Osteoprotegerin inhibits prostate cancer-induced osteoclastogenesis and prevents prostate tumor growth in the bone.

Prostate cancer (CaP) forms osteoblastic skeletal metastases with an underlying osteoclastic component. However, the importance of osteoclastogenesis in the development of CaP skeletal lesions is unknown. In the present study, we demonstrate that CaP cells directly induce osteoclastogenesis from osteoclast precursors in the absence of underlying stroma in vitro. CaP cells produced a soluble form of receptor activator of NF-kappaB ligand (RANKL), which accounted for the CaP-mediated osteoclastogenesis. To evaluate for the importance of osteoclastogenesis on CaP tumor development in vivo, CaP cells were injected both intratibially and subcutaneously in the same mice, followed by administration of the decoy receptor for RANKL, osteoprotegerin (OPG). OPG completely prevented the establishment of mixed osteolytic/osteoblastic tibial tumors, as were observed in vehicle-treated animals, but it had no effect on subcutaneous tumor growth. Consistent with the role of osteoclasts in tumor development, osteoclast numbers were elevated at the bone/tumor interface in the vehicle-treated mice compared with the normal values in the OPG-treated mice. Furthermore, OPG had no effect on CaP cell viability, proliferation, or basal apoptotic rate in vitro. These results emphasize the important role that osteoclast activity plays in the establishment of CaP skeletal metastases, including those with an osteoblastic component.

Employing a transgenic animal model to obtain cementoblasts in vitro.

BACKGROUND: Proper formation of cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. Further, the presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. Despite the significance of cementum to general oral health, the mechanisms controlling development and regeneration of this tissue are not well understood and research has been hampered by the lack of adequate in vitro experimental models. METHODS: In an effort to establish cementoblast cell populations, without the trappings of a heterogeneous population containing periodontal ligament (PDL) cells, cells were obtained from the root surface of first mandibular molars of OC-TAg transgenic mice. These mice contain the SV40 large T-antigen (TAg) under control of the osteocalcin (OC) promoter. Therefore, only cells that express OC also express TAg and are immortalized in vitro. Based on results of prior in situ studies, OC is expressed by cementoblasts during root development, but not by cells within the PDL. Consequently, when populations are isolated from developing molars using collagenase/trypsin digestion, only cementoblasts, not PDL cells, are immortalized and thus, will survive in culture. RESULTS: The resulting immortalized cementoblast population (OC/CM) expressed bone sialoprotein (BSP), osteopontin (OPN), and OC, markers selective to cells lining the root surface. These cells also expressed type I and XII collagen and type I PTH/PTHrP receptor (PTH1R). In addition to expression of genes associated with cementoblasts, OC/CM cells promoted mineral nodule formation and exhibited a PTHrP mediated cAMP response. CONCLUSIONS: This approach for establishing cementoblasts in vitro provides a model to study cementogenesis as required to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.

Response of immortalized murine cementoblasts/periodontal ligament cells to parathyroid hormone and parathyroid hormone-related protein in vitro.

Cementum is an essential component of the periodontium, but the mechanisms involved in regulating the activity of this tissue are poorly understood. As one approach to better defining the cellular and molecular properties of cementum and the associated ligament, immortalized murine cell populations expressing gene markers associated with both cementoblasts (CM) and periodontal ligament cells (PDL), termed CM/PDL cells, were established. To further characterize these cells, their responsiveness to parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) was examined. CM/PDL cells were tested for the presence of steady state PTH-1 receptor mRNA using Northern blot analysis. In addition, the ability of PTH and PTHrP to stimulate cAMP production and c-fos mRNA expression in CM/PDL cells was determined, using a cAMP-binding assay and northern blot hybridization, respectively. Rat osteosarcoma cells (ROS 17/2.8) were used as a positive control and human periodontal ligament cells as a negative control. Northern blot analysis demonstrated that cells within the CM/PDL cell population expressed PTH-1 receptor mRNA. Both PTH (1-34) and PTHrP (1-34) increased cAMP and c-fos mRNA in CM/PDL cells. Furthermore, PTHrP treatment for either 24 or 48 h downregulated expression of transcripts for bone sialoprotein, osteocalcin and PTH-1 receptor by CM/PDL cells and abolished CM/PDL cell-mediated mineralization in vitro. These results indicate that cells within the CM/PDL population are targets for PTH and PTHrP action and that PTHrP may play an important part in regulating the biomineralization of cementum.

