Functional characterization of the parathyroid hormone 1 receptor in human periodontal ligament cells.

OBJECTIVES: Intermittent parathyroid hormone (PTH) exerts anabolic effects on bone and has been approved for osteoporosis therapy. The dual actions of PTH are mediated primarily through the parathyroid hormone 1 receptor (PTH1R). Upon ligand binding, PTH1R activates diverse signaling pathways, including cAMP/protein kinase A (PKA)- and phospholipase C/protein kinase C (PLC/PKC)-dependent pathways. PTH1R has been abundantly studied in bone cells. Knowledge on PTH1R characteristics and physiology in periodontal ligament (PDL) cells is still in its infancy. MATERIALS AND METHODS: We characterized PTH1R in PDL cells in terms of its cellular localization, binding affinity, and signal transduction and compared these characteristics to those of MG63 osteoblast-like cells. RESULTS: PTH1R mRNA/protein was identified in PDL and MG63 cells. PTH1R was mainly localized on the plasma membrane, in vesicular structures inside the cell, and, to some extent, in the nucleus of both cell types. Binding characteristics of PTH1R were cell type specific, with PDL cells demonstrating a lower binding affinity. The response of cAMP and active PKC production in MG63 cells was dose dependent with increasing PTH(1-34) concentration, whereas in PDL cells, it was regulated biphasically. However, we observed a cross talk between the cAMP/PKA and PLC/PKC signaling pathways, which were regulated diametrically opposed at a given concentration of PTH(1-34). CONCLUSION: These data indicate that, albeit the similarity in its subcellular distribution, PTH1R in PDL cells exhibits characteristics different from those in MG63 cells, pointing to the cell type specificity of this receptor. CLINICAL RELEVANCE: The findings further elucidate the characteristics of PTH action in dental tissues and widen the theoretical basis for the development of anabolic treatment strategies.

Autofluorescent characteristics of human periodontal ligament cells in vitro.

The periodontal ligament (PDL) is a connective tissue surrounding the tooth root and attaching it to its bony socket. Being the predominant cell type of this connective tissue, PDL fibroblasts are thought to be responsible for PDL homeostasis and regeneration. Autofluorescence is found in abundant natural substances and many cells exhibit some intrinsic level of autofluorescence depending on the cell type and function. Thus, autofluorescence often makes immunofluorescence staining and flow cytometric analysis difficult. Therefore, in the present study, we have investigated the autofluorescent characteristics of human periodontal ligament (PDL) cells and compared them to cells of epithelial and mesenchymal origin. The results of fluorescence immunostaining and flow cytometric analysis revealed that PDL cells exhibit an extremely high level of autofluorescence in the short wave length region including FITC emission spectra which exceeded those of HEK293 cells, oral keratinocytes and MG63 osteoblast-like cells. This observation reflects the high capacity of PDL cells to synthesize extracellular matrix proteins and collagens. In the long wavelength region, the autofluorescent intensity of PDL cells was the lowest amongst the investigated cell lines. A permeabilization step with Triton X-100 also reduced the autofluorescence of PDL cells in the FITC channel. Our data indicate that conjugated antibodies with emission spectra in the red fluorescence region might be the proper choice for studies using immunofluorescence staining and flow cytometric analysis of human PDL cells. Fixation and permeabilization of the cells might also be beneficial in this respect.

Effect of intermittent PTH(1-34) on human periodontal ligament cells transplanted into immunocompromised mice.

Residual periodontal ligament (PDL) cells in the damaged tissue are considered a prerequisite for a successful regeneration of the periodontal architecture with all its components, including gingiva, PDL, cementum, and bone. Among other approaches, current concepts in tissue engineering aim at a hormonal support of the regenerative capacity of PDL cells as well as at a supplementation of lost cells for regeneration. Here, we investigated how far an anabolic, intermittent parathyroid hormone (iPTH) administration would enhance the osteoblastic differentiation of PDL cells and the cellular ability to mineralize the extracellular matrix in an in vivo transplantation model. PDL cells were predifferentiated in a standard osteogenic medium for 3 weeks before subcutaneous transplantation into CD-1 nude mice using gelatin sponges as carrier. Daily injections of 40 µg/kg body weight PTH(1-34) or an equivalent dose of vehicle for 4 weeks were followed by explantation of the specimens and an immunohistochemical analysis of the osteoblastic marker proteins alkaline phosphatase (ALP), osteopontin, and osteocalcin. Signs of biomineralization were visualized by means of alizarin red staining. For verification of the systemic effect of iPTH application, blood serum levels of osteocalcin were determined. The osteogenic medium stimulated the expression of ALP and PTH1-receptor mRNA in the cultures. After transplantation, iPTH resulted in an increased cytoplasmic and extracellular immunoreactivity for all markers investigated. In contrast to only sporadic areas of mineralization under control conditions, several foci of mineralization were observed in the iPTH group. Blood serum levels of osteocalcin were elevated significantly with iPTH. These data indicate that the osteoblastic differentiation of human PDL cells and their ability for biomineralization can be positively influenced by iPTH in vivo. These findings hold out a promising prospect for the support of periodontal regeneration.

