Immune efficacy of a porcine circovirus type 2 vaccine purified using Gram-positive enhancer matrix surface display technology.

AIMS: Purification of porcine circovirus type 2 (PCV2) using Gram-positive enhancer matrix (GEM) surface display technology and immunogenicity evaluation of the purified antigen. METHODS AND RESULTS: A recombinant bifunctional protein containing a protein anchor domain and a 'virus anchor' domain was designed as a protein linker (PL) between PCV2 and GEM particles. By incubating with PL and GEM particles sequentially, PCV2 could be purified and enriched through a simple centrifugation process with GEM surface display technology. Our data showed that one unit (2·5 × 109 particles) of GEM particles with 80 µg PL could purify 100 ml of PCV2-containing culture supernatant (viral titre: 106·5 TCID50 per ml-1 ) with a recovery rate up to 99·6%. The impurity removal efficiency of this method, calculated according to decreased total protein content during purification, was approximately 98%. Furthermore, in vivo experimentation showed that piglets immunized with purified PCV2 could elicit strong immune responses to prevent against PCV2 infection. CONCLUSION: Porcine circovirus type 2 could be efficiently purified and enriched with GEM display technology via a crucial PL, and the purified PCV2 could elicit effective immune responses against PCV2 infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The GEM-based purification method established here is cost-efficient and high-throughput, and may represent a promising large-scale purification method for PCV2 vaccine production.

Modulation of osteogenic potential by recombinant human bone morphogenic protein-2 in human periodontal ligament cells: effect of serum, culture medium, and osteoinductive medium.

BACKGROUND AND OBJECTIVE: Bone morphogenic proteins are known, in animal models, to promote many developmental processes, including osteogenesis. Clinical trials are currently underway to evaluate the potential of bone morphogenic proteins to promote bone and periodontal regeneration in humans. The aim of this study was to establish an optimal cell culture condition for using to study the biological effects of recombinant human bone morphogenic protein-2 on periodontal ligament cells. MATERIAL AND METHODS: The roles of serum concentration, types of culture medium (alpha-modified essential medium or Dulbecco's modified Eagle's medium), the presence of osteoinductive medium (including dexamethasone, ascorbic acid and beta-glycerophosphate), and timing of addition of the osteoinductive medium and recombinant human bone morphogenic protein-2, on the expression of alkaline phosphatase were investigated in cultured periodontal ligament cells. Cytochemical stainings and biological assay of alkaline phosphatase were also demonstrated. RESULTS: Our results suggested that an increased concentration of serum might mask the effect of recombinant human bone morphogenic protein-2 on the expression of alkaline phosphatase in periodontal ligament cells. alpha-Modified essential medium was found to induce a stronger cytochemical staining of the alkaline phosphatase than Dulbecco's modified Eagle's medium under similar culture conditions. Pre-incubation of cells with osteoinductive medium before the addition of various concentrations of recombinant human bone morphogenic protein-2 enhanced greater alkaline phosphatase expression than the simultaneous presence of both osteoinductive medium and recombinant human bone morphogenic protein-2. CONCLUSION: The findings of this study suggest that the effect of recombinant human bone morphogenic protein-2 on periodontal ligament cells could be efficiently investigated after the proper selection of culture variables and temporal sequence of adding bioactive factors. The optimal culture condition identified in this study might be useful in further studies to elucidate the regulatory mechanism of periodontal ligament cells in periodontal regeneration after stimulation with recombinant human bone morphogenic protein-2.

Autologous transplantation of gingival fibroblast-like cells and a hydroxylapatite complex graft in the treatment of periodontal osseous defects: cell cultivation and long-term report of cases.