Growth factors regulate expression of osteoblast-associated genes.

BACKGROUND: The goal of periodontal regenerative therapies is to reconstruct periodontal tissues such as bone, cementum, and periodontal ligament cells (PDL). The need to establish predictable treatment modalities is important for reconstruction of these tissues. The aim of this study was to determine the effects of a low molecular extract of bovine bone protein (BP) containing bone morphogenetic proteins (BMPs) 2, 3, 4, 6, 7, 12, and 13, alone or in combination with platelet-derived growth factor (PDGF) and/or insulin-like growth factor (IGF) on osteoblast differentiation in vitro. METHODS: BP, mixed with a collagen matrix, was added to a poly (DL-lactide-co-glycolide) polymer (PLG) and placed at orthotopic sites in the skullcaps of Sprague-Dawleys rats. At day 28, rats were sacrificed for histological analysis. All sites treated with the polymer/BP produced bone while control sites (without BP) showed no bone formation. Having established the biological activity of BP, in vitro studies were initiated using MC3T3-E1 cells, a mouse osteoprogenitor cell line. The ability of BP and other growth factors to alter cell proliferation was determined by Coulter counter, and differentiation was determined by Northern analysis for specific genes. RESULTS: When compared with cells treated with 2% serum alone, PDGF enhanced cell numbers at 10 and 20 ng/ml; IGF produced no significant effect at these doses; and BP at 10 and 20 microg/ml decreased cell proliferation. Northern analysis revealed that PDGF blocked gene expression of osteopontin (OPN) and osteocalcin (OCN), while BP and IGF promoted gene expression of bone sialoprotein (BSP) and OPN. The combination of BP and IGF enhanced expression of OPN beyond that of either BP or IGF alone. PDGF was able to block the effects of IGF on gene expression, but not those of BP. CONCLUSIONS: These results indicate that BP, PDGF, and IGF influence cell activity differently, and thus raise the possibility that combining factors may enhance the biological activity of cells.

Immortalized cementoblasts and periodontal ligament cells in culture.

Cementum, a mineralized tissue lining the surface of the tooth root, is required for formation of a functional periodontal ligament attachment during development. Additionally, during regeneration of tissues after disease, cementum is thought to play a critical role in the reparative process. Research efforts aimed toward understanding mechanisms involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production, i.e., cementoblasts. Using classical techniques for osteoblast isolation, immortalized, heterogeneous cementoblast/periodontal ligament cell (CM/PDL) populations were established from cells lining the tooth root surface of: 1) CD-1 mice, where cells were immortalized using SV40, or 2) H-2KbtsA58 "immorto" mice, where cells containing an immortalizing transgene were removed and cultured. CM/PDL populations were derived from tissues adherent to developing tooth root surfaces, while tissues adherent to the surrounding alveolar bone were specifically excluded from the population. Immortalized CM/PDL cells were characterized to ensure their phenotype reflected that previously demonstrated in situ and in primary, nonimmortalized cultures. Proteins/mRNAs associated with bone/cementum and known to be expressed by root lining cementoblasts, but not by PDL cells, in situ, e.g., bone sialoprotein, osteopontin, and osteocalcin, were expressed by cells within the immortalized populations. Furthermore, CM/PDL cells, in vitro, attached to bone sialoprotein in an arginine-glycineaspartic acid (RGD)-dependent manner, promoted mineral nodule formation and exhibited a PTH/PTHrP-mediated cAMP response. These immortalized heterogeneous populations, containing both CM and PDL cells, provide a unique opportunity to study cells involved in cementogenesis and to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.

Evolution of periodontal regeneration: from the roots' point of view.

