Meta-regression Analysis of Study Heterogeneity for Systemic Outcomes after Periodontal Therapy.

INTRODUCTION: The contribution of periodontal disease to adverse systemic consequences remains controversial. This analysis examined 2 well-investigated conditions independently and combined-adverse pregnancy outcomes and glycemic control for patients with diabetes-based on shared pathogenic mechanisms of periodontal infection and inflammation. It was proposed that inconsistencies in study design significantly contribute to outcome discrepancies found between periodontal intervention studies undergoing meta-analysis. METHODS: Meta-analyses evaluating periodontal interventions on the rate of preterm birth and changes in glycated hemoglobin A1c in type 2 diabetes populations were conducted based on a systematic review of randomized controlled trials. Meta-regression covariates for exploring heterogeneity included sample size, level of medical management, and bias risk as moderator variables in a random-effects meta-regression. RESULTS: Systematic review identified 17 studies of diabetes and 13 of pregnancy outcomes. Analyses of these studies identified 0.50% reduction in HbA1c and 0.78 odds ratio for preterm births. The heterogeneity associated with the models was high (I2 = 92.4 and I2 = 62.7%, respectively). The adjusted models evaluating each systemic condition separately accounted for 52.2% of the effect for diabetes and 81.4% for pregnancy outcome effects independently, and 63.5% collectively, across interventional studies. CONCLUSION: This systematic review with meta-regression analysis of heterogeneity demonstrates that disparate results seen in randomized controlled trials of periodontal therapy affecting systemic outcomes may be explained in large part by study design, specifically stringency in consideration of medical management and sample size. The potential for confounding factors to influence outcomes remains a concern in understanding the implications of oral health on systemic conditions. KNOWLEDGE TRANSFER STATEMENT: The findings of this study demonstrate that much of the benefits seen from periodontal therapy on adverse systemic outcomes for diabetes and pregnancy are due to limitations in study design.

Glycemic control and implant stabilization in type 2 diabetes mellitus.

Diabetes mellitus is considered a relative contra-indication for implant therapy. However, the effect of glycemic level on implant integration in persons with diabetes remains poorly understood. The hypothesis of this research was that poor glycemic control is directly related to short-term-impairment implant stabilization. This prospective clinical study evaluated 10 non-diabetic individuals (12 implants) and 20 persons with type 2 diabetes (30 implants). Glycated hemoglobin (HbA1c) levels ranged from 4.7-12.6%. Implant stability was assessed by resonance frequency analysis over 4 months following placement. Minimum stability levels were observed 2-6 weeks following placement for all 42 implants. Persons with HbA1c > or = 8.1% had a greater maximum decrease in stability from baseline and required a longer time for healing, as indicated by return of stability level to baseline. This study demonstrates alterations in implant stability consistent with impaired implant integration for persons with type 2 diabetes mellitus in direct relation to hyperglycemic conditions.

Dielectric functions of a growing silver film determined using dynamic in situ spectroscopic ellipsometry.

The dielectric functions of plasma deposited silver on SiO2 through all stages of Volmer-Weber growth at room temperature and 150 degrees C were determined unambiguously by applying a model-independent inversion method to dynamic in situ spectroscopic ellipsometric data. The results show large differences in the localized plasmon resonance and the percolation threshold at the two temperatures. Using these model-independent dielectric functions we assess the effectiveness of modelling the plasmon resonance by fitting a Lorentz oscillator. The methods show agreement for the position of the plasmon resonance below the percolation threshold and for the effective film thickness up to 5.6 nm at room temperature and 11.5 nm at 150 degrees C, however the line shape of the resonance is described by the Lorentzian only in the early stages of film growth.

Dynamic spectroscopic ellipsometry determination of nanostructural changes in plasmonic silver films.

