Orthodontic cell stress modifies proinflammatory cytokine expression in human PDL cells and induces immunomodulatory effects via TLR-4 signaling in vitro.

OBJECTIVE: Biomechanical orthodontics loading of the periodontium initiates a cascade of inflammatory signaling events that induce periodontal remodeling and finally facilitate orthodontic tooth movement. Pattern recognition receptors such as toll-like receptors (TLRs) have been well characterized for their ability to induce the activation of inflammatory, immunomodulatory cytokines. Here, we examined whether the cellular response of human periodontal ligament (hPDL) cells to mechanical stress involves TLR-4 signaling in vitro. MATERIALS AND METHODS: Confluent hPDL cells were cultured in the presence of 5 µg/ml TLR-4 antibody (TLR-4ab) for 1 h prior to the induction of compressive forces by the use of round glass plates for 24 h. At harvest, interleukin-6 and interleukin-8 (IL-6, IL-8) mRNA and protein expression were analyzed by real-time PCR and ELISA. The immunomodulatory role of mechanical cell stress and TLR-4 signaling was addressed in co-culture experiments of hPDL and THP-1 cells targeting monocyte adhesion and by culturing osteoclastic precursors (RAW 264.7) in the presence of the conditioned medium of hPDL cells that had been mechanically loaded before. RESULTS: Basal expression of IL-6 and IL-8 was not affected by TLR-4ab, but increased significantly upon mechanical loading of hPDL cells. When cells were mechanically stressed in the presence of TLR-4ab, the effect seen for loading alone was markedly reduced. Likewise, monocyte adhesion and osteoclastic differentiation were enhanced significantly by mechanical stress of hPDL cells and this effect was partially inhibited by TLR-4ab. CONCLUSIONS: The results of the present study indicate a proinflammatory and immunomodulatory influence of mechanical loading on hPDL cells. Intracellular signaling involves a TLR-4-dependent pathway. CLINICAL RELEVANCE: These findings hold out the prospect of interfering with the cellular response to mechanical cell stress in order to minimize undesired side effects of orthodontic tooth movement.

Heat shock protein 70 dampens the inflammatory response of human PDL cells to mechanical loading in vitro.

BACKGROUND AND OBJECTIVE: Previously, we demonstrated an inflammatory response of human PDL (hPDL) cells to mechanical loading. The cellular reaction was dampened by heat pre-treatment suggesting a protective role for heat shock proteins (HSP) during stress-induced ischemia. Here we explored if HSP70, which has already been documented in the pressure zone of tooth movement, might be regulatorily involved in the attenuation of the inflammatory response. MATERIALS AND METHODS: Fifth passage hPDL cells were mechanically loaded in the presence of the HSP70 inhibitor VER155008. Cell morphology, HSP70 expression, viability, IL-6 and IL-8 expression were determined by means of microscopy, realtime-PCR and ELISA. The conditioned medium of mechanically loaded and pre-treated hPDL cells was used to culture monocytes to identify a potential impact on adhesion and osteoclastic differentiation capacity. RESULTS: Mechanical cell stress resulted in a significant increase of pro-inflammatory parameters. HSP70 inhibition led to a further enhancement of cytokine expression. The conditioned medium of mechanically loaded hPDL cells significantly increased monocyte adhesion and differentiation along the osteoclastic pathway. VER155008 pronounced this effect significantly. CONCLUSION: The results indicate a regulatory role for HSP70 in the control of the inflammatory hPDL cell response to mechanical loading and identify HSP70 as a target in the attempt to attenuate tissue damage during orthodontic tooth movement. Furthermore, the present findings point to the risk of increased periodontal destruction when medication targeting HSP70 is applied for severe medical conditions during orthodontic tooth movement.

Role of HSP70 protein in human periodontal ligament cell function and physiology.