Autogenous cell transplantation via hydroxylapatite (HA) vehicle has been reported to have beneficial effects on the treatment of human periodontal osseous defects. The aim of this study was to explore the possibility of using gingival fibroblast-like cells in the therapy of osseous defects caused by inflammatory periodontitis by reporting long-term results of gingival fibroblast-coated hydroxylapatite (GF-HA) grafting for healing these defects. Gingival fibroblasts were cultured from healthy gingivae of treated subjects. Growth of cells on HA particles was established in vitro, and then the GF-HA complex was transplanted into the periodontal osseous defects. Clinical parameters of gingival and plaque indices, probing depth, and periapical x-ray were monitored at baseline and at various periods from 50 months to 6 years after surgery. Grafting with only HA in the osseous defects of the same patient was used for comparison. The present study shows that GF-HA-treated sites could achieve marked pocket reduction and probing attachment gain at reentry and later recalls. Good clinical bone filling of osseous defects in GF-HA-treated sites was also demonstrated in periapical radiographs (increased bone height and reappearance of the crestal cortex) and in some reentry sites. One HA-treated site was filled with connective tissue only, and the absence of new bone formation was noted during a reentry operation. Another HA-treated site exhibited a comparable increase in radiographic density, while part of HA particles were gradually lost in longer recalls. These limited observations conclude that GF-HA grafting may provide a treatment modality leading to regeneration of periodontal tissues in periodontitis-affected osseous defects. Further studies including more cases and demonstration of the deposition of differentiated periodontal tissues are necessary before further application of this therapy.

Expression of functional type 1 protease-activated thrombin receptors by mouse primary palatal mesenchymal cells in vitro.

Development of the primary palate involves a series of processes including cell growth, differentiation, and morphogenesis. To study the molecular and cellular processes during mouse primary palatogenesis, mesenchymal cells were isolated from the primary palate of BALB/cBy embryos (day-11, hour 20). Most of the primary palatal mesenchymal (PPM) cells were morphologically similar to fibroblasts. The population doubling time was about 36 h. At concentrations of 5 and 10 unit/ml, alpha-thrombin significantly stimulated the proliferation of these palatal cells by 2- to 2. 4-fold compared to untreated controls over a 72 hour incubation period. Reverse transcriptase-polymerase chain reaction using primers based on the mouse type 1 protease-activated thrombin receptor (PAR1) detected PAR1 mRNA in the PPM cells, the authenticity of which was confirmed by partial DNA sequencing. Blocking of the alpha-thrombin proteolytic site with the highly specific inhibitor D-phenylalanyl-prolyl-arginyl chloromethyl ketone significantly suppressed the mitogenic effect of thrombin on the PPM cells by 71%. These results suggest that PAR1 is present on PPM cells in the mouse embryo and that serine protease activity is important for the receptor activation.

Biological effects of cementum and bone extracts on human periodontal fibroblasts.

BACKGROUND: Non-collagenous proteins of mineralized tissues play important roles in bone induction during mineralization and in regulating the activity of many types of mesenchymal cells. This study was conducted to determine the effects of acetic acid extracts of bone and cementum on alkaline phosphatase (ALPase) activity and in vitro mineralization of cultured human periodontal fibroblasts (hPF). METHODS: Alveolar bone and cementum obtained from clinically healthy subjects were extracted by a solution containing 0.5 M acetic acid and enzyme inhibitors. Osteoblastic phenotypes of hPF were assayed by ALPase activity, gene expression of bone marker proteins, and the ability to produce in vitro mineralization in culture media containing 50 microg/ml ascorbic acid, 10 mM sodium beta-glycerophosphate, and 10(-7) M dexamethasone. The effects of cementum and bone extracts on the expression of osteoblastic phenotypes in hPF were also determined. RESULTS: Many protein components, varying in molecular weight from 10 to 14 to 120 kDa, were detectable in 10% SDS-PAGE of both cementum and alveolar bone extracts. The hPF cells were found to exhibit a moderate ALPase activity when compared with rat osteosarcoma (ROS) 17/2.8 cells under the same experimental conditions. Gene expression for ALPase, osteocalcin bone sialoprotein, osteopontin, and BMP-7 at mRNA message was detected by RT-PCR in hPF and ROS 17/2.8 cells. The confluent hPF and ROS 17/2.8 cells showed evidence of calcium deposition in the extracellular milieu at 30 and 15 to 30 days' cultures, respectively, under a mineralization medium. The hPF appeared to form mineralized foci with morphological characteristics different from the mineralized nodules produced by ROS 17/2.8 cells. The addition of low concentrations (5 microg/ml) of either cementum or bone extract produced an increase in the size and number of mineralization spots, as well as greater ALPase activity in both hPF and ROS 17/2.8 cultures during the observation periods. CONCLUSIONS: These results suggest that hPF possess certain mineralizing phenotypes, and that acetic acid extracts of bone and cementum contain components capable of stimulating osteogenic differentiation of hPF.