Tissues lost as a consequence of periodontal diseases, i.e. bone, cementum and a functional periodontal ligament (PDL), can be restored to some degree. Nevertheless, results are often disappointing. There is a need to develop new paradigms for regenerating periodontal tissues that are based on an understanding of the cellular and molecular mechanisms regulating the development and regeneration of periodontal tissues. As one approach we have developed strategies for maintaining cementoblasts in culture by first determining the gene profile for these cells in situ. Next, cells were immortalized in vitro using SV 40 large T antigen (SV40 Tag) or by using mice containing transgenes enabling cellular immortality in vitro. Cementoblasts in vitro retained expression of genes associated with mineralized tissues, bone sialoprotein and osteocalcin, that were not linked with periodontal fibroblasts either in situ or in vitro. Further, cementoblasts promoted mineralization in vitro as measured by von Kossa and ex vivo using a severely compromised immunodeficient (SCID) mouse model. These cells responded to growth factors by eliciting changes in gene profile and mitogenesis and to osteotropic hormones by evoking changes in gene profile and ability to induce mineral nodule formation in vitro. The ultimate goal of these studies is to provide the knowledge base required for designing improved modalities for use in periodontal regenerative therapies.

Protein extracts of dentin affect proliferation and differentiation of osteoprogenitor cells in vitro.

Proteins associated with the mineral phase of dentin are considered to have the potential to alter cell function within the local environment, during development and regeneration of tooth/periodontal tissues. Cells that may be altered include osteoblasts, ameloblasts, periodontal ligament cells, odontoblasts, and cementoblasts. However, specific factors within dentin controlling cell activity have not been elucidated. To investigate further the role of dentin proteins in regulating cell behavior, MC3T3-E1 cells, a mouse osteoprogenitor cell line, were exposed to guanidine/EDTA extracts of dentin (G/E-D) prepared from bovine teeth. Cells, with or without G/E-D (2 to 50 microg/ml), were evaluated for proliferative activity and for mRNA expression of bone-associated genes. Results indicated that G/E-D suppressed cell proliferation and caused striking morphological changes, including the conversion of cuboidal cells into fibroblastic, spindle-shaped cells. Markers of osteoblast differentiation, osteocalcin and bone sialoprotein mRNA were decreased, while osteopontin mRNA was enhanced in cells exposed to G/E-D. Since transforming growth factor beta (TGFbeta1) has been reported to influence cells in a similar fashion, G/E-D were examined for the presence of and concentration of TGFbeta using slot blot analysis and enzyme immunoassay (ELISA), respectively. These analyses demonstrated that G/E-D contained 6.6 ng/mg of TGFbeta1. Next, cells were exposed to G/E-D in conjunction with anti-TGFbeta1,2,3 antibody. When cells were exposed to antibody, G/E-D-mediated changes in morphology and gene expression were blocked. These results suggest that TGFbeta1 and perhaps other factors in dentin can regulate cell behavior and, therefore, can influence development, remodeling, and regeneration of mineralized tissues.

Expression of type I and XII collagen during development of the periodontal ligament in the mouse.

The purpose (of this study) was to determine the temporal and spatial pattern of type XII collagen expression during murine tooth/root development. Using in situ hybridization techniques, expression of type XII collagen was compared with that of type I collagen, the most abundant collagen in periodontal tissues. Mouse first mandibular molars were examined at the following developmental periods: pre-root formation, early root formation, initial alignment of the periodontal ligament (PDL) fibres, and PDL maturation as the tooth erupted and attained occlusal function. Transcripts for type I collagen were identified in bone cells and odontoblasts at all times but not in the dental follicle before root formation. As root formation progressed, type I collagen expression became apparent within cells of the dental follicle and forming PDL. During early stages of tooth development, signal for type XII collagen was not observed in any cells/tissues. Type XII collagen expression was first detected in the dental follicle/PDL region during tooth eruption and increased in the PDL as the molar tooth erupted into the mouth and achieved occlusal contact. These findings suggest that type XII expression is timed with the alignment and organization of PDL fibres and is limited in tooth development to cells within the periodontal ligament.

Isolation of murine cementoblasts: unique cells or uniquely-positioned osteoblasts?