OBJECTIVE: Heat pre-treatment of mechanically loaded human periodontal ligament cells (hPDL) dampens the inflammatory cellular response, as evidenced by a reduced expression of pro-inflammatory cytokines, inhibition of monocyte adhesion and osteoclastic differentiation. These findings imply heat shock proteins (HSP) as cell protective molecules acting in the PDL that are up-regulated upon ischemia caused by mechanical loading. HSP70 and its inhibition by VER155008 as the active agent in several pharmaceuticals are established targets and strategies, respectively, in the treatment of neoproliferative diseases. However, the effect of both players on periodontal remodeling in unknown. Therefore, we analyzed the role of HSP70 and its frequently used inhibitor VER155008 in the regulation of physiological hPDL cell functions and immune cell interaction. MATERIALS AND METHODS: Fifth passage hPDL cells were cultured in the presence of 25µm HSP70 inactivating agent VER155008. At harvest, HSP70 expression, cell proliferation, and parameters of cell interaction, colony formation and wound healing were analyzed by means of real-time PCR, immunohistochemistry, Western blot, biochemical MTS assay, microscopy, and functional assays for monocyte adhesion and differentiation. RESULTS: Basal HSP70 expression and hPDL cell morphology were not affected by HSP70 inhibitor VER155008. In contrast, cell proliferation, tissue defect healing, and colony formation were reduced significantly following HSP70 inhibition, whereas apoptosis and necrosis, monocyte adhesion and osteoclastic differentiation were markedly increased. CONCLUSIONS: The present data indicate a regulatory role for HSP70 protein in hPDL cell biology. CLINICAL RELEVANCE: These findings identify HSP70 as a promising target in the attempt to modify periodontal remodeling and point to potential periodontal side effects of HSP70 pharmaceutical usage.

CD8+ T cells mediate the regenerative PTH effect in hPDL cells via Wnt10b signaling.

It was the aim of the present investigation to examine whether the stimulating effect of parathyroid hormone (PTH) on human periodontal ligament (hPDL) cell proliferation and differentiation would be enhanced by hPDL/T-cell interaction involving Wnt10b signaling as a mediating pathway. hPDL cells were cultured from healthy premolar tissues of three adolescent orthodontic patients and exposed to PTH(1-34) in monocultures or co-cultures with CD8+ T cells. At harvest, proliferation, alkaline phosphatase-specific activity (ALP), and osteocalcin production were determined by immunofluorescence cytochemistry, real-time PCR, biochemical assay, and ELISA. Wnt10b signaling was analyzed by the use of a specific WNT10b neutralizing antibody. PTH(1-34) stimulation of T cells significantly increased Wnt10b expression and production. Wnt10b exposure of hPDL cells enhanced proliferation and differentiation. PDL cells co-cultured with T cells showed a Wnt10b-dependent regulation of proliferation and differentiation parameters. The addition of a Wnt10b-neutralizing Ab to the co-culture medium resulted in a significant inhibition of the PTH(1-34) effect on proliferation, ALP-specific activity, and osteocalcin protein expression. Our findings provide novel insight into the mechanism of action of PTH on hPDL cells and establish the interplay of T cells and hPDL cells via the Wnt10b pathway as a modulating factor for the anabolic properties of the hormone in 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.

Histopathological Verification of Osteoimmunological Mediators in Peri-Implantitis and Correlation to Bone Loss and Implant Functional Period.

