Osteoblast proliferation and differentiation on dentin slices are modulated by pretreatment of the surface with tetracycline or osteoclasts

BACKGROUND: Implant surface roughness and chemical composition, as well as other factors, affect the ability of osteogenic cells to form bone adjacent to an implant. The same principles may also apply to the tooth root and some reports have shown that surface modification of the root may lead to improved restoration of the periodontal apparatus. The most common of these surface modification techniques involves demineralization with citric acid or treatment with tetracycline to expose collagen fibrils. In addition, during normal bone remodeling, osteoclasts demineralize the extracellular matrix, leaving resorption pits and exposed collagen fibrils. In this study, the effect of different dentin surface-preparation techniques on osteoblasts were compared. METHODS: Slices of sperm whale dentin were mechanically polished and surfaces were treated with tetracycline-HCl (TCN) or were cultured with mouse bone marrow cells to create a surface with osteoclast (OC) resorption pits or left untreated. Profilometry, x-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were used to evaluate the 3 different dentin surfaces. MG63 osteoblast-like cells were cultured on the 3 different surfaces and the effect of dentin surface preparation technique on MG63 cell proliferation (cell number), differentiaton (alkaline phosphatase specific activity of isolated cells and cell layer lysates; osteocalcin production), and local factor production (transforming growth factor (TGF)-beta1 and prostaglandin E2 (PGE2) compared. RESULTS: Profilometry showed the polished and TCN surfaces were smooth with comparable Ra values, whereas the OC surfaces were slightly rougher due to resorption pits which covered 3.7% of the surface. XPS measurements showed that TCN treatment reduced the Ca and P content of the surface, indicating that it had dissolved the mineral. Osteoclast-resorption also reduced the Ca and P content, but to a lesser extent. MG63 cell proliferation on polished dentin and tissue culture polystyrene was equivalent. In contrast, cells grown on the TCN- and OC-treated surfaces exhibited increased proliferation. No effect of surface treatment on cell alkaline phosphatase activity was observed, but activity in the cell layer lysates was increased on the TCN- and OC-treated surfaces. Osteocalcin production was reduced on all dentin surfaces, but the greatest reduction was found on the TCN-treated surface. Production of both TGF-beta1 and PGE2 was increased on the treated surfaces. All effects were greatest in cultures grown on the TCN-treated dentin. CONCLUSIONS: These data indicate that demineralization of the dentin surface promotes proliferation of osteoblasts and early differentiation events like production of alkaline phosphatase and autocrine mediators such as PGE2 and TGF-beta1. However, later differentiation events like osteocalcin production are decreased. Osteoclast-mediated bone resorption elicits similar responses; less than 4% of the dentin surface resulted in approximately 75% of the response caused by TCN treatment. These observations suggest that greater attention should be paid to the effects of osteoclastic resorption in designing methods for enhancing bone and cementum formation adjacent to root surfaces.     

Effects of enamel matrix derivative on proliferation and differentiation of primary osteoblasts

OBJECTIVE: The aim of this study was to investigate the effects of enamel matrix derivative (EMD) on proliferation, protein synthesis, and mineralization in primary mouse osteoblasts. STUDY DESIGN: Osteoblasts were obtained from mouse calvaria by enzymatic digestion and grown in monolayer together with EMD (2-100 microg/ml). Metabolic activity and cell proliferation were determined by tetrazolium salt assay (MTT) and by 5-bromo-2'-deoxyuridine (BrdU) incorporation. For differentiation studies, a 3-dimensional organoid culture system was used. Osteoblastic differentiation was estimated by alkaline phosphatase (ALP) activity and calcium content. Collagen synthesis was assessed by [(3)H]-proline incorporation. Morphologic observations were made by electron microscopy. RESULTS: EMD treatments increased metabolic cell activity and BrdU incorporation. In the organoid cultures, ALP activity and calcium accumulation were enhanced by EMD treatment, but [(3)H]-proline incorporation was not. Morphologically, an increased deposition of mineralized nodules was found. CONCLUSIONS: EMD treatment enhanced cellular activities of primary osteoblasts and might support the regeneration of periodontal bony defects.     

