PTH(1-34) affects osteoprotegerin production in human PDL cells in vitro

Since periodontal ligament (PDL) cells exhibit several osteoblastic traits, we hypothesized that human PDL cells will respond to hormonal stimulation in an osteoblast-like manner. Confluent and pre-confluent PDL cells from six patients were challenged with PTH(1-34). Cell number, ALP, osteocalcin, osteoprotegerin, and RANKL expression were determined. Intermittent PTH(1-34) treatment of confluent PDL cells caused a significant increase in proliferation, whereas differentiation and osteoprotegerin production decreased significantly. In pre-confluent PDL cells, this treatment regimen induced a biphasic decrease in proliferation, but a biphasic increase in differentiation and osteoprotegerin production. Continuous PTH(1-34) exposure enhanced proliferation but inhibited osteocalcin production in confluent cells and stimulated osteoprotegerin production in pre-confluent PDL cells. RANKL was hardly detectable and unaffected by PTH(1-34) treatment. These results indicate that human PDL cells respond to PTH(1-34) in an osteoblast-like manner, and that the PTH(1-34) effect depends on the maturation state of the cells and on the mode of administration.     

Aging affects the phenotypic characteristics of human periodontal ligament cells and the cellular response to hormonal stimulation in vitro

Lossdörfer S, Kraus D, Jäger A. Aging affects the phenotypic characteristics of human periodontal ligament cells and the cellular response to hormonal stimulation in vitro. J Periodont Res 2010; 45: 764–771. © 2010 John Wiley & Sons A/S Background and Objective: Aging modulates the proliferative activity and organic matrix production of cells in vivo and in vitro. Here, we explore how aging affects the phenotypic characteristics of human periodontal ligament cells and their response to hormonal stimulation. Material and Methods: Fifth passage periodontal ligament cells from subjects aged 12–14 (group 1), 41–55 (group 2) and 61–70 years (group 3) were characterized for the expression of mesenchymal marker genes and proteins by real‐time PCR and flow cytometry. Confluent cultures were exposed to 10^?12 m parathyroid hormone(1–34) [PTH(1–34)] intermittently for three cycles. At harvest, cell number, alkaline phosphatase activity and osteocalcin production were determined by cell count, biochemical assay and ELISA. Results: The characterization of the cells revealed a decreased expression of osteoblast‐specific marker genes along with a lower percentage of cells presenting the respective proteins with age. An intermittent exposure of the cultures to 10^?12 m PTH(1–34) induced an increase of the cell number as opposed to a significant decrease of alkaline phosphatase activity and osteocalcin production. The cellular response to PTH(1–34) was strongest in group 1. Basal osteoprotegerin levels were highest in the cultures from the oldest donors and inhibited by intermittent PTH(1–34) in all groups. Conclusion: Our data indicate that periodontal ligament cells from older subjects display a less differentiated phenotype and a reduced response to intermittent PTH, suggesting a compromised ability to maintain tissue homeostasis and a limited possibility to support periodontal repair processes with age. The high basal osteoprotegerin expression in older subjects might serve as a compensatory mechanism.     

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.     

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.     

Short-term heat pre-treatment modulates the release of HMGB1 and pro-inflammatory cytokines in hPDL cells following mechanical loading and affects monocyte behavior

Objective

Heat shock proteins (HSP) act as cell-protective molecules that are upregulated upon thermal insult, hypoxia, and ischemia. Such ischemic conditions can be found during tissue remodeling associated with orthodontic tooth movement or trauma when compression forces lead to cell necrosis and subsequent clearance of cellular debris by immune competent cells. Host immune overreaction can result in undesired side effects such as tooth root resorption. Here, we analyzed whether heat pre-treatment would affect the initially catabolic host immune response induced by mechanical loading of human periodontal ligament (hPDL) cells, which represent major constituents of the tooth supporting apparatus involved in the regulation of periodontal remodeling.