Peri-implantitis (PI) is characterized by inflammation and bone resorption eventually leading to implant failure, but the characteristic pathologic determinants are undefined to date. This study aims to elucidate the parameters involved in PI pathogenesis, including intraoral implant retention time, extent of bone loss, smoking history, and identification of osteoimmunological markers for inflammation and bone loss. Peri-implant tissues (n = 21) displaying clinically diagnosed PI from patients with vertical bone loss ranging from 0-12 mm and implant function period between 1 and 60 months were evaluated by histochemistry and immunohistochemistry for TRAP, CD3, RANK, RANKL, OPG, and TNF-α. Statistical analyses were performed with the Welch test and correlation coefficients were calculated. Most bone resorption occurred during the first 12 months of implant function and correlated with the extent of inflammation, although histological signs of inflammation strongly varied between samples from minimal appearance of inflammatory cells to extended infiltrates. Implant function period and smoking history did not significantly affect the degree of inflammation. Higher RANK levels emerged in the first 12 months of implant function compared to longer retention times and were negatively correlated to the occurrence of RANKL. Additionally, histological signs of inflammation were about two-fold higher in specimens with bone resorption up from 5 mm compared to under 5 mm. CD3(+) cells were more prevalent in extensive inflammatory infiltrates and samples derived from smokers. Our analyses proved that PI-induced bone loss is differentially influenced by the parameters evaluated in this study, but a distinct interconnection between disease severity and implant retention time can be established.

Anabolic properties of high mobility group box protein-1 in human periodontal ligament cells in vitro.

High mobility group box protein-1 (HMGB1) is mainly recognized as a chemoattractant for macrophages in the initial phase of host response to pathogenic stimuli. However, recent findings provide evidence for anabolic properties in terms of enhanced proliferation, migration, and support of wound healing capacity of mesenchymal cells suggesting a dual role of the cytokine in the regulation of immune response and subsequent regenerative processes. Here, we examined potential anabolic effects of HMGB1 on human periodontal ligament (PDL) cells in the regulation of periodontal remodelling, for example, during orthodontic tooth movement. Preconfluent human PDL cells (hPDL) were exposed to HMGB1 protein and the influence on proliferation, migration, osteogenic differentiation, and biomineralization was determined by MTS assay, real time PCR, immunofluorescence cytochemistry, ELISA, and von Kossa staining. HMGB1 protein increased hPDL cell proliferation, migration, osteoblastic marker gene expression, and protein production as well as mineralized nodule formation significantly. The present findings support the dual character of HMGB1 with anabolic therapeutic potential that might support the reestablishment of the structural and functional integrity of the periodontium following periodontal trauma such as orthodontic tooth movement.

Analysis of the torque capacity of a completely customized lingual appliance of the next generation.

INTRODUCTION: In lingual orthodontic therapy, effective torque control of the incisors is crucial due to the biomechanical particularities associated with the point of force application and the tight link between third order deviations and vertical tooth position. AIM: The aim of the present in vitro investigation was to analyze the torque capacity of a completely customized lingual appliance of the next generation (WIN) in combination with different finishing archwire dimensions. METHODS: Using a typodont of the upper arch carrying the WIN appliance, slot filling and undersized individualized ß-titanium archwires were engaged. Horizontal forces ranging from 0 to 100 cN were applied at the central incisor by means of spring gauges. The resulting angular deviations were recorded and the corresponding torque moments were calculated. RESULTS: For fullsize archwires (0.018"×0.018" ß-titanium and 0.018"×0.025" ß-titanium), an initial torque play of 0-2° had to be overcome prior to the development of an effective torque moment. Thereafter, a linear correlation between torque angle and torque moment developed for both archwire dimensions with steeper slopes calculated for the specimens with the larger dimension. A torque moment of 2 Nmm required for effective torque correction was noted after a minimum of 2-3° of twist for the 0.018"×0.018" ß-titanium wires as compared to 2-4° for the 0.018"×0.025" ß-titanium study sample. When undersized archwires were analyzed (0.0175"×0.0175" ß-titanium), the measured torque play ranged from 5-7°. After 8-12° of torque angle, the threshold of 2 Nmm was reached. A linear relationship between twist angle and torque moment in which the steepness of the slopes was generally flatter than the ones calculated for the slot filling archwires was noted. CONCLUSIONS: Given the high precision of the bracket slot-archwire-combination provided with the WIN appliance, an effective torque control can be clinically realized.

Regulation of high mobility group box protein 1 expression following mechanical loading by orthodontic forces in vitro and in vivo.

