C. E. Markopoulou

Role of growth factors on periodontal repair.

Regeneration of periodontal structures lost during periodontal diseases constitutes a complex biological process regulated among others by interactions between cells and growth factors. Growth factors are biologically active polypeptides affecting the proliferation, chemotaxis and differentiation of cells from epithelium, bone and connective tissue. They express their action by binding to specific cell-surface receptors present on various target cells including osteoblasts, cementoblasts and periodontal ligament fibroblasts. The observation that growth factors participate in all cell functions led to exogenous application during periodontal tissue repair aiming to their use as an alternative therapeutic approach to periodontal therapy. Cell types and cultures conditions, dose, carrier materials, application requirements are of critical importance in the outcome of periodontal repair. The purpose of this article is to review the literature with respect to the biological actions of PDGF, TGF, FGF, IGF and EGF on periodontal cells and tissues, which are involved in periodontal regeneration.

Oestrogen regulates proliferation, osteoblastic differentiation, collagen synthesis and periostin gene expression in human periodontal ligament cells through oestrogen receptor beta

OBJECTIVE The present study was designed to examine how oestrogen regulates proliferation, osteoblastic differentiation, collagen synthesis and periostin gene expression in primary human periodontal ligament (hPDL) cells. DESIGN The short interfering RNA (siRNA) technique was used to inhibit oestrogen receptor beta (ERβ) expression hPDL cells. hPDL cell were isolated and fully characterized. A colorimetric assay was applied for the determination of alkaline phosphatase (ALP). An ELISA kit was used to detect osteocalcin (OCN) levels. Collagen synthesis was determined by measuring the incorporation of L-[3H] praline. RT-PCR was performed to detection of periostin mRNA relative gene expression. RESULTS ERβ mRNA was expressed in hPDL cells and significant inhibition of mRNA expression and ERβ mature protein of the ERβ was evident in the siRNA group. At 72h, there was a significant increase in non-transfected hPDL cell proliferation after estradiol stimulation. Addition of 17β-estradiol significantly enhanced ALP activity and production of OCN in non-transfected cells but had no effect on collagen synthesis. A clear increase in periostin mRNA expression levels was observed after incubating hPDL cells with estradiol. In hPDL-siERβ cells, the application of estradiol did not produce any evident differences in periostin mRNA expression CONCLUSIONS ERβ may play important roles in oestrogen-induced effects on hPDL cell proliferation, osteoblastic differentiation and expression of key molecules for the functional and structural integrity of the periodontium (i.e. periostin).

Chemical modification of an implant surface increases osteogenesis and simultaneously reduces osteoclastogenesis: an in vitro study

OBJECTIVES The present study investigated the effect of a chemical modification of the SLA surface (SLActive surface) on human periodontal ligament (hPDL) cell (1) adhesion, (2) proliferation, (3) osteogenic differentiation (core binding factor α-1 [Cbfa-1], bone morphogenetic protein-7 [BMP-7] gene expression and alkaline phosphatase [ALP] activity) and (4) osteoclast formation and activity (osteoprotegerin [OPG] and receptor activator of nuclear factor-κ B ligand [RANKL] gene expression). The above activities were based on the hypothesis that the expression of such molecules might be dependent on the characteristics of the implant surface. MATERIAL AND METHODS hPDL cells were isolated and characterized for their mesenchymal origin, fibroblastic and osteoblastic phenotype. hPDL cells were cultured on smooth, SLA and SLActive implant surfaces (chemically modified). Cell attachment and proliferation were assessed for 5, 24, 72 h, 5 and 7 days. Cbfa-1, BMP-7, OPG and RANKL gene expression was assessed by RT-PCR and a colorimetric assay for ALP activity was applied. RESULTS hPDL cells grown on SLActive surfaces demonstrated increased proliferation rates (24 h, 5 and 7 days of the incubation period), and ALP activity was found to be significantly upregulated (5, 72 h and 7 days) as compared with the SLA surfaces. After 7 days of culture, the gene expression of BMP-7, Cbfa-1 and OPG by hPDL cells was significantly upregulated, while RANKL gene expression was significantly downregulated in response to the SLActive surface. CONCLUSION Chemical modification of a previously roughened implant surface increases hPDL proliferation and upregulates osteoblastic differentiation. It can also suppress osteoclastogenesis regulating the RANKL-RANK-OPG axis. Hence, an osteoprotective microenvironment is created around chemically modified implants that may enhance osseointegration.

