Attachment and proliferation of human osteoblast-like cells on guided bone regeneration (GBR) membranes in the absence or presence of nicotine: an in vitro study.

PURPOSE: To compare in vitro the attachment and proliferation of human osteoblast-like cells (MG63) on tissue culture plates and guided bone regeneration (GBR) membranes in the absence or presence of nicotine. MATERIALS AND METHODS: Membrane samples were fixed to wells and the cell number (CN) was counted after 24 hours (attachment assay) or 5 days (proliferation assay). The ratio of cell count (RCC) (CN at 5 days/CN at 24 hours) was calculated. The study had three parts: First, five different resorbable GBR membranes were compared (Resolut Adapt LT [RALT], Biocollagen [BC], Bio-Gide [BG], OsseoGuard [OG], and Demokritos Human Tissue Bank [DEM]). Next, cells were cultured on tissue culture plates with five different concentrations of nicotine. Finally, cells were cultured on the membrane that had demonstrated the highest RCC and CN in part 1 with four different concentrations of nicotine. RESULTS: At 24 hours, BG showed the highest CN and OG showed the lowest CN. At 5 days, BG showed the highest CN. The order of RCC was BG > OG > DEM > RALT ~ BC. At 24 hours, lower nicotine concentrations (0.3 and 3 µg/mL) showed higher CNs versus the control, whereas CNs for high nicotine concentrations (30 and 300 µg/mL) were lower than for the control. CN at 5 days and RCC were lowest with 300 µg/mL nicotine. At 24 hours and 5 days, all differences among wells with membrane were statistically insignificant. Nevertheless, CN at 5 days and RCC were highest with the lowest nicotine concentration (3 µg/mL) and lowest with high concentrations. CONCLUSIONS: Membrane materials influence attachment and proliferation of bone cells and, therefore, could affect the outcomes of GBR. On both tissue culture plates and membranes, there is a tendency toward a biphasic effect of nicotine, with stimulatory effects at low concentrations and inhibitory effects at high concentrations.

RANKL and OPG mRNA level after non-surgical periodontal treatment.

Recent research evidence shows that the receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) play an important role in osteoclastogenesis and the inflammatory bone loss during periodontitis. Bone remodeling process is dependent on the balance of these two proteins while a high ratio of RANKL/OPG characterizes the increased osteolytic process and it has been reported in inflammatory diseases including the periodontal disease. The purpose of this study was to determine the OPG and RANKL mRNA levels in periodontal tissues derived from patients with advanced chronic periodontitis after non-surgical periodontal therapy (SRP) and to compare the RANKL/OPG ration with that in healthy persons. Gingival biopsies were obtained from subjects with clinically healthy periodontium (H) (N = 11) and patients with advanced chronic periodontitis (CP) (N = 14). Total RNA was isolated from the gingival samples and 1 microg RNA was reverse transcribed to cDNA, followed by polymerase chain reaction (PCR) using specific primers for OPG and RANKL. The efficiency of reverse transcription was verified by the amplification of the GAPDH gene. The intensity of RT-PCR products was analyzed by a densitometer and was normalized to the intensity of the band for the housekeeping gene GAPDH. Immunohistochemical evaluation of the RANKL and OPG expression was also performed. The expression of RANKL as well as of OPG was reduced in CP specimens in comparison to that of healthy persons in a statistical significant way. However, the RANKL/OPG ratio showed to be slightly elevated in CP compared to H specimens but this finding was not of statistical significance. The immunohistochemical analysis revealed a non-uniform expression pattern for both proteins. Although further investigation is needed to identify the specific role of RANKL and OPG protein in periodontitis progression, our data after SRP might indicate the possible involvement of these proteins in the activation of pathways, which regulate the repair of the periodontal tissues.

Platelet-rich plasma effect on periodontally affected human gingival fibroblasts: an in vitro study.

OBJECTIVE: The purpose of this study was to evaluate the gingival fibroblast proliferative response derived from patients with chronic and aggressive periodontitis to homologous platelet-rich plasma (PRP). MATERIALS AND METHODS: Gingival fibroblasts derived from nine patients with chronic periodontitis, aggressive periodontitis and healthy periodontium were grown. Medium was replaced with DMEM containing 0.5% FBS in which cells remained for two days. Cells were incubated and cultured with medium containing 50 microl/ml homologous platelet-rich plasma (PRP) or not containing PRP (control) for 24 and 48 hours. PRP originated from three donors. Cell proliferation effect was evaluated at 24 and 48 hours. Cell viability was assessed with a hemocytometer. Viable cells were counted under a phase contrast microscope. RESULTS: The results revealed that incubation of human gingival fibroblasts, derived from healthy and intact periodontium, chronic periodontitis and aggressive periodontitis, in culture medium containing homologous PRP statistically significantly increased the cell proliferation at 24 and 48 hours of culture. CONCLUSION: The addition of PRP to human gingival fibroblast cultures significantly increased the proliferative response, irrespective of the presence of periodontitis, type of periodontitis and PRP donor.

Time- and dose-dependent mitogenic effect of basic fibroblast growth factor combined with different bone graft materials: an in vitro study.

OBJECTIVES: In periodontal regeneration, the growth factor concentrations and the delivery system used are of great importance. In an attempt to assess the mitogenic effect of basic fibroblast growth factor (bFGF) on periodontal ligament (PDL) cells combined with different bone replacement materials, two allografts of cortical (DFDBA) and cancellous (DFBA) bone and an anorganic bovine material with a synthetic peptide (ABM P-15) were used. The purpose of this study was to evaluate the in vitro mitogenic effect of different doses of bFGF alone or in combination with DFDBA, DFBA and ABM P-15 on human PDL cells in a time-dependent mode. MATERIAL AND METHODS: PDL cell cultures were derived from the mid-root of four maxillary premolars. Cells were grown and reached confluence. On day 2 of quiescence, new medium was added along with (1) 1, 5, 10 and 25 ng/ml of bFGF alone, (2) 10 mg of DFDBA, DFBA and ABM P-15 alone and (3) their combination. The mitogenic effect was determined at 24 and 48 h of culture by using a hemocytometer chamber. The cells were counted under a phase contrast microscope. RESULTS: The results revealed that bFGF at the highest concentrations and after 48 h exerted a significant mitogenic effect on PDL cells, and also DFDBA and DFBA supported cell proliferation. Furthermore, DFDBA and DFBA enriched with bFGF had a significant mitogenic effect after 48 h of culture. ABM P-15 with 10 and 25 ng/ml of bFGF up-regulated PDL cell proliferation after 48 h of incubation. CONCLUSIONS: The findings of this study demonstrate the beneficial role of bFGF combined with DFDBA and DFBA as carriers in periodontal repair.