Dynamic in situ spectroscopic ellipsometry is used to probe post-deposition nano-structural changes in silver films at room temperature in the pre- and post-coalescence stages of Volmer-Weber growth. In the island growth phase the Maxwell-Garnett theory is used to determine structural changes in the island film. Changes in the plasmon resonance frequency indicate an increased distance between islands which explain pre-coalescence resistivity changes. Post-coalescence changes in the resistivity are determined to be due to grain growth. A reduction in film thickness of 0.2 - 0.3 nm is also observed. The results are used to evaluate recent competing theories based on in situ stress measurements.

Persistent acute inflammation at the implant-abutment interface.

The inflammatory response adjacent to implants has not been well-investigated and may influence peri-implant tissue levels. The purpose of this study was to assess, histomorphometrically, (1) the timing of abutment connection and (2) the influence of a microgap. Three implant designs were placed in the mandibles of dogs. Two-piece implants were placed at the alveolar crest and abutments connected either at initial surgery (non-submerged) or three months later (submerged). The third implant was one-piece. Adjacent interstitial tissues were analyzed. Both two-piece implants resulted in a peak of inflammatory cells approximately 0.50 mm coronal to the microgap and consisted primarily of neutrophilic polymorphonuclear leukocytes. For one-piece implants, no such peak was observed. Also, significantly greater bone loss was observed for both two-piece implants compared with one-piece implants. In summary, the absence of an implant-abutment interface (microgap) at the bone crest was associated with reduced peri-implant inflammatory cell accumulation and minimal bone loss.

Long-term changes in soft tissue height on the facial surface of dental implants.

The success of osseous healing around dental implants has allowed for an increased emphasis on soft tissue healing and esthetic results. However, there is limited information profiling the long-term healing of the soft tissues following prosthesis placement. The purpose of this study was to assess the long-term changes in the position of the facial soft tissue margins following restoration of a one-stage implant system. One hundred and six one-stage ITI implants were evaluated in 39 patients. Implants were placed in maxillary and mandibular anterior regions. Clinical assessment of the soft tissues on the midfacial aspect of the implants was performed over a 2-year period, at 3 and 6 month intervals, following placement of the final restoration. A total of 63 implants were placed as multiple units in the mandible, 23 as single units in the maxilla, and 20 as multiple units in the maxilla. There were no implant failures over this time period. Overall, on the facial aspect of 61% of the 106 implants there was 1 mm or more of soft tissue recession, whereas 19% of the implants showed 1 mm or more of gain in soft tissue height. There was a significantly (P < 0.01) greater number of implants showing a gain in soft tissue levels in the mandibular implants compared with the maxillary implants. Of the 39 patients assessed, 24 showed a loss and five showed a gain of 1 mm or more of the soft tissue levels around the implants. Overall, there was a significant decrease in the mean levels of tissue height of 0.6 mm within the first 6 months, with relatively little change afterward. However, in evaluating only patients showing a loss in tissue height around one or more implants, the mean loss in tissue height was 1.6 mm after 24 months. These results suggest that the potential for significant changes in soft tissue levels after completion of restorative therapy need to be considered for implant therapy in esthetic areas.

Clinical, radiographic and biochemical assessment of IL-1/TNF-alpha antagonist inhibition of bone loss in experimental periodontitis.