Expression of MMPs and TIMP-1 in smoker and nonsmoker chronic periodontitis patients before and after periodontal treatment.

BACKGROUND AND OBJECTIVE Nonsurgical periodontal treatment controls periodontal inflammation. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are implicated both in the destruction and in the healing of periodontal tissues. The aim of the present study was to compare the mRNA expression of MMP-1, -3, -8, -9 and -13 and TIMP-1 in chronic periodontitis before and after initial periodontal treatment. MATERIAL AND METHODS Ninety gingival samples were harvested from 30 patients with chronic periodontitis (15 nonsmokers and 15 smokers) before and after nonsurgical treatment and from 30 periodontally healthy control subjects (15 nonsmokers and 15 smokers). Clinical parameters were assessed before and after treatment. Total RNA was isolated, and mRNA expression of MMPs and TIMP-1 was assessed by RT-PCR. RESULTS Periodontal treatment significantly increased TIMP-1 expression and decreased the ratios of MMPs/TIMP-1. Post-treatment, nonsmokers with periodontitis had significantly higher MMP-8 and TIMP-1 expression than healthy nonsmokers, and smokers with periodontitis had significantly higher MMP-13 and TIMP-1 expressions than healthy smokers. Post-treatment, smokers had significantly higher TIMP-1 expression and lower MMP-8/TIMP-1 ratio than nonsmokers. Post-treatment, there was no correlation among MMPs, and the expression of MMPs and TIMP-1 was not correlated with clinical measurements. CONCLUSION Periodontal treatment increased TIMP-1 expression and decreased the ratios of MMPs/TIMP-1 in chronic periodontitis. The post-treatment increase in TIMP-1 expression was higher for smokers. The TIMP-1 expression was higher post-treatment than in health. Post-treatment, MMP-8 expression was higher in nonsmokers with periodontitis than in healthy nonsmokers, whereas MMP-13 expression was higher in smokers with periodontitis than in healthy smokers.

The effect of smoking on the mRNA expression of MMPs and TIMP-1 in untreated chronic periodontitis patients: a cross-sectional study.

BACKGROUND AND OBJECTIVE Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important for extracellular matrix. Expression of MMPs has been evaluated in gingiva without studying smoking. The aim of this study was to explore the effect of smoking on mRNA expression of MMP-1, -3, -8, -9 and -13 and TIMP-1 in untreated chronic periodontitis and in periodontal health. MATERIAL AND METHODS Gingival samples were harvested from 30 subjects with untreated chronic periodontitis (15 nonsmokers and 15 smokers) and 30 periodontally healthy subjects (15 nonsmokers and 15 smokers). Full-mouth plaque score, gingival index, bleeding on probing, probing depth and clinical attachment level were recorded. Total RNA was isolated, and the mRNA expression of MMPs and TIMP-1 was assessed by RT-PCR. RESULTS Periodontitis groups were comparable in clinical measurements. Nonsmoker subjects with periodontitis had statistically significantly higher MMP-1, lower MMP-9 and TIMP-1 expression and higher MMP-1/TIMP-1 ratio than smokers; and higher MMP-8 expression and MMP-8/TIMP-1 and MMP-1/TIMP-1 ratios than healthy nonsmokers. Healthy nonsmokers had statistically significantly higher MMP-13 expression than healthy smokers. Smoker periodontitis and healthy subjects had similar expression levels of MMPs and TIMP-1 and MMPs/TIMP-1 ratios. There was correlation among the MMPs only for smoker periodontitis subjects. Expression of MMP-13 was correlated with mean clinical attachment level. CONCLUSION Within its limits, this study demonstrated that smoking affected mRNA expression of MMPs and TIMP-1, MMPs/TIMP-1 ratios and relationships among MMPs in untreated chronic periodontitis and expression of MMPs in health. In the absence of smoking, chronic periodontitis affected expression of MMPs and MMPs/TIMP-1 ratios.

