, a mineralized tissue located on tooth root surfaces, is considered critical for development and regeneration of a functional periodontal apparatus. Studies to date have been limited to examining the properties of cementum in situ. Advanced technologies now enable us to determine regulators of cementoblasts using both in vitro and in vivo models, allowing for improved strategies for re-engineering periodontal tissues. Based on studies to date using a murine tooth root model the investigators hypothesize: 1) that cells lining the root surface, cementoblasts, secrete a matrix required for biomineralization of cementum and respond to putative periodontal regenerative factors by expressing genes linked with biomineralization; 2) the bone sialoprotein (BSP), expressed by these cells, is required for formation of cementum; and 3) that cementoblasts exhibit a protein profile different from that of cells associated with bone, dentin, and/or enamel. These hypotheses will be tested under three specific aims: 1) Clonal populations of cementoblasts, obtained from immorto-mice molars, exposed to putative periodontal regenerative factors, IGF-I, PDGF and BMP-7, will be evaluated for changes in gene expression and in proliferation rate; 2) The ability of cementoblasts (plus and minus agents) to promote biomineralization and the importance of BSP in this process will be determined, in vitro by von Kossa/TEM and in vivo using a rat periodontal window defect. Sense and anti-sense technology will be used to overexpress and/or disrupt synthesis of BSP in cementoblasts. As factors/cells promoting biomineralization are identified, they will be incorporated into biodegradable polymers and tested for ability to foster regeneration, in vivo. The mechanical integrity of newly formed cementum will be determined using atomic force microscopy; and 3) The molecular profile of cementoblasts will be determined using mRNA differential display. The information obtained from these studies will fill major gaps in our knowledge of cellular and molecular mechanisms regulating root surface formation. This foundation is required for understanding various pathologies, e.g., hypophosphatasia, cementomas, and Paget's associated hypercementosis, and for designing effective clinical procedures for regeneration of periodontal tissues, as well as other mineralized tissues.

DESCRIPTION: (provided by the applicant) The 7th International Conference on the Chemistry and Biology of Mineralized Tissues is planned for November 9, 2001, and will take place at the Sawgrass Marriott, Ponte Vedra Beach, Florida. Health of bones, cartilage and teeth is essential for the activities of normal daily life (locomotion, eating, conversation); yet, the factors and their complex interactions responsible for the formation of these essential organs and those which contribute to their degeneration during aging and disease and to their repair/regeneration during healing remain unknown. The purpose of these International Conferences is to forecast future directions of research into these questions with the ultimate goal of designing improved therapies for treatment of individuals with mineralized tissue related diseases. The setting of these meetings provides an excellent means for achieving these goals. Participants, usually around 200-250, include crystallographers, geneticists, cell and molecular biologists, engineers and clinicians, and the meeting is organized with state-of-the-art presentations in these fields, with ample time provided for discussions, e.g. breakout groups and meal time. The major impact of previous International Conferences (101-106) can be seen from the frequency with which the meeting proceedings are cited and from the number of new collaborations, which have been established by people attending the conferences.

DESCRIPTION (provided by applicant): Periodontal diseases, among the most prevalent of human chronic diseases, result in marked destruction of soft and hard connective tissues and in tooth loss if left untreated. Recognition that periodontal tissues have the capacity to form new bone and cementum with a functional periodontal ligament (PDL) attachment has resulted in increased efforts focused on understanding the regulators of these tissues. In this regard, we noted a dramatic increase in cementum formation (with normal formation of bone and PDL) and in OPN expression in ank mutant mice when compared with wild-type littermates. ANK is a multipass transmembrane protein, reported to transport intracellular pyrophosphate to the extracellular matrix. ANK mutant mice exhibit generalized progressive arthritis, accompanied by mineral deposition and joint destruction. This data, coupled with the knowledge that mice null for tissue non-specific alkaline phosphatase (TNAP), a PPi-ase enzyme, have limited cementum, the following hypotheses are set forth: 1) Phosphate is a critical modulator of cementoblasts/cementum formation; 2) ANK and TNAP work in concert to regulate the extracellular ratio of PPi to Pi; 3) Expression of OPN is increased in response to increased levels of PPi/Pi, thereby preventing fusion of root with surrounding bone, ankylosis; and 4) Molecules that can regulate phosphate levels at local sites can be used therapeutically in attempts to regenerate mineralized tissues. Three aims will address these hypotheses. The first aim will establish the role of ank in modulating genes associated with cementum formation, in situ. Under the second aim the temporal expression of mineral associated genes in cementoblasts exposed to phosphate in vitro will be identified and the phosphate specific promoter regions and transcription factors associated with the OPN promoter determined. The third aim will prove that modulating the levels of PPi/Pi at sites of periodontal wound healing using a rodent window defect will promote cementum formation. The information gained from these studies is critical for determining the etiology of diseases associated with ectopic calcification and also, will improve the effectiveness of periodontal regenerative therapies (neocementogenesis).

[unreadable] DESCRIPTION (provided by applicant): For normal biological mineral deposition to proceed, a tight balance is required between the levels of extracellular inorganic phosphate (Pi and pyrophosphate (PPi) The periodontium, the collection of upportive structures of the tooth root, is especially sensitive to P\/PP\ balance, as evidenced by dramatic cementum phenotypes resulting from mutations that alter local levels of PI and PP|. A markedly increased cementum was demonstrated on the root surfaces of fully developed teeth in mice presenting mutations in either the multiple-pass transmembrane ankylosis protein (ANK) or plasma cell membrane glycoprotein-1 (PC-1); both mutations result in reduced extracellular PPi though via different mechanisms. Conversely, mice and humans with defective tissue nonspecific alkaline phosphatase (TNAP) present locally increased levels of PP|, leading to defective formation of acellular cementum, and subsequently, premature tooth loss. In contrast to these cementum phenotypes, the underlying mineralized tissue, dentin, seems to develop normally in conditions of altered P/PP|. Based on these observations, we hypothesize that periodontal ligament (PDL) cells adjacent to the tooth root cementum are more sensitive to changes in Pi levels than pulp cells adjacent to the dentin. In order to resolve the differential regulation of these two mineralized tissues, the following aims are set: (1) To determine basal differences in gene/protein expression in pulp vs. PDL cells in healthy and hypophosphatasia-diagnosed (HPP) subjects, focusing on genes/proteins associated with differentiation/mineralization and P/PPi metabolism; (2) To determine the effect that Pi treatment exerts on pulp vs. PDL cells, in vitro; and (3) To demonstrate that knocking down TNAP function in pulp and PDL cells harvested from healthy subjects results in a more dramatic change in PDL vs. pulp cell behavior, in vitro. Establishing the mechanisms by which P/PPi influence the periodontium is a critical step towards understanding the development of these tissues, with the ultimate goal of applying further insight to the design of therapies to regenerate periodontal tissues. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. OAR funding for India Project. AIDS research

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Support for AIDS ressearch.