OBJECTIVES: To assess clinical, radiographic, and biochemical markers as diagnostic indicators of disease activity by comparing ligature-induced bone loss in the presence or absence of IL-1/TNF-alpha antagonist inhibition of bone loss in a primate model. MATERIAL AND METHODS: 6 animals with a naturally-occurring gingivitis were evaluated over a 6-week time period following the placement of silk ligatures and initiation of a soft diet. Three animals received intrapapillary injections of soluble receptors (blockers), capable of blocking the biologic activity for both IL-1 and TNF-alpha, and 3 animals received vehicle (control) injections. Injections were given 3X per week over the course of the study. Clinical assessments included a gingival index and quantification of gingival crevicular fluid (GCF) levels. Collected GCF samples were then used in the biochemical assessment of pyridinoline (PYD) and bone alkaline phosphatase (BAP). Radiographic assessment was made using computer-assisted subtraction radiography to measure both bone density (CADIA) values and linear changes in crestal bone height. RESULTS: Significant (p<0.01) changes using both radiographic measures occurred between 2 and 4 weeks following initiation of disease in this model. The use of the blockers significantly (p<0.01) reduced the levels of radiographic bone loss by approximately 50% over that found in the control sites. Both biochemical markers showed the greatest increase during the first two weeks of the study with PYD levels increased 35-fold over baseline levels after 1 week. This difference in response was significantly (p<0.05) greater than the levels found in the non-ligated teeth or in the ligated teeth receiving blockers injections. BAP levels showed significant increases in ligated teeth compared to non-ligated teeth, but failed to show any significant differences between animals treated with vehicle and those treated with IL-1/TNF antagonists. In contrast to these radiographic and biochemical effects, there were no significant differences detected between animals treated with antagonists and the control group for any of the clinical measures. CONCLUSIONS: The results of this study demonstrate that both subtraction radiography and PYD crevicular fluid levels can detect relative differences in periodontal disease progression, while BAP crevicular fluid levels cannot.

Characterization of proliferation and cellular wound fill in periodontal cells using an in vitro wound model.

BACKGROUND: The therapeutic success of periodontal regenerative therapy may be compromised by our limited understanding of the wound healing process. Wound healing requires the coordination of complex cellular and molecular interactions. Recently, using an in vitro wound model, our laboratory has shown that gingival fibroblasts (GF) fill an in vitro wound more rapidly than periodontal ligament cells (PDL). This suggests that there may be differences in the levels of proliferation for these 2 cell types during the wound healing process. Such specific cell type differences may be significant in clinical outcomes of regenerative therapy. Therefore, the aim of this research was to characterize and compare the levels of both proliferation and cellular wound fill between GF and PDL using our in vitro wound model. METHODS: Primary cultures of human PDL and GF cells were established from explanted tissue, and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer across the diameter of the wells. The wells were then incubated for 2, 6, or 9 days in media containing either 0.1% or 10% fetal bovine serum (FBS). At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU), fixed, and nuclei stained to measure DNA synthesis (as a measure for proliferation). Cells were counter stained with cytoplasmic stain to measure cell number. Quantitative analysis distant from (area of interest [AOI 1]), next to (AOI 2), and within the wound boundaries (AOI 3 and 4) was accomplished using computer-assisted histomorphometry. RESULTS: The levels of proliferation and cellular fill for each cell type were assessed relative to time and AOI. Overall, the PDL displayed greater (P <0.01) levels of proliferation than the GF. For both cell types, proliferation was found to be significantly (P<0.001) greater at day 2 compared to other time points. PDL displayed greater levels of proliferation than GF in all AOI, with this difference reaching significance (P<0.02) within the cell layer (AOI 1 and 2). When comparing levels of cellular fill in 10% FBS, GF displayed greater wound fill than the PDL. This difference was significant at day 6 (P <0.05) for both the marginal (AOI 3) and central (AOI 4) portions of the wound. CONCLUSIONS: These findings, demonstrating unique differences between PDL and GF with respect to proliferation and wound fill in an in vitro model, suggest that there may be cell-specific differences in cellular activity critical to periodontal wound healing. In addition, the results of this study show that the cellular proliferation response may not accurately reflect the overall wound healing response.

The effects of platelet-derived growth factor-BB on periodontal cells in an in vitro wound model.