Gel-filtrated fractions of alveolar bone extract contain factors promoting cell attachment and a mitogenic effect on periodontal ligament fibroblasts.

The purpose of this study was to investigate the effect of acetic acid-extracted bone proteins on human periodontal ligament fibroblasts (hPF) with respect to mitogenic and cell attachment promoting activity. Alveolar bone was harvested from healthy donors and subjected to 0.5 M acetic acid extraction, dialysis and lyophilization, and gel filtration. Promotion of cell attachment and stimulation of DNA synthesis by the crude extract and gel-filtrated fractions were studied in cultured hPE Many protein components, varying in molecular weight from 10-14 to 120 kDa, were detectable in 10% SDS-PAGE of the extract. Gel filtration of bone extract disclosed four fractions with molecular weights of 55, 34, 29 and 19-20 kDa. Both the 34 and 55 kDa fractions at a concentration of 5 microg/ml, but not the 29- or 19-20 kDa fractions, were found to promote cell attachment while only the 55 kDa fraction (5 microg/ml) stimulated DNA synthesis of hPF, Both mitogenic activity and the promotion of the cell attachment by gel-filtrated active fractions were resistant to thermal treatment (70 degrees C) and pH (4 to approximately 8) changes. These findings suggest that acetic acid extract of alveolar bone may contain components which are capable of modulating cell attachment and mitogenesis of hPF.

Bacterial morphotypes and early cellular responses in clinically infected and non-infected sites after combination therapy of guided tissue regeneration and allograft.

OBJECTIVES: To compare the bacterial morphotypes and early cellular responses in periodontally treated sites with and without pus formation after a combination of guided tissue regeneration (GTR) and allograft therapy. METHODS: 45 subjects with 80 sites having periodontal lesions with moderate to deep pockets and angular bone defects participated. 28 treated sites in 25 patients were included in the studies. 14 sites suffered from symptoms and signs of infection with pus formation during the healing period were assigned to the pus (P) group. Another 14 sites had asymptomatic healing and were assigned to the non-pus (NP) group. The GTR membranes were retrieved 4-6 weeks after surgery and processed for SEM examination. The bacterial morphotypes on the membranes were observed and photographed. Bacterial adhesion score (BAS, 0-5) and the presence of leukocytes and fibroblasts were estimated from photographs. RESULTS: The results showed that large numbers of bacteria (high BAS) were present on both sides of the coronal 2/3 of the membrane in both groups, irrespective of clinical conditions. At the apical 1/3 of the membrane, moderate numbers of bacteria were still found on the outer side in the P group. The BAS of rod-shaped bacteria were significantly higher in the P group than that of the NP group on the outer coronal 2/3 of the membrane. The frequency of the presence of fibroblasts (18.5%) at the apical 1/3 of the inner (tooth facing) side of the P group was much lower than that of the same location (28.6-29.6%) in the NP group. The presence of leukocytes and fewer numbers of fibroblasts on the GTR membrane were associated with greater BAS for rod- and filament-shaped bacteria. CONCLUSIONS: GTR membranes are commonly colonized by oral bacteria during retention, even on uncomplicated and tissue covered portions. The overt infection clinically (pus group) of the membrane-allograft treated sites is associated with a significantly elevated BAS of rod-shaped bacteria, and may be closely related to the occurrence of its adverse early healing responses (inflammation, pus formation, fewer fibroblasts and greater accumulation of leukocytes).