SUMMARY INTRODUCTION: The chromatin-binding protein high mobility group box protein 1 (HMGB1) can be released into the extracellular milieu by necrotic and damaged cells and functions as an alarmin that is recognized by the innate immune system to initiate and modulate tissue repair. However, little is known about the function of HMGB1 within orthodontic tooth movement. Therefore, it was the aim of the present study to investigate HMGB1 expression by periodontal ligament (PDL) cells challenged by mechanical loading similar to force levels being applied in orthodontic treatment in vitro and to transfer these findings to an in vivo microenvironment in an animal model of tooth movement in rats. In addition, we addressed the question of whether the regulation of HMGB1 expression is modulated in a time-dependent manner in zones of compressive forces. METHODS AND RESULTS: Protein analysis revealed a basal HMGB1 expression in PDL cell cultures and an increased expression when orthodontic forces were applied. In a rat model of tooth movement including 25 animals that received orthodontic tooth movement for 3, 6, 9, and 12 days, HMGB1 protein expression was demonstrated to be regulated in a time-dependent manner as determined by means of immunohistochemistry and histomorphometrical analyses. CONCLUSION: These data indicate a potential role for HMGB1 protein originating from PDL cells in the regulation of orthodontic tooth movement and the periodontal remodelling process by modifying the local microenvironment.

High-mobility group box protein-1 released by human-periodontal ligament cells modulates macrophage migration and activity in vitro.

Recent studies have demonstrated the interplay of human periodontal ligament cells (hPDLs) with immune cells, such as macrophages, during tissue repair. High-mobility group box protein-1 (HMGB1) is released into the extracellular milieu by damaged cells and functions as an alarmin to mediate the inflammatory host response. The present study addressed the role of HMGB1 released by hPDLs in the regulation of macrophage differentiation, migration and activity. The aim was to examine the inflammatory potential of HMGB1 itself and in combination with other mediators. The induction of sterile necrosis by thermal insult of hPDLs resulted in HMGB1 translocation from the nucleus to the cytoplasm and on to the extracellular space, as determined by immunocytochemistry/ELISA. Exposure of human macrophages to the conditioned PDL cell medium increased the expression of macrophage differentiation/activation markers CD14, CD23, CD64 and CD163. Chemotactic migration and osteoclastic differentiation of macrophages were also enhanced. Supplementation of the conditioned medium with a saturating concentration of HMGB1-Ab reduced these effects. Challenge with recombinant HMGB1 protein induced less migration and osteoclast differentiation than thermal insult. These data point to the immune modulatory capacity of hPDLs by the release of mediators, including HMGB1, which modify macrophage differentiation, migration and activity during periodontal repair, and indicate an enhanced HMGB1 activity when acting in concert with other mediators.

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.

Control of lower incisor inclination with a completely customized lingual appliance for dentoalveolar compensation of class III malocclusion.

OBJECTIVE: Bracket slots and orthodontic archwires offering high dimensional precision are needed for fully customized lingual appliances. We aimed to investigate whether high-precision appliances of this type enable dentoalveolar compensation of class III malocclusion so that lower incisor inclination at the end of treatment will closely match the anticipated situation as defined in a pretreatment setup. MATERIALS AND METHODS: This retrospective study included a total of 34 consecutive patients who had worn a fully customized lingual appliance to achieve dentoalveolar compensation for class III malocclusion by intermaxillary elastics, or proximal enamel reduction, or extraction of teeth in one or both jaws. Casts fabricated at different points in time were three-dimensionally scanned to analyze how precisely the lower incisor inclinations envisioned in the setup were implemented in clinical practice. RESULTS: Aside from minor deviations of ±3.75°, the lower incisor inclinations were clinically implemented as planned even in patients with major sagittal discrepancies. CONCLUSION: Treatment goals predefined in a setup of dentoalveolar compensation for class III malocclusion can be very precisely achieved via a customized lingual appliance. Correct planning can prevent undesirable lingual tipping of the lower incisors. This finding should not encourage a more liberal use of dentoalveolar compensation, but it should heighten clinicians' awareness of how essential it is to sufficiently consider the individual anatomy of the dentoalveolar complex during treatment planning.