BACKGROUND: Platelet-derived growth factor (PDGF-BB) has been shown to enhance periodontal regeneration. Principles of guided tissue regeneration dictate that one of the goals of therapy is to modulate the wound healing processes to favor repopulation of the wound with cells derived from the periodontal ligament rather than from the gingival tissues. Using an in vitro wound model, gingival fibroblasts (GF) have been shown to fill a wound space significantly faster than periodontal ligament cells (PDL). There are no data reported directly comparing the response of these 2 cell types to PDGF-BB within such a wound model. Therefore, the aims of this research were: 1) to characterize both the proliferative and wound fill (WF) effects of PDGF-BB within an in vitro model and 2) to compare specific growth factor effects between GF and PDL. METHODS: Primary cultures of both human PDL and GF were derived from explanted tissues and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer across the diameter of the wells. The wells were then incubated for 2, 6, and 9 days in media containing 0.1% fetal bovine serum (FBS) and 1 of 5 concentrations of PDGF-BB. At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU) fixed, and nuclei were stained to measure BrdU incorporation (as a measure for proliferation). Cells were counter-stained with cytoplasmic stain to measure cell number. Quantitative analyses within the wound boundaries, marginally (area of interest [AOI] 1) and centrally (AOI 2), were accomplished using computer-assisted histomorphometry. RESULTS: PDL exhibited a significantly greater proliferative response to PDGF-BB in both AOI when compared to GF (P <0.0001). The PDL exhibited increased levels of proliferation at concentrations of PDGF-BB greater than or equal to 10 ng/ml. By contrast, GF displayed no increase in proliferation in response to stimulation with PDGF-BB at any of the concentrations tested when compared to negative controls. The wound fill (WF) responses to PDGF-BB were similar between PDL and GF, with both cell types responding in an all or none fashion when measured at day 2, and in a concentration-dependent manner at later time points. The only significant difference in WF between PDL and GF occurred in AOI 2 in negative control medium (0 ng/ml of PDGF-BB), with GFs having greater (P <0.01) levels of WF over the 9 days. CONCLUSION: The findings from this study demonstrate differing effects of PDGF-BB on the proliferation of PDL and GF in this in vitro model. These results suggest that there may be cell-specific differences critical to periodontal wound healing that may be exploited in the development of new therapies.

In vitro wound healing responses to enamel matrix derivative.

BACKGROUND: Enamel matrix derivative (EMD) contains a variety of hydrophobic enamel matrix proteins and is extracted from developing embryonal enamel of porcine origin. EMD has been associated with the formation of acellular cementum and it has been found to stimulate periodontal regeneration. The present study was established to investigate the influence of EMD on human periodontal ligament (PDL) cells, gingival fibroblasts (GF), and osteosarcoma (MG-63) cells on wound-fill rates using an in vitro wound model. METHODS: Wounds were created by making 3 mm incisions in cell monolayers across the length of tissue culture plates. The wounded PDL, GF, and MG-63 cell monolayers were treated with media containing EMD over a concentration range of 5 to 100 microg/ml, platelet-derived growth factor (PDGF-BB) at 20 ng/ml as a positive control and insulin-like growth factor (IGF-I) at 100 ng/ml as a negative control. PDL cell wounded monolayers also were treated in EMD coated tissue culture plates. After an incubation period (up to 9 days), the cells were fixed and stained and cellular fill was measured across the width of the wound by computer-assisted histomorphometry. RESULTS: When PDL, GF, and MG-63 cells were exposed to EMD in culture medium, an enhanced wound-fill was observed for all cells compared to untreated conditions. At early time points, PDL wound-fill rates in the presence of EMD were statistically greater than the rates of GF and MG-63 treated with EMD (P<0.001). There were no significant differences in wound-fill rates of PDL cells treated with EMD in medium versus EMD coated on culture plates. At days 3 and 6 post-wounding, PDL cells showed a significantly greater response to EMD than to PDGF-BB (P <0.001). EMD also had a greater effect on GF wound-fill rates than PDGF-BB at days 6 and 9. MG-63 cells were less responsive to PDGF-BB and EMD than PDL cells and GF. All 3 cell types treated with IGF-I showed no significant increase of wound-fill rates. CONCLUSION: The present data support the concept that clinical application of enamel matrix derivative may enhance periodontal wound regeneration by specifically modifying periodontal ligament cell proliferation and migration.