Comparison of Nd:YAG laser versus scaling and root planing in periodontal therapy.

BACKGROUND: The Nd:YAG laser has recently been used in the treatment of periodontal disease. However, although a clinical reduction of probing depth and gingival inflammation to this new approach has been reported, it has not been fully evaluated. Interleukin-1 beta (IL- 1beta), a potent stimulator of bone resorption, has been identified in gingival crevicular fluid (GCF), which is closely associated with periodontal destruction. The aim of this study was to compare the effects of Nd:YAG laser treatment versus scaling/root planing (SRP) treatment on crevicular IL-1beta levels in 52 sampled sites obtained from 8 periodontitis patients. METHODS: One or 2 periodontitis-affected sites with a 4 to 6 mm probing depth and horizontal bone loss from 3 adjacent single-root teeth in each of 4 separate quadrants were selected from patients for clinical documentation and IL-1beta assay. Sampling site(s) from each diseased quadrant was randomly assigned to one of the following groups: 1) subgingival laser treatment (20 pps, 150 mJ) only; 2) SRP only; 3) laser treatment first, followed by SRP 6 weeks later; or 4) SRP first, followed by laser therapy 6 weeks later. The GCF was collected and the amount of IL-1beta was assayed by enzyme-linked immunosorbent assay (ELISA). Clinical parameters and GCF were measured at baseline and biweekly after therapy for 12 weeks. RESULTS: An obvious clinical improvement (marked decrease in the number of diseased sites with gingival index > or =2) and reduction of crevicular IL- 1beta were found in all groups. The level of IL- 1beta was significantly lower in the SRP group (P = 0.035) than in the laser therapy group for the duration of the 12 weeks. The laser combined SRP therapy group showed a further reduction of IL- 1beta (6 to 12 weeks after treatment) than either laser therapy alone or SRP combined laser therapy. CONCLUSIONS: Our data suggest that laser therapy appeared to be less effective than traditional SRP treatment. Of the 4 treatment modalities, inclusion of SRP was found to have a superior IL- 1beta response, when compared to other therapies without it. In addition, no additional benefit was found when laser treatment was used secondary to traditional SRP therapy.

The response of cAMP and DNA synthesis in rat osteosarcoma cells to mechanically deformed culture dishes.

The purpose of this research was to document the biochemical response of rat osteosarcoma cells (ROS 17/2.8) to mechanical stress in vitro. The influence of mechanical stress on ROS 17/2.8 cells was studied using a stress application device. Briefly, the device was fabricated by bonding an orthodontic expansion screw outside the bottom of a plastic culture dish with self-curing acrylic resin. Irreversible deformation of the culture dish was produced by activating an expansion screw. The resulting mechanical stress was transferred to the cells which attached to the culture dish. The response in terms of cyclic adenosine monophosphate (cAMP) of ROS 17/2.8 cells to mechanical stress was measured using a competitive protein-binding method. The effect of mechanical stress on cellular growth was assessed through the incorporation of 3H-thymidine after different periods of mechanical stress application. The results revealed that mechanical stress could increase the production of cAMP in ROS 17/2.8 cells at an early phase after stress stimulation. This change in the cAMP level was dependent on the duration of stress application, and the maximal response occurred when the mechanical stress was applied for one hour. Although the cellular incorporation of 3H-thymidine decreased 40-60% in ROS 17/2.8 cells subjected to mechanical stress for 1 hour, this reaction recovered from the inhibition effect to 80-85% of the baseline when the mechanical stress lasted for 24 hours. The observations in this study indicate that mechanical stress stimulates the production of cAMP and inhibits the 3H-thymidine incorporation of ROS 17/2.8 cells at an early phase of stress application.