While cementoblasts express a number of mineral-related proteins, including bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin (OC), these proteins do not appear to be expressed by cells of the intermediate dental follicle/periodontal ligament (PDL). This information was utilized in an experimental strategy to isolate presumptive cementoblasts from the root surface of day 24 murine mandibular first molars. Using microscopic dissection techniques, molars were carefully extracted from their alveolar crypts and subjected to trypsin-collagenase digestion to remove adherent cells. Primary cultures were established and assayed for expression of proteins known to be expressed by cementoblasts at this timepoint in vivo (i.e. BSP, OPN, OC) and also an odontoblast-specific protein (i.e. DSP) to rule out contamination by pulpal cells. A subgroup of cells were found to express Type I collagen (89% of cells), BSP (46%), OPN (23%) and OC (30%); DSP was not detected within these cultures. We propose that cells within this heterogeneous population, which express this profile of osteogenic proteins, represent cementoblasts. The availability of a cementoblast cell line will make possible rigorous and controlled in vitro analysis of these cells and allow for determination of the unique characteristics of these cells not shared with other cells, particularly osteoblasts.

Expression of bone associated markers by tooth root lining cells, in situ and in vitro.

Periodontal disease is marked by inflammation and subsequent loss and/or damage to tooth-supporting tissues including bone, cementum, and periodontal ligament. A key tissue in the initial process of periodontal development as well as regeneration following periodontal disease is cementum. Research efforts aimed toward understanding mechanisms involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production (i.e., cementoblasts). Much has been learned regarding the processes and mechanisms involved in bone formation and function from experiments using bone cell cultures. Therefore, the purpose of this study was to develop a strategy whereby cementoblasts could be isolated, cultured, and characterized. As a first step, using in situ hybridization, we determined the timed and spatial expression of mineral-associated proteins during first molar root development in CD-1 mice. These proteins included dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), and type I collagen. During root development in mice BSP, OPN, and OCN mRNAs were expressed selectively by cells lining the tooth root surface--cementoblasts--with high levels of expression at day 41. Importantly, at this time point BSP, OPN, and OCN mRNAs were not expressed throughout the periodontal ligament. These findings provided us with markers selective to root-lining cells, or cementoblasts, in situ, and established the time (day 41) for isolating cells for in vitro studies. To isolate cells from tissues adherent to the root surface, enzymatic digestion was used, similar to what are now considered classical techniques for isolation of osteoblasts. To determine whether cells in vitro contained root-lining cells and cementoblasts, cultured cells were analyzed for expression of mineral-associated proteins. Cells within this heterogeneous primary population expressed type I collagen, BSP, OPN, and OCN as determined by in situ hybridization. In contrast, cells within this population did not express dentin sialoprotein, an odontoblast-specific protein. These procedures have provided a means to obtain root-lining cells in vitro that can now be cloned and used for studies directed at determining the properties of root-lining cells, or cementoblasts, in vitro.

Expression of adhesion molecules during tooth resorption in feline teeth: a model system for aggressive osteoclastic activity.

Tooth resorption, a common feline dental problem, is often initiated at the cemento-enamel junction and hence is called cat 'neck' lesion. Studies have demonstrated that osteoclasts/odontoclasts are increased and activated at resorption sites, and that areas of resorption are partly repaired by formation of tissues resembling bone, cementum, and possibly dentin. However, the cellular/molecular mechanisms/factors involved in resorption and repair are unknown. In this study of tissues from cats with 'neck' lesions, we used specific antibodies and immunohistochemical analyses to examine adhesion molecules associated with mineralized tissues, bone sialoprotein (BSP) and osteopontin (OPN), and a cell-surface receptor linked with these molecules, alpha v beta 3, for their localization in these lesions. In addition, to determine general cellular activity during repair, we performed in situ hybridization using a type I collagen riboprobe. Results showed OPN localized to resorption fronts and reversal lines, while BSP was localized to reversal lines. However, some osteoclasts and odontoblasts "sat" on mineralized surfaces not associated with OPN. The cell-surface receptor, alpha v beta 3, was localized to surfaces of osteoclasts/odontoclasts. Type I collagen mRNA was expressed where osteoblasts attempted to repair mineralized tissue. In contrast, odontoblasts did not express mRNA for type I collagen. This study suggests that osteoclastic resorption is the predominant activity in 'neck' lesions and that this activity was accompanied, at least in part, by increased concentrations of OPN and an associated integrin, alpha v beta 3, at resorption sites. Lack of collagen expression by odontoblasts indicates that odontoblasts do not play an active role in attempts at repair.