Effects of bisphosphonate on matrix metalloproteinase enzymes in human periodontal ligament cells.

BACKGROUND: The host response is a critical component in the pathogenesis of periodontitis. In fact, the clinical benefits associated with regulating the host response have been demonstrated in studies using several different classes of drugs. Biophosphates are one host-modulating class of drugs that has demonstrated this ability. These drugs are clinically effective at reducing bone resorption and have shown the ability to inhibit host degradative enzymes, specifically the matrix metalloproteinases (MMPs). Therefore, the purpose of this study was to investigate the regulatory effects of a bisphosphonate, tiludronate, on MMP levels and activity in human periodontal cells. METHODS: MMP-1 and MMP-3 were assessed in cultured human periodontal ligament cells treated with a bisphosphonate, tiludronate. Reverse transcription-polymerase chain reaction was used to identify mRNA levels for both enzymes, and also for tissue inhibitors (TIMP-1). Enzyme immunoassay (EIA) and immunocytochemistry were used to assess MMP proteins in these cell cultures. Enzyme activity was assessed using FITC-conjugated substrates and quantitated using spectrophotofluorometry. RESULTS: Tiludronate significantly inhibited both MMP-1 and MMP-3 activity in a concentration-dependent manner. A maximal reduction in activity of 35% was achieved for each of the enzymes at a 10(-4) M concentration. Tiludronate did not have a significant effect on the mRNA levels for MMP-1, MMP-3, or TIMP-1. Similarly, there were no effects noted for either MMP-1 or MMP-3 on the protein level. CONCLUSIONS: This study demonstrates an inhibitory effect of tiludronate on the activity of both MMP-1 and MMP-3. These effects appear to occur without altering either mRNA or protein levels for these enzymes, supporting a possible mechanism of action that involves the ability of bisphosphonates to chelate cations from the MMPs. Furthermore, these results support the continued investigation of these drugs as potential therapeutic agents in periodontal disease.

Development of an in vitro wound healing model for periodontal cells.

BACKGROUND: Periodontal wound healing and regeneration are influenced by a multitude of factors. While many in vitro investigations have compared the proliferation of periodontal ligament (PDL) cells and gingival fibroblasts (GF), there are no reports directly comparing the abilities of these 2 cell types to fill a wound site. As such, the goals of this research were: 1) to develop an in vitro model of wound healing which would allow for the investigation of the biologic basis of periodontal wound healing and regeneration and 2) to compare the rates of PDL cells and GF to fill an in vitro wound site. METHODS: Using both human PDL cells and GF confluent cultures, in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer. Wounded cultures were then incubated for time periods up to 12 days in media containing fetal bovine serum (FBS) concentrations (0, 0.1, 1, 5, 10, and 20%) as appropriate for each experiment. Slides were fixed, stained, and cells quantified within the wound boundaries by computer-assisted histomorphometry. The effect of wounding a cell layer was determined by comparing wounded cells as described above with a cell layer margin created without physically disrupting the cell layer. RESULTS: The in vitro model for periodontal wound healing established in this study showed that GF fill in the wound site at a significantly (P <0.0025) faster rate than PDL cells over 12 days of healing. In addition, PDL cells and GF were found to have unique concentration-dependent responses to FBS (P<0.0025). It was also shown that wounding resulted in a significant delay (P <0.01) in the initial healing response of an in vitro wound. CONCLUSION: This in vitro model demonstrated that the characteristics of wound healing are dependent on cell type, disruption (wounding) of the cell layer, and serum concentration. In addition, this model has incorporated both proliferation and migration to provide the first direct evidence demonstrating GF has a significantly greater ability to fill a wound site than PDL cells. This in vitro model may be utilized in future investigations of the biologic basis of periodontal wound healing.