Regulation of macrophage migration and activity by high-mobility group box 1 protein released from periodontal ligament cells during orthodontically induced periodontal repair: an in vitro and in vivo experimental study.

OBJECTIVE: Recent studies have shown that periodontal ligament (PDL) cells interact with macrophages from the immune system during orthodontically induced repair of periodontal tissue. Hypothesizing that high-mobility group box 1 (HMGB1) protein is released by mechanically stressed PDL cells into the extracellular space and has a role in mediating the local immune response by acting as an "alarmin", this study was performed to further elucidate these cellular interactions, with a special focus on the impact of proinflammatory mediators secreted by PDL cells on macrophage physiology. MATERIALS AND METHODS: The study included an in vivo part in which orthodontic stress was induced in rats and their PDL analyzed for expression of HMGB1 by immunohistochemistry after 5 days of tooth movement. In the in vitro part, human PDL cells were subjected to compressive loading, followed by stimulating human macrophages with conditioned supernatants of these stressed PDL cells and analyzing how mediators that had been released by these cells into the medium would impact macrophage physiology. Assays for macrophage migration and osteoclast differentiation were used in addition to immunohistochemistry, enzyme-linked immunosorbent assays, and western blotting. RESULTS: Induction of mechanical stress was found to upregulate HMGB1 expression both in vivo and in vitro. At the same time, translocation HMGB1 from nuclei into cytoplasm was observed. Culturing macrophages in conditioned PDL cell medium was associated with enhanced chemotactic migration and osteoclast differentiation. Addition of anti-HMGB1 antibodies to inhibit HMGB1 in the conditioned medium was found to significantly attenuate these effects. A less marked increase of migration and osteoclast differentiation by macrophages was observed after isolated addition of HMGB1, at its observed pathological concentration, to nonconditioned medium. CONCLUSION: This study clearly indicates an immunomodulatory potential of human PDL cells via release of mediators, including HMGB1 protein. Our finding that these mediators modify the migration and differentiation of macrophages as a function of periodontal repair during orthodontic treatment broadens the theoretical basis toward developing interventional strategies to avoid orthodontically induced root resorption.

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.

PTH(1-34)-induced changes in RANKL and OPG expression by human PDL cells modify osteoclast biology in a co-culture model with RAW 264.7 cells.

Parathyroid hormone (PTH) is widely accepted as an anabolic agent when administered intermittently. Here, we explored the influence of intermittent PTH(1-34) on the expression of local factors by human periodontal ligament (PDL) cells that modify osteoclast biology. This approach aimed at a further elucidation of the role of the hormone and of PDL cells in the regulation of periodontal tissue homeostasis and of repair processes. In a co-culture model of mature PDL cells and RAW 264.7 cells, intermittent PTH(1-34) induced an increased gene expression for tartrate-resistant acid phosphatase (+84%), cathepsin K (+56%), and vitronectin-receptor (+56%); and an enhanced resorptive activity of differentiated osteoclasts (+154%). These findings were correlated with a reduction of the osteoprotegerin (OPG)/receptor activator of nuclear factor kappaB ligand (RANKL) ratio in the presence of PTH(1-34; -44%). Similar results were obtained when RAW cells were cultured with the conditioned medium of PTH(1-34)-stimulated PDL cells. In contrast, when less mature PDL cells were co-cultured with RAW cells, PTH(1-34) induced an inhibition of osteoclastic differentiation (TRAP, -35%; cathepsin K, -28%; vitronectin-receptor, -35%), a reduction of the resorbed substrate area (-77%) and an increase of the OPG/RANKL ratio (+11%). The conditioned medium of PTH(1-34)-pretreated less mature PDL cells led to a down-regulation of the number and activity of multinucleated cells. These data indicate that intermittent PTH(1-34) modifies the expression of membrane-bound and secreted factors by PDL cells which then in turn alter osteoclast biology. The PDL cell response to PTH(1-34) is specific in terms of cell maturation and the mechanism involved.