Intermittent PTH(1-34) signals through protein kinase A to regulate osteoprotegerin production in human periodontal ligament cells in vitro.

Periodontal ligament (PDL) cells have been associated with the regulation of periodontal repair processes by the differential expression of osteoprotegerin and RANKL in response to intermittent parathyroid hormone (PTH) resulting in a modified activity of bone-resorbing osteoclasts. Here, we examined the intracellular signaling pathways that PDL cells use to mediate the PTH(1-34) effect on osteoprotegerin production and hypothesized that those would be dependent on the cellular maturation stage. Two stages of confluence served as a model for cellular maturation of 5th passage human PDL cells from six donors. Intermittent PTH(1-34) (10(-12) M) and PTH(1-31), the latter lacking the protein kinase C (PKC) activating domain, induced a significant decrease of osteoprotegerin production in confluent cultures, whereas the signal-specific fragments PTH(3-34) and PTH(7-34), which both are unable to activate protein kinase A (PKA), had no effect. The addition of the PKA inhibitor H8 antagonized the PTH(1-34) effect, whereas the PKC inhibitor RO-32-0432 did not. In pre-confluent, less mature cultures, intermittent PTH(1-34) resulted in a significant increase of osteoprotegerin. Similar results were obtained when PTH(1-31) substituted for PTH(1-34) as opposed to a lack of an effect of PTH(3-34) and PTH(7-34). Likewise, in confluent cultures, H8 inhibited the PTH(1-34) effect in pre-confluent cultures contrasted by RO-32-0432 which had no effect. These findings indicate that PTH(1-34) signaling targeting osteoprotegerin production in PDL cells involves a PKA-dependent pathway. The PTH(1-34) effect is dependent on cell status, whereas intracellular signal transduction is not. Clinical trials will have to prove whether those in vitro data are of physiological relevance for interference strategies.

Bone morphogenetic protein-7 modifies the effects of insulin-like growth factors and intermittent parathyroid hormone (1-34) on human periodontal ligament cell physiology in vitro.

BACKGROUND: Components of the insulin-like growth factor (IGF) system and intermittent parathyroid hormone (PTH) were demonstrated to exert anabolic effects on the periodontal ligament (PDL) and, thereby, contribute to the regeneration of structures that were lost because of inflammatory periodontal disease. METHODS: In the present study, a PDL cell culture is created to examine whether a combination of IGFs and intermittent PTH would enhance the cellular response elicited by each factor alone and addressed the question of whether a pretreatment of the cells with bone morphogenetic protein (BMP)-7 alters the PDL cell phenotype and behavior on IGF-PTH stimulation. RESULTS: IGF-I and IGF-II enhanced proliferation and differentiation in preconfluent and confluent cultures. A BMP-7 pretreatment inhibited those effects in preconfluent cells but not in confluent cultures. Intermittent PTH (1-34) inhibited the proliferation but stimulated the differentiation of preconfluent cells, whereas the opposite effect was observed in confluent cultures. BMP-7 prestimulation made preconfluent cells respond like confluent cultures on intermittent PTH (1-34) challenge. A combined administration of PTH (1-34) and IGF-I or IGF-II mostly mirrored the effects seen for one of the individual agents alone, but synergistic effects were not observed. CONCLUSIONS: The present data indicate that both PTH (1-34) and IGF-I or IGF-II exert a maturation stage-dependent effect on PDL cell proliferation and differentiation. Combining PTH (1-34) and IGF does not seem to potentiate the effects seen for the agents alone. Furthermore, our data suggest that BMP-7 induces preconfluent cells to acquire a more differentiated phenotype and to respond to intermittent PTH (1-34) accordingly. Confluent cultures seem to be less susceptible to BMP-7.