Microbiological response of localized sites with recurrent periodontitis in maintenance patients treated with tetracycline fibers.

BACKGROUND: Whether adjunctive tetracycline fibers can provide an additive effect to scaling and root planing in treating non-responsive sites in maintenance subjects is still controversial. Recolonization of the bacteria from untreated sites or from the extracrevicular region may explain the insignificant response to local therapy. The purpose of the present study was to evaluate the microbiological response of sites treated with tetracycline fibers combined with scaling and root planing. METHODS: The study was conducted in a split-mouth design. Thirty patients on maintenance therapy having at least 2 non-adjacent sites in separate quadrants with probing depths between 4 to 8 mm with bleeding on probing, or aspartate aminotransferase enzyme levels > 800 microIU in the gingival crevicular fluid, were treated with scaling and root planing plus tetracycline fibers or with scaling and root planing only. Subgingival plaque samples were collected at baseline, and 1, 3, and 6 months following treatment. A. actino-mycetemcomitans, C. rectus, B. forsythus, E. corrodens, F. nucleatum, P. gingivalis, and P. intermedia were detected by culture, immunofluorescence, or PCR technique. RESULTS: There was a reduction of total bacterial cell count, as well as of certain periodontal pathogens, following treatment. The prevalence of A. actinomycetemcomitans, B. forsythus, and P. gingivalis and the mean proportions of C. rectus, P. intermedia, F. nucleatum, and P. gingivalis decreased after therapy, but there was no statistically significant difference between the 2 treatment groups with respect to bacterial proportions or the number of positive sites. Besides, the pathogens could not be eliminated from the periodontal pocket, and recolonization of the pocket was noted at 3 months post-treatment. CONCLUSIONS: Bacteria located within the cheek, tongue mucosa, saliva, or untreated sites may contribute to reinfection of the pockets and explain the insignificant response to local tetracycline therapy.

Characterization of dental follicle cells in developing mouse molar.

Dental follicle has been implicated as the origin of alveolar bone, cementum and periodontal ligament, but there is no direct evidence of their cellular lineage. The present pilot study was designed to characterize the phenotype of cultured cells obtained from the dental follicle of neonatal mouse molars. Developing mandibular molars from 6-day-old CD-1 mice were subjected to 1% trypsin in Hank's balanced salt solution. After trypsinization, the dental follicle was enucleated from the tooth germ and separated from the associated epithelial root sheath. Pure dental follicle tissue was cultured in alpha-minimal essential medium containing 10% fetal bovine serum and antibiotics. The nature of the cultured follicle cells was determined in situ by immunocytochemical staining for type I and III collagen, fibronectin, and alkaline phosphatase expression. Earlier phenotypic markers for mineralization such as bone sialoprotein and osteopontin were also examined by in situ hybridization of matched molar tissues. The extracellular matrix proteins (such as type I collagen and fibronectin) were moderately expressed cytochemically. However, type III collagen was strongly stained. Gene expression of bone sialoprotein and osteopontin was detected in sections of mouse molars of similar age. The ALPase activity showed moderate to strong intensity in these primary cultured cells and responded to 1,25(OH)2 vitamin D3 treatment. Cytokeratin stains were not noted in these cells. In conclusion, the 6-day-old dental follicle cells exhibit partial characteristics of a mineralized tissue-forming phenotype even though the expression of osteopontin, type I collagen and fibronectin was low at this stage.

Interleukin 1beta-secreting cells in inflamed gingival tissue of adult periodontitis patients.

Interleukin 1beta (IL-1beta) is a cytokine with a wide range of biological activities. It is produced by various cell types including macrophages, fibroblasts, and neutrophils. The inflammatory responses mediated by IL-1beta play an important role in periodontal tissue destruction. The purposes of this study were: (1) to determine the location of IL-1beta in inflamed human gingival tissues by the immunofluorescence method; and (2) to correlate this location to the concomitant presence of macrophage or neutrophils by immunohistochemistry. Five patients with moderate to advanced adult periodontitis receiving periodontal phase I therapy were included in this study. One month after phase I therapy, 15 sites with a probing pocket depth >/=5 mm and gingivitis index >/=1 were arranged for modified Widman flap operation. Another three sites with a probing pocket depth

Relationship of the subgingival microbiota to a chairside test for aspartate aminotransferase in gingival crevicular fluid.