In Vitro Evaluation of Demineralized Freeze‐Dried Bone Allograft in Combination With Enamel Matrix Derivative

Background: Preclinical and clinical studies suggest that a combination of enamel matrix derivative (EMD) with demineralized freeze‐dried bone allograft (DFDBA) may improve periodontal wound healing and regeneration. To date, no single study has characterized the effects of this combination on in vitro cell behavior. The aim of this study is to test the ability of EMD to adsorb to the surface of DFDBA particles and determine the effect of EMD coating on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. Methods: DFDBA particles were precoated with EMD or human blood and analyzed for protein adsorption patterns via scanning electron microscopy. Cell attachment and proliferation were quantified using a commercial assay. Cell differentiation was analyzed using real‐time polymerase chain reaction for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen 1α1, and mineralization was assessed using alizarinred staining. Results: Analysis of cell attachment revealed no significant differences among control, blood‐coated, and EMD‐coated DFDBA particles. EMD significantly increased cell proliferation at 3 and 5 days after seeding for both osteoblasts and PDL cells compared to control and blood‐coated samples. Moreover, there were significantly higher messenger ribonucleic acid levels of osteogenic differentiation markers, including collagen 1α1, alkaline phosphatase, and osteocalcin, in osteoblasts and PDL cells cultured on EMD‐coated DFDBA particles at 3, 7, and 14 days. Conclusion: The results suggest that the addition of EMD to DFDBA particles may influence periodontal regeneration by stimulating PDL cell and osteoblast proliferation and differentiation.     

Participation of endogenous IGF-I and TGF-beta 1 with enamel matrix derivative-stimulated cell growth in human periodontal ligament cells

Previous studies have provided the biological basis for the therapeutic use of enamel matrix derivative (EMD) at sites of periodontal regeneration. A purpose of this study is to determine effects of EMD on cell growth, osteoblastic differentiation and insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta 1) production in human periodontal ligament cells (HPLC). We also examined participation of endogenous IGF-I and TGF-beta 1 with EMD-stimulated cell growth in these cells. HPLCs used in this study were treated with EMD alone or in combination with antihuman IGF-I antibody (anti-hIGF-I) or anti-hTGF-beta 1, recombinant human bone morphogenetic protein-2 (rhBMP-2), 1,25-dihydroxyvitamin D3[1,25(OH)2D3], rhTGF-beta 1 or rhIGF-I. After each treatment, cell growth, the production of IGF-I and TGF-beta 1 and the expression of osteoblastic phenotypes were evaluated. EMD stimulated cell growth in dose-dependent and time-dependent manners. EMD was also stimulated to express IGF-I and TGF-beta 1 at protein and mRNA levels. The EMD-stimulated cell growth was partially suppressed by cotreatment with anti-hIGF-I or anti-hTGF-beta 1, and cell growth was also stimulated by treatment with rhIGF-I or rhTGF-beta 1. rhBMP-2 stimulated alkaline phosphatase (ALPase) activity and ALPase mRNA expression, and 1,25(OH)2D3 stimulated ALPase and osteocalcin mRNA expression. However, EMD showed no effect on the osteoblastic phenotypes expression. These results demonstrated that EMD has no appreciable effect on osteoblastic differentiation, however it stimulates cell growth and IGF-I and TGF-beta 1 production in HPLC, and that these endogenous growth factors partially relate to the EMD-stimulated cell growth in HPLC.     

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative

Objectives

Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials.

Aims

This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells.

Materials and methods

EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining.

Results

Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points.

Conclusion

The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings.

Clinical relevance

The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.     