Expression of bone sialoprotein mRNA by cells lining the mouse tooth root during cementogenesis.

Adhesion molecules are considered to have an active role in controlling cell differentiation, although the mechanisms involved have yet to be determined. The developing tooth provides an excellent model to use for determining the factors/processes regulating cell differentiation. The studies presented here focused specifically on the timed and spatial expression of a bone-associated adhesion molecule, bone sialoprotein, during tooth root development. Mandibular tissues in the first molar region of CD-1 mice, at sequential stages of development, were analysed by in situ hybridization. The results demonstrate distinct expression of bone sialoprotein in surrounding bone at early stages of tooth development. At stages of active cementogenesis, bone sialoprotein transcripts were specific to cells lining the root surface, with limited expression in the surrounding connective tissue (periodontal ligament) region. The strong expression of bone sialoprotein, a mineral-specific protein having the capacity to act as a nucleator of hydroxyapatite in vitro, by cells lining the root surface at early stages of cementogenesis suggests that this molecule is operative in the cell/matrix events that accompany cementum formation.

Agents with periodontal regenerative potential regulate cell-mediated collagen lattice contraction in vitro.

A variety of pharmaceutical agents has been proposed for use in periodontal therapy to inhibit loss of alveolar bone and to promote regeneration of tissues lost to disease. The purpose of this study was to determine the effects of such agents on periodontal cell-mediated gel contraction, an in vitro process considered representative of wound contraction and remodeling in vivo. Human gingival fibroblasts were cultured in a type I collagen lattice, and contraction was quantified by means of a computer-assisted video imaging system. Cell-gel combinations were prepared with cells both pre-exposed and non-exposed to agents; non-anchored cell-gels were then incubated with agents for various time periods. Agents tested included Demecolcine (an inhibitor of cytoskeletal contraction), growth factors (i.e., TGF-beta 1, PDGF, and IGF-1), and non-steroidal anti-inflammatory drugs (NSAIDs) (indomethacin, ibuprofen, naproxen, and flurbiprofen). While Demecolcine inhibited gel contraction, TGF-beta 1 (1-20 ng/mL), PDGF (100 ng/ML), IGF-1 (1000 ng/mL), and [PDGF + IGF], all of which have been reported to enhance wound healing in vivo, promoted lattice contraction in this system. In contrast, NSAIDs inhibited cell-gel contraction. Ethanol, used to solubilize two specific NSAIDs, also inhibited cell proliferation and gel contractile ability, even at very low concentrations. These findings indicate that periodontal cells respond differently to various agents in vitro and may be adversely affected by alcohol. Furthermore, the results of this study suggest that the cell-lattice contraction system holds potential as a method for screening agents for positive or negative effects on cell activity.

Localization and expression of osteopontin in mineralized and nonmineralized tissues of the periodontium.

To summarize results from various studies focusing on determining the expression/localization of BSP and OPN during tooth root development, there is general agreement that OPN is expressed/localized to the root surface during cementogenesis and is also seen throughout the PDL region. The expression/localization of OPN to odontoblasts and its role in dentinogenesis is less apparent. Recent studies directed at establishing odontoblast cell lines should help to resolve this conflict. Studies on BSP expression during tooth root formation indicate a very precise expression and localization of this molecule during cementogenesis indicating that this molecule may play an important role in the formation of this mineralized tissue. However, as with OPN, the expression of BSP and its role in dentin formation is not clearly defined.

Role of two mineral-associated adhesion molecules, osteopontin and bone sialoprotein, during cementogenesis.