Chief complaints of patients seeking treatment for periodontitis.

BACKGROUND: Hardly any data are available concerning the chief complaints, or CCs, of patients with periodontitis. The authors conducted a study to determine the most common CCs among a group of subjects with periodontitis. METHODS: The authors examined the dental records of 191 patients with periodontitis to determine what CCs they orally reported having at an initial examination. Patients were referred mainly by other members of the dental health team. Eighty percent of the patients were diagnosed with moderate or moderate-to-severe periodontitis. The authors recorded the frequency of different CCs to determine the most common complaints. RESULTS: The authors recorded 336 CCs from the records of 191 subjects with periodontitis. There were 21 different CCs. The most common CC reported was, "I was told I have gum disease." The second most common CC reported was, "I would like to save my teeth." Neither of these CCs are true periodontitis symptoms. Bleeding gums--a true periodontitis symptom--was the third most common CC. Only 6.2 percent of the subjects reported having painful gingiva, and only 29.3 percent of the subjects reported having dental-emergency-related CCs. CONCLUSIONS: The authors found that the motivation to seek periodontal treatment was most commonly based on information given to the subjects by a member of the dental health team, rather than a periodontitis symptom. CLINICAL IMPLICATIONS: Renewed efforts and increased responsibility of the dental health team members to inform patients about the presence of periodontitis are needed, as well as emphasizing to the public the risk of losing teeth as a result of periodontitis.

PDGF-alpha receptor subunit expression down-regulated by IL-1beta in human periodontal ligament cells.

The responses of cells to the distinct PDGF isoforms have been correlated directly to the relative numbers of specific PDGF receptor subunits on the cell surface. The modulation of PDGF-alpha receptor subunits, the major subunit expressed in human periodontal ligament (PDL) cells, by cytokines present in the periodontal wound site, such as interleukin-1 (IL-1), may be an important factor influencing regenerative outcomes. The purpose of the present study was to examine the effects of IL-1 beta on PDGF-alpha receptor subunit expression in human PDL cells. Primary cultures of human PDL cells were treated with IL-1 beta over a range of concentrations. We assessed PDGF-alpha receptor subunits by examining the mitogenic responses of cells to PDGF-AA, specific binding of 125I-labeled PDGF-AA, immunofluorescent analysis of PDGF-alpha receptor subunits, and PDGF-alpha receptor subunit mRNA levels using Northern blot analysis. The results demonstrate a significant concentration-dependent decrease in 3H-thymidine incorporation in response to PDGF-AA following IL-1 beta treatment (p < 0.001). This decreased response correlated directly with IL-1-induced decreases in 125I-labeled PDGF-AA binding (p < 0.01), the numbers of immunolabeled PDGF-alpha receptor subunits, and in PDGF-alpha receptor subunit mRNA levels. However, when combined with TGF-beta, IL-1 beta did not show additional down-regulation in proliferative response to PDGF-AA or PDGF-alpha receptor subunits beyond that achieved with these factors individually. These experiments identify IL-1 beta, along with TGF-beta, as significant inhibitors of PDGF stimulation in human PDL cells, acting through the down-regulation of PDGF-alpha receptor subunit expression.

Effects of interleukin-1 beta on matrix metalloproteinase-3 levels in human periodontal ligament cells.