BACKGROUND: The aim of the present study was to evaluate the association between the occurrence of certain specific periodontal pathogens and aspartate aminotransferase (AST) levels in gingival crevicular fluid (GCF). METHODS: Thirty systemically healthy subjects with moderate to advanced periodontitis were selected. Within each subject, the AST contents of GCF from sites with probing depth between 5 mm and 7 mm were measured using a chairside colorimetric test. AST-positive site refers to one that had an AST level > or = 800 microIU. Subgingival plaque samples from one AST-positive and one negative site were collected for microbiological examination. One site with probing depth < or = 3 mm and no gingival inflammation was selected as a healthy control. Clinical parameters of the chosen sites, including the plaque index and gingival index scores, probing depth, and clinical attachment level were measured. Culture and immunofluorescence (IF) were used for detecting common periodontal pathogens, including Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Capnocytophaga species, Prevotella intermedia, Prevotella melaninogenica, and Porphyromonas gingivalis. Logistic regression was used to analyze the correlation between the AST test and certain specific pathogens. RESULTS: The GCF scores and total cultivable bacterial counts were higher in AST-positive sites than either AST-negative or healthy sites. The prevalence and proportions of specific periodontal pathogens such as C rectus, E. corrodens, F. nucleatum, Capnocytophaga species, P. intermedia, and P. gingivalis were significantly higher in positive than in negative sites. In analyzing the correlation of the proportion of 6 pathogens with the AST test by logistic regression, only P. gingivalis showed a significant positive correlation. The odds ratio of having a high proportion of P. gingivalis in the presence of a positive AST test was 1.21. CONCLUSIONS: The present study showed that at AST-positive sites, there is a higher prevalence and higher proportion of certain periodontal pathogens. Although only the correlation of P. gingivalis and AST values was statistically significant, the results imply that certain periodontal pathogens may be associated with elevation of AST levels in GCF.

Clinical response of localized recurrent periodontitis treated with scaling, root planing, and tetracycline fiber.

The purpose of this study was to compare the clinical efficacy of scaling and root planing alone versus tetracycline fiber therapy used adjunctively with scaling and root planing in the treatment of nonresponsive active periodontitis in patients under supportive periodontal therapy. Thirty patients who were receiving supportive treatment and had at least two nonadjacent periodontitis sites with a probing depth of between 4 and 8 mm and bleeding on probing, or had aspartate aminotransferase (AST) levels above 800 microIU in the gingival crevicular fluid in separate quadrants participated in this study. For each patient, the test sites were treated with scaling and root planing plus tetracycline fibers while the control site was treated with scaling and root planing only. Probing depths, clinical attachment levels, gingival recession, AST levels, and bleeding on probing were recorded and subgingival plaque samples were collected at baseline and 1, 3, and 6 months following treatment. At 3 months after treatment, there was a reduction of bleeding on probing and probing depth, and a gain of clinical attachment in both test and control sites. The mean reduction in probing depth of the test sites was 1.38 mm and the attachment gain was 0.8 mm after 6 months. The clinical response obtained at 3 months following therapy was maintained throughout the 6-month follow-up period. However, there were no statistically significant differences between sites treated with scaling and root planing alone and those treated with combined tetracycline therapy. Most of the reductions of probing depths in the fiber group were attributed to gingival recession. The present study did not confirm the efficacy of adjunctive tetracycline fibers in treating nonresponsive sites in maintenance subjects with regard to probing depth reduction or clinical attachment gain. Reinfection of the pockets from untreated sites and extra-crevicular regions may explain the insignificant response to local tetracycline therapy.