Effect of rhTGF-β1 combined with bone grafts on human periodontal cell differentiation

Various techniques and materials have been proposed for the treatment of periodontal defects. In periodontal regeneration, periodontal ligament (PDL) cell differentiation as well as certain growth factors and their delivery system applied are critical. The purpose of this study was to evaluate the in vitro effect of recombinant human transforming growth factor-beta 1 (rhTGF-β1) combined with two different bone grafts on human PDL (hPDL) cell differentiation. The hPDL cells were treated with TGF-β1 alone or in combination with a calcified freeze-dried bone allograft (FDBA) and a porous biphasic calcium phosphate (BC) bone graft. Cell differentiation effect was estimated by measuring alkaline phosphatase (ALPase) activity and osteocalcin secretion. Results demonstrated that rhTGF-β1 alone or in combination with FDBA and BC provoked a significant (p < 0.05) increase in ALPase activity as compared with controls. The findings of this study confirmed the beneficial role of rhTGF-β1 combined with FDBA and BC as carriers in periodontal regeneration.

Ability of a bovine bone graft, alone or enriched with PDGF-BB or rhBMP-2, to promote human periodontal ligament (PDL) cells proliferation. A preliminary study

One of the most important goals of the periodontal therapy procedures is to stimulate the formation of new bone into osseous defects resulted from periodontal disease. A wide range of grafting materials is used to achieve this aim. Recently, the Human Tissue Bank of the National Center for Scientific Research ‘Demokritos’ in Athens (Greece) has prepared, in a preliminary study, a cancellous bovine-derived bone matrix (BBM). The purpose of the present work was to investigate the role of this bovine bone material in the periodontal regeneration, by studying the rate of human periodontal ligament (PDL) cells proliferation in the presence of this matrix alone, or after the addition of the growth factors, platelet-derived growth factor-BB (PDGF-BB) or recombinant human bone morphogenetic protein-2 (rhBMP-2).Bovine bone graft was prepared using the ‘know how’ acquired by the 30 years continuous preparation and delivery of lyophilized human bone grafts by the ‘Demokritos’ Bank.PDL cells cultures were derived from the mid root of two maxillary premolars. The teeth were caries-free and were extracted for orthodontic reasons from 1 adult female patient. Cells were grown in 24-well dishes in the presence of 20 mg BBM. On day 2 of quiescence, new medium was added with 10 ng/ml of PDGF-BB or 50 ng/ml of rhBMP-2. To determine the effects of the test agents on cell proliferation, DNA synthesis was estimated by measuring [3H] thymidine incorporation. After 48 h of incubation the cells were processed to subject to scintillation counting. Counts per minute (cpm/well) were determined for each sample.The results revealed that this BBM has the ability to maintain PDL cells proliferation and could be used as an alternative graft material. PDGF-BB when added improved the cell proliferative response resulting in a more active BBM, while the presence of rhBMP-2 did not support cell mitosis.

Effect of rhBMP-7 combined with two bone grafts on human periodontal ligament cell differentiation

The purpose of this study was to evaluate the in vitro effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) combined with demineralised freeze-dried bone allograft (DFDBA) and an inorganic bovine material with a synthetic peptide (PepGen P-15) on human periodontal ligament (hPDL) cell differentiation, in a time-dependent manner. hPDL cells were cultured and treated with: (1) 500 ng/ml of rhBMP-7, (2) 10 mg of DFDBA or PepGen P-15 and (3) their combination. Cell differentiation was estimated after 48 and 72 h by measuring alkaline phosphatase (ALPase) activity and osteocalcin (OC) secretion. The presence of rhBMP-7, DFDBA, PepGen P-15, rhBMP-7 + DFDBA and rhBMP-7+ PepGen P-15 promoted a significant increase of ALPase activity after 48 and 72 h. The combination of rhBMP-7 with DFDBA or PepGen P-15 did not lead to significant OC secretion. The results of this study imply that rhBMP-7 stimulates the early osteoblastic differentiation of hPDL cells and that DFDBA and PepGen P-15 could serve as carriers for rhBMP-7.