MATRIX METALLOPROTEINASE-3 (MMP-3), or stromelysin-1, is an enzyme responsible for the degradation of a wide range of extracellular matrix proteins. Increases in MMP-3 activity have been found in several chronic inflammatory diseases, and this increased activity is thought to be mediated by interleukin-1 beta (IL-1 beta). Because IL-1 beta has been strongly associated with inflammatory periodontal disease, the purpose of this in vitro study was to investigate the role of IL-1 beta on the regulation of MMP-3 levels in cells derived from the human periodontal ligament (PDL). Human PDL cell cultures were treated with IL-1 beta at varying concentrations (0.01-1.0 ng/ml) for 24 hour prior to analysis at either transcript or protein levels. Following the isolation of total RNA, the relative levels of MMP-3 mRNA were determined using reverse transcription-polymerase chain reaction (RT-PCR) with 32P-end-labeled primers. Immunocytochemical detection of MMP-3 protein was performed using polyclonal antibodies to human MMP-3. The results of RT-PCR analysis demonstrated a concentration-dependent increase in MMP-3 mRNA expression, with IL-1 beta treatments of 0.1 and 1.0 ng/ml significantly (P < 0.01) increased over those cells not treated with IL-1 beta. This increase in mRNA expression was paralleled by significant (P < 0.001) changes at the protein level, with an average of 27.6% of the cells stained positive for MMP-3 following IL-1 beta treatment (1.0 ng/ml), compared with control cells showing no positive staining for MMP-3. In conclusion, the results of this study demonstrate that IL-1 beta upregulates MMP-3 in human PDL cells on both an mRNA and a protein level. These findings suggest possibly important roles for IL-1 beta and MMP-3 in both normal turnover and maintenance of the PDL and in the connective tissue degradation associated with periodontal disease.

Long-term prognosis following resectional and regenerative periodontal procedures.

The ultimate goal of both resective and regenerative periodontal procedures is the creation of soft- and hard-tissue architecture that is consistent with periodontal health. Osseous resective procedures predictably produce minimal clinical probing depth, but sacrifice periodontal support. An alternative method to treat anatomic defects not easily managed through resection is guided tissue regeneration (GTR). GTR provides clinicians with the opportunity to reverse the disease-related loss of periodontal attachment. However, at present, the outcomes of GTR procedures have not been shown to be predictable. Continued improvements in techniques and materials, and identification of patient-related factors significant to the success of the GTR procedures, should enhance the consistency of the clinical outcomes. An evidence-based approach to the use of both regenerative and resective therapies will enhance the clinical results achieved through these procedures.

Development and characterization of a transformed human periodontal ligament cell line.

Periodontal ligament (PDL) cells are thought to be important for establishing and maintaining a stable interface between bone and teeth. In addition, PDL cells are thought to play critical roles in both the pathogenesis of periodontal disease and the regeneration of periodontal ligament tissues. The purpose of this study was to develop a continuous or stable human PDL cell line as an in vitro model for the investigation of cellular mechanisms involved in periodontal regeneration and destruction. Human PDL cells, derived from a primary cell culture, were transfected with simian virus 40 (SV40) T antigen-containing virus with a neomycin resistance gene. The transformed cells expressed the SV40 T antigen mRNA as assayed by reverse transcription polymerase chain reaction (RT-PCR). This cell line was also characterized for morphological changes and growth characteristics compared to primary PDL cell cultures. The transformed cells were shown to form a multilayer pattern and distinct colonies on tissue culture surfaces. However, no colony formation was found in soft agar. The transformed PDL cell line was found to have a greater rate of proliferation in 10% fetal bovine serum than primary culture, and continued to proliferate in low serum concentrations capable of producing quiescence in primary cells. Interleukin-1 beta (IL-1 beta) was shown to produce a 7-fold elevation in collagenase (MMP-1) mRNA levels, consistent with primary PDL cells. In addition, IL-1 beta was shown to produce a decrease in alkaline phosphatase activity in a concentration-dependent manner. The transformed cell line has been maintained for over 30 generations of cell culture. In conclusion, a stable human PDL cell line has been established to serve as a model for future in vitro investigations into periodontal pathogenic mechanisms and to evaluate therapies directed at the regeneration of periodontal ligament.

Bone cell interactions and regulation by inflammatory mediators.