Relationships between clinical parameters, Interleukin 1B and histopathologic findings of gingival tissue in periodontitis patients.

IL-1 beta is one of the cytokines which can induce bone resorption and connective tissue destruction. Previous studies demonstrated that the amount of IL-1 beta in gingival crevicular fluid (GCF) was closely related to the severity of gingival index (GI) and probing depth (PD). To further investigate whether the amount of IL-1 beta in GCF and the tissues adjacent to the periodontal pockets can reflect the degree of inflammation and destruction of these tissues, the GCF and inflamed gingival specimens were harvested from 14 periodontal patients and IL-1 beta in these samples was measured by ELISA. Clinical parameters of the sampled teeth were also recorded. Possible relations among periodontal parameters, GCF or tissue IL-1 beta and the degrees of inflammation in tissue sections were analysed statistically. The results showed that with increasing GI and PD, there was an elevation of GCF and tissue IL-1 beta activity. GCF flow also showed a tendency to increase with a higher percentage of inflammatory infiltrate. The GCF and tissue IL-1 beta activity were significantly correlated with clinical parameters (GI, PD and GCF flow), as well as with the percentage of inflammatory infiltrate and tissue alterations. With the exception of a significantly negative correlation with the extent of inflammation, no obvious relation between the GCF or tissue concentration of IL-1 beta and the state of tissue pathology could be detected. In conclusion, measurements of IL-1 beta activity in GCF or diseased tissues based on the classification of clinical parameters can reflect the degree of inflammation within periodontal disease tissue. These observations indicate that clinical parameters can provide reliable means of evaluation of the severity of periodontal disease, and that IL-1 beta plays a pivotal role in the pathogenic mechanism of periodontal tissue destruction.

Long-term effects of gingival fibroblast-coated hydroxylapatite graft on periodontal osseous defects.

This study was designed to examine the long-term effects of gingival fibroblast-coated hydroxylapatite (GF-HA) grafting on the healing of osseous defects caused by inflammatory periodontitis. Gingival fibroblasts were cultured from biopsy specimens of a healthy portion of gingiva in a periodontal therapy patient. Growth of cells on hydroxylapatite (HA) particles was established in vitro and the GF-HA complex was transplanted into the periodontal osseous defects. Clinical data of the gingival and plaque index, probing depth and periapical X-ray were monitored at baseline and at regular intervals up to 28 months after surgery. Postoperative plaque control and regular maintenance were meticulously performed. In addition to disappearance of gingival inflammation, results showed that the GF-HA treated sites had a marked pocket reduction and probing attachment gain at reentry and later recalls. Clinical bone fills of osseous defects were also demonstrated in periapical radiographs. These observations suggest that GF-HA grafting treatment may lead to regeneration of periodontal tissue in periodontitis-affected osseous defects.

Modulation of periodontal ligament fibroblasts by cementum and alveolar bone extracts.

This study investigated the effect of acetic acid extracted proteins of bone and cementum on periodontal ligament fibroblasts. Alveolar bone and cementum obtained from clinically healthy subjects were subjected to 0.5 M acetic acid extraction, dialysis and lyophilization. Effects of both extracts on cell attachment and stimulation of DNA synthesis were studied in fibroblast-like cells cultured from human periodontal ligament tissue. A wide spectrum of proteins, such as 11-14, 17, 19, 22, 24, 27, 30, 34-44, 47 and 55 kDa, were detected in 10% SDS-polyacrylamide gel electrophoresis of both extracts. A few tissue-specific bands (19, 22, 47 kDa in cementum extract; 34-44, 55 kDa in bone extract) were noted. These extracts were found to promote cell attachment and stimulate DNA synthesis of periodontal fibroblasts. Cementum extract appeared to have a higher activity in stimulating DNA synthesis than bone extract, while promotion of cell attachment was similar with both extracts. Addition of serum evoked a synergistic effect on the promotion of DNA synthesis of both extracts. These preliminary observations suggest that acetic acid extracts from bone and cementum contain distinct components capable of modulating cellular activities of periodontal fibroblasts.