Materials and methods

Fifth passage hPDL cells were exposed to an elevated temperature of 43° for 1 h prior to mechanical loading. Cell morphology, high mobility group box protein 1 (HMGB1), interleukin (IL)-6, and IL-8 expression were analyzed microscopically and by ELISA. The physiological relevance for monocyte behavior was tested in monocyte adhesion and osteoclast differentiation assays.

Results

Short-term heat pre-treatment did not show any visible effect on hPDL cell morphology, but resulted in a significant downregulation of pro-inflammatory cytokines when being additionally loaded mechanically. Supernatants of heat-exposed hPDL cell cultures demonstrated a reduced impact on monocyte adhesion and osteoclastic differentiation.

Conclusions

Heat pre-treatment of hPDL cells induces cell-protective mechanisms towards mechanical stress and favors the reduction of cell stress associated effects on monocyte/macrophage physiology.

Clinical relevance

These data present the induction of heat shock proteins as a promising treatment option to limit undesired side effects of periodontal remodeling.

     

Potential role of high mobility group box protein 1 and intermittent PTH (1–34) in periodontal tissue repair following orthodontic tooth movement in rats

Objectives

Recent studies indicate that high mobility group box protein 1 (HMGB1) can be released by necrotic and damaged cells and functions as an alarmin that is recognized by the innate immune system. Little is known about the role of HMGB1 within the periodontal ligament (PDL). Therefore, we examined HMGB1 expression by PDL cells in vitro and compared the findings to an in vivo model of orthodontically induced tooth root resorption. In addition, we addressed the question of whether a potentially anabolic intermittent administration of parathyroid hormone (iPTH) would modulate the expression of HMGB1.

Materials and methods

In confluent PDL cell cultures, HMGB1 messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction. In a rat model comprising 25 animals, mechanical loading for 5 days was followed by administration of either iPTH (1–34) systemically or sham injections for up to 56 days. HMGB1 expression was determined by means of immunohistochemistry and histomorphometry.

Results

The in vitro experiments revealed an inhibitory effect of iPTH on basal HMGB1 mRNA expression in confluent PDL cells. In vivo, the mechanical force-induced enhanced HMGB1 protein expression declined time dependently. Intermittent PTH further inhibited HMGB1 expression. The significantly higher basal HMGB1 protein expression in the former compression side was followed by a more pronounced time- and iPTH-dependent decline in the same area.

Conclusions

These data indicate a major role for HMGB1 in the regulation of PDL wound healing following mechanical load-induced tissue injury.

Clinical relevance

The findings point to the potential benefit of iPTH in the attempt to support these immune-associated reparative processes.     

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.     

Immunohistochemical localization of receptor activator of nuclear factor kappaB (RANK) and its ligand (RANKL) in human deciduous teeth

Osteoblasts and osteoclasts are involved in bone formation and resorption. RANK and RANKL [receptor activator of nuclear factor kappaB (ligand)], two cytokine-like proteins of the tumor necrosis factor superfamily, are localized on these bone cells and are crucial for the regulation of osteoclastic cell differentiation from hematopoietic precursors and also for the upregulation of mature osteoclasts mediated by cell-to-cell contact and a subsequent cascade of diverse intracellular signaling processes in the osteoclasts. It was the aim of this study to examine the sites of expression of RANKL and RANK in the corresponding cells of human dental hard and periodontal tissues using immunohistochemical light microscopical methods on tissue sections of 15 paraffin-embedded human deciduous teeth undergoing root resorption. We detected granular cytoplasmic RANKL-immunoreactivity in odontoblasts, pulp fibroblasts, periodontal ligament fibroblasts, and in single odontoclasts, the latter finding suggesting an autocrine/paracrine role. RANK-positive cells were identified as multinucleated odontoclasts localized near the dentine surface in resorption lacunae or as mononucleated precursors. These findings indicate that human dental cells express key mediators of hard tissue resorption and, though the RANK/RANKL-system may not be the sole regulator of tooth root resorption, these factors could at least contribute to this complex process under both physiological and pathological conditions.