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.

The role of periodontal ligament cells in delayed tooth eruption in patients with cleidocranial dysostosis.

OBJECTIVE: The clinical appearance of patients with cleidocranial dysplasia (CCD), which is caused by mutations in the RUNX2 gene, is characterized by anomalies of the clavicles, thorax, spine, pelvis and extremities and by disturbances of the skull and tooth development. Of orthodontic relevance are multiple supernumerary teeth associated with delayed tooth eruption. The present investigation is based on the hypothesis that an altered phenotypic expression of periodontal ligament (PDL) cells from CCD patients and a reduced ability of those cells to support the differentiation of bone-resorbing osteoclasts might contribute to delayed tooth eruption. MATERIALS AND METHODS: To test this hypothesis, PDL cells from healthy donors and from two patients with clinically and molecular biologically diagnosed CCD were characterized for the basal and induced mRNA expression of osteoblast marker genes. The physiological relevance of the findings for the differentiation of osteoclasts was examined in an osteoclast assay, as well as in a co-culture model of PDL cells and osteoclast precursors. RESULTS: Both CCD patients displayed missense mutations of the RUNX2 gene. The in vitro experiments revealed an unaltered expression of RUNX2 mRNA, however especially in CCD patient 2 there was a reduced basal expression of mRNA for the key regulatory gene for bone remodeling RANKL. Furthermore, compared to the control cells from healthy donors, these factors were less inducible by stimulation of the cultures with 1alpha,25(OH)(2)D(3). In the osteoclast assays as well as in the co-culture experiments, PDL cells from the CCD patients showed a reduced capacity to induce the differentiation of active osteoclasts. CONCLUSIONS: These data indicate that PDL cells from CCD patients express a less distinctive osteoblastic phenotype resulting in an impaired ability to support osteoclastogenesis which might, in part, account for the delayed tooth eruption that can be observed clinically.

Immunohistochemical characterization of nanocrystalline hydroxyapatite silica gel (NanoBone(r)) osteogenesis: a study on biopsies from human jaws.

OBJECTIVES: Bone substitute biomaterials may be osteogenic, osteoconductive or osteoinductive. To test for these probable characteristics in a new nanoporous grafting material consisting of nanocrystalline hydroxyapatite embedded in a porous silica gel matrix (NanoBone(s)), applied in humans, we studied biopsies from 12 patients before dental implantation following various orofacial augmentation techniques with healing times of between 3.5 and 12 months. MATERIAL AND METHODS: Sections from decalcified specimens were investigated using histology, histochemistry [periodic acid Schiff, alcian blue staining and tartrate-resistant acid phosphatase (TRAP)] and immunohistochemistry, with markers for osteogenesis, bone remodelling, resorption and vessel walls (alkaline phosphatase, bone morphogenetic protein-2, collagen type I, ED1, osteocalcin, osteopontin, runx2 and Von-Willebrand factor). RESULTS: Histologically, four specific stages of graft transformation into lamellar bone could be characterized. During early stages of healing, bone matrix proteins were absorbed by NanoBone(s) granules, forming a proteinaceous matrix, which was invaded by small vessels and cells. We assume that the deposition of these molecules promotes early osteogenesis in and around NanoBone(s) and supports the concomitant degradation probably by osteoclast-like cells. TRAP-positive osteoclast-like cells were localized directly on the granular surfaces. Runx2-immunoreactive pre-osteoblasts, which are probably involved in direct osteogenesis forming woven bone that is later transformed into lamellar bone, were attracted. Graft resorption and bone apposition around the graft granules appear concomitantly. CONCLUSIONS: We postulate that NanoBone(s) has osteoconductive and biomimetic properties and is integrated into the host's physiological bone turnover at a very early stage.