Adhesion molecules and their cell membrane receptors are known to play important regulatory roles in cell differentiation. Consequently, the following experiments were conducted to determine the role of two adhesion molecules, bone sialoprotein (BSP) and osteopontin (OPN) in tooth root formation. Developing murine molar tooth germs at sequential stages of development (developmental days 21-42) were analyzed using immunohistochemical and in situ hybridization techniques. While BSP was localized to alveolar bone and odontoblasts early in development, BSP was distinctly localized to the cemental root surface at latter periods coincident with the initiation of root formation and cementogenesis. Conversely, OPN was distributed in a nonspecific fashion throughout the PDL and the eruption pathway of the forming tooth. In situ hybridization confirmed that cells lining the root surface express BSP. The fact that BSP is specifically localized to the cemental surface suggests that this protein is involved in cementoblast differentiation and/or early mineralization of the cementum matrix. Localization of OPN to non-mineralized tissues further suggests that OPN functions as an inhibitor of mineralization during periodontal ligament formation. These findings collectively suggest that BSP and OPN are intimately involved in the sequence of cellular and molecular events accompanying cementogenesis.

Models for the study of cementogenesis.

Cementum is a mineralized tissue that acts to connect the periodontal ligament to the tooth root surface. Its composition is very much like bone, being comprised mainly of type I collagen, inorganic mineral and noncollagenous proteins, however the origin of the cells and factors necessary for cementum formation have yet to be elucidated. Our laboratory has focused on the role that adhesion molecules, and their cell surface receptors, play in the formation of cementum and tooth root. In order to study this, we used a mouse molar as a model system. This system enabled us to study the formation of four distinct mineralized tissues; bone, cementum, dentin and enamel at various stages of their development. For these studies, we initiated experiments to examine potential cementoblast progenitor cells, in vitro. As a first step, we show that dental papilla and dental follicle cells, n vitro, obtained from molar tissues at day 21 of development, induce mineralized nodules, in vitro. In addition, we obtained tissues from mice where defects in root development may exist and determined bone sialoprotein (BSP) protein expression, a mineralized tissue specific adhesion molecule, in such tissues. As discussed here, we found that osteopetrotic (op/op) mice have delayed and/or defective root development and BSP does not localize in the dental tissues, at day 33 of development. In addition, dentin formation was defective and odontoblasts appeared immature, based on morphological examination. In contrast, the day 33 control molars demonstrated positive staining for BSP localized to root cementum, with normal formation of dentin.

Bone sialoprotein is localized to the root surface during cementogenesis.

Bone sialoprotein (BSP), an RGD-containing protein with cell attachment properties, is believed to play a regulatory role in the biomineralization of various connective tissues. To determine its possible role in tooth root formation, murine dentoalveolar tissues at sequential phases of development were analyzed immunohistochemically for the presence of BSP. BSP was localized to alveolar bone and cementum at time points associated with initial mineralization of these tissues. In addition, northern blot analyses of dental follicle tissue at day 27 of tooth development indicated that BSP mRNA is expressed by dental follicle cells at a time point coincident with the initiation of cementogenesis on the peripheral tooth root surface. Collectively, these findings indicate that BSP may play an important role in the formation and mineralization of cementum.

Increased osmolal gap in alcoholic acidosis.

We studied a patient with alcoholic acidosis and an increased osmolal gap. Ethyl alcohol and other compounds that are known to increase serum osmolality in alcoholics were not detected. However, the levels of glycerol, acetone, and the acetone metabolites acetol and 1,2-propanediol were increased in the serum of this patient. On admission and 3 and 7 hours after admission, the combined serum osmolality of glycerol, acetone, acetol, and 1,2-propanediol accounted for 48%, 92%, and 62% of the increase in the osmolal gap above the highest normal level of 10 mOsm/kg H2O. The disappearance of the osmolal gap correlated with the correction of the acidosis and the concomitant reduction in serum glycerol and acetone levels. Elevations of endogenous glycerol, acetone, and acetone metabolite levels should now be added as causes for an increased osmolal gap in the alcoholic patient. Ingestion of toxic alcohols can no longer be assumed to be the only cause for an increased osmolal gap in alcoholic patients.