Guided tissue regeneration demineralized freeze-dried bone allograft: treatment of furcation defects in mandibular molars.

The present study evaluated the effects of combined guided tissue regeneration (GTR) and demineralized freeze-dried bone allograft (DFDBA) therapy on the healing of grade III furcation lesions in mandibular molars of seven periodontitis patients. De novo surgical debridement of furcation roofs by fine diamond bur was introduced. Routine presurgical preparation of teeth and a strict plaque control program were performed for at least six weeks before surgery. A papillary conserved full thickness mucoperiosteal flap was used in all cases. In addition to conventional debridement, odontoplasty was performed on the furcation areas with a diamond bur to eradicate inaccessible fissures or grooves and ensure calculus-free root surfaces. Following debridement, the bony defects were filled with DFDBA and covered with polytetrafluoroethylene (ePTFE) membranes. The flaps were then closed by interproximal sutures coronally positioned through the contact point. The ePTFE membranes were removed 6 to 7 weeks after operation. Clinical parameters such as probing depth (PD), gingival recession (GR), probing attachment level (PAL), tooth mobility (TM), and periapical x-ray were recorded at the baseline and 0, 3, 6, 9, and 12 months after removal of the ePTFE membrane. The results showed a significant increase in the probing attachment level and radiographic evidence of bone fill at the furcation sites. Thus, the addition of fine diamond bur debridement on the furcation in the GTR procedure with DFDBA grafting may be effective in the treatment of grade III furcation involvement.

Expression of matrix proteins in cloned fibroblasts derived from periodontal tissue under different cell growth densities.

Extracellular matrix (ECM) proteins have been implicated in the attachment, migration and differentiation of cells during periodontal wound healing. This study was designed to investigate the expression of ECM proteins in gingival fibroblasts (GF) and periodontal ligament fibroblasts (PF) at different cell growth densities. Both mass-cultured and cloned cells were derived from explants of healthy human gingivae and periodontal ligament. The grown cells, in situ, were subjected to immunofluorescent staining for the expression of collagen type I (CI), III (CIII) and fibronectin (Fn), and then measured by microfluorimetry. Partial biochemical measurement of the ECM proteins secreted in the medium was done by SDS-PAGE and densitometry. These data indicated that CI, CIII and Fn were expressed in both mass-cultured and cloned GF and PF. There were variations in the expression of these ECM proteins among the clones. PF and most of its clones produced a greater amount of total cellular and ECM proteins than did GF. The expression of these proteins was found to be greater in areas of low, as opposed to high, cell density. Parallel results were also noted in limited biochemical analyses. Thus, PF differed from GF with regard to the production of cellular and matrix proteins, and protein metabolism was affected by cell growth density. These data tend to support the previous hypothesis that PF is the essential cell type contributing to periodontal regeneration.

DNA content and alkaline phosphatase expression in cells of different gingival overgrowths.

The present study compared the alkaline phosphatase (ALPase) expression and DNA content at specific periods in cultured cells derived from non-inflamed enlarged gingivae of idiopathic gingivofibromatosis (IGF) and phenytoin-induced hyperplasia (PHG). Cultured cells from healthy gingiva or periodontal ligament (PDL) were used as controls. The DNA assay, ALPase assay and cytochemical staining for ALPase in cultured cells were performed at four, seven, and nine days. The presence of intense ALPase activity was a prominent feature in cultured IGF cells, whereas very low ALPase activity was detected in PHG cells. The cell lines tested showed no significant differences in DNA content. The expression of ALPase in these cells was population density-dependent. The observation that cells isolated from both types of gingival overgrowth exhibited a different ALPase profile at variance with normal gingival fibroblasts suggested that a distinct pathogenic mechanism may be involved in each type of gingival overgrowth.