The loss of alveolar bone tissue associated with periodontal disease appears to be related to local factors that alter the physiologic balance between bone formation and resorption. Physiologic bone metabolism requires the coupled activities of bone resorption and bone formation. Recent investigations of this coupling phenomenon have been directed at understanding the regulatory mechanisms involved in osteoblast-osteoclast interactions. These regulatory mechanisms utilize multiple cytokines, which function in context with numerous other factors, including cell-cell interactions, extracellular matrix molecules, and levels of differentiation. Local alterations of these factors may disrupt the physiologic balance in bone metabolism and lead to the pathologic loss of alveolar bone. Future strategies for altering bone loss found in the periodontal diseases will be directed at modulating the balance of bone resorption and bone formation at the local site.

Receptor binding of PDGF-AA and PDGF-BB, and the modulation of PDGF receptors by TGF-beta, in human periodontal ligament cells.

The growth factors PDGF-AA and PDGF-BB have previously been shown to be potent mitogens for human periodontal ligament (hPDL) cells in vitro. Additionally, the mitogenic response to PDGF-AA has been shown to be specifically inhibited by TGF-beta. The purpose of the present investigation was to examine the binding of PDGF-AA and PDGF-BB, and the modulation of PDGF binding by TGF-beta, in hPDL cells. Scatchard analysis identified an average of 32,000 PDGF-AA high-affinity binding sites per cell with a dissociation constant (Kd) of 0.66 nM and an average of 36,000 PDGF-BB binding sites per cell with a dissociation constant (kd) of 0.44 nM. After treatment with TGF-beta, the receptor number for PDGF-AA was found to specifically decrease by approximately 50%, with no change in binding affinity. This reduced number of binding sites was shown to correlate with both a decrease in levels of receptor tyrosine phosphorylation and a decreased number of alpha receptor subunits. Northern blot analysis identified the TGF-beta-mediated decrease in PDGF alpha receptor subunit mRNA levels. PDGF-BB showed little change in the number of binding sites or in the binding affinity with TGF-beta treatment, and the data were consistent with an increase in the number of beta receptor subunits. These results demonstrate nearly equivalent numbers of receptors for both PDGF-AA and PDGF-BB in hPDL cells. Also, modulation of PDGF binding, by TGF-beta, was shown to result in a reduced number of alpha receptor subunits with an increase in the number of beta receptor subunits.

Mitogenic effects of growth factors on human periodontal ligament cells in vitro.

Periodontal regeneration is thought to require the migration and proliferation of periodontal ligament cells. Evidence suggests that the polypeptide growth factors PDGF, IL-1, and TGF-beta are mediators of these cellular events in wound healing. The purpose of this study was to determine the effects of these growth factors on human periodontal ligament (PDL) cell mitogenesis, and to identify the regulatory influences of TGF-beta on the response to PDGF and IL-1. Confluent, quiescent human PDL cells were cultured in vitro and treated with the polypeptide growth factors PDGF-AA and -BB, IL-1 beta, and TGF-beta in both a dose and time-dependent manner. Mitogenic activity, as a measure of proliferative potential, was determined by the quantitation of 3H-thymidine incorporation during DNA synthesis. The results of this study demonstrated that both PDGF-AA and -BB enhance mitogenic activity in a dose-dependent manner over a concentration range of 1.0 to 50.0 ng/ml. IL-1 beta (0.01 to 1.0 pM) resulted in no mitogenic enhancement, and at high concentrations (10.0 to 100.0 pM) demonstrated an inhibitory effect. TGF-beta produced a significant increase (P < 0.01) in mitogenic activity (although relatively much less than PDGF) in a delayed, bimodal, dose-dependent manner over a concentration range of 0.01 to 20.0 ng/ml, with a maximal response at a concentration of 1.0 ng/ml. Additionally, incubation with TGF-beta at 1.0 ng/ml prior to the addition of PDGF significantly enhanced (P < 0.01) the mitogenic response to both PDGF-AA and PDGF-BB.(ABSTRACT TRUNCATED AT 250 WORDS)