Enamel matrix derivative promotes human periodontal ligament cell differentiation and osteoprotegerin production in vitro

Enamel matrix derivative (EMD) has been used successfully to aid periodontal repair. We sought to elucidate the mechanism of action of EMD and hypothesized that combined exposure to EMD and parathyroid hormone (PTH), which acts anabolicly when administered intermittently, would enhance periodontal ligament cell proliferation, differentiation, and local factor production. Confluent human periodontal ligament cells were exposed to EMD continuously or to PTH(1-34) intermittently, or a combination of both. Cell number, alkaline phosphatase activity, osteocalcin, and osteoprotegerin production were determined. Continuous challenge with EMD resulted in an increase of the differentiation parameters and osteoprotegerin production, while simultaneously inhibiting proliferation. Intermittent PTH(1-34) administration exerted opposite effects. Combined administration of EMD and PTH(1-34) weakened or even nullified the effects seen for the agents alone. These results suggest that EMD promotes periodontal ligament cell differentiation and osteoprotegerin production, potentially resulting in a microenvironment supporting periodontal repair, whereas combining EMD and PTH(1-34) failed to prove beneficial in this respect.     

Study on proliferation and function of periodontal ligament fibroblasts and osteoblastic cells under hypoxia

There have been many reports recognizing vascular changes on pressure side of periodontal tissues during orthodontic tooth movement. The vascular changes cause local hypoxia which seems to affect the phenotypes of periodontal tissue cells. In order to clarify the effect of hypoxia on proliferation and function of periodontal tissue cells, DNA content, alkaline phosphatase (ALP) activity and prostaglandin E2 (PGE2) production under a hypoxic condition in both periodontal ligament fibroblasts (PLF) and osteoblastic cells (MC3T3-E1 cells) were examined in vitro. PLF were cultured from human periodontium and identified by both morphologic characterization and presence of ALP. The results obtained were as follows: 1. Under 10% O2 condition, the activity of proliferation in PLF did not change but that of osteoblasts was inhibited. 2. ALP activity in PLF was stimulated but that of osteoblasts was inhibited under the hypoxic condition. 3. Production of PGE2 in osteoblasts increased after 7 days of hypoxia though that in PLF decreased. In addition, the enhancement of PGE2 production in osteoblasts was due to activation of both phospholipase A2 and PGE2-synthesizing enzymes. 4. From the orthodontic point of view, hypoxia on the pressure side may induce bone resorption by inhibiting mineralization activity of osteoblasts and enhancing production of PGE2 in osteoblasts.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic differentiation in cells isolated from human periodontal ligament.

Periodontal ligament cells may play an important role in the successful regeneration of the periodontium. We investigated the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2), one of the most potent growth factors that stimulates osteoblast differentiation and bone formation, on cell growth and osteoblastic differentiation in human periodontal ligament cells (HPLC) isolated from four adult patients. rhBMP-2 induced no significant changes in cell growth in any of the HPLCs. rhBMP-2 at concentrations over 50 ng/mL significantly stimulated alkaline phosphatase (ALPase) activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in the HPLCs. rhBMP-2 (500 ng/mL) also slightly enhanced the level of PTH/PTH-related peptide receptor mRNA expression in these cells. While interleukin-1 beta enhanced ALPase activity stimulated with rhBMP-2, tumor necrosis factor-alpha inhibited the rhBMP-2-stimulated activity. Interleukin-6 induced no significant changes in ALPase activity stimulated with rhBMP-2. Although HPLCs, whether treated with rhBMP-2 or not, could not produce measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced osteocalcin mRNA expression and protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HPLCs at both the mRNA and protein levels. These results suggest that rhBMP-2 provides an anabolic effect on periodontal regeneration by stimulation of osteoblastic differentiation in human periodontal ligament cells, and that this stimulatory effect is differentially modulated by inflammatory cytokines during the course of periodontal regeneration.     

釉基质衍生物对人牙周膜干细胞成骨分化的影响

目的研究釉基质衍生物对人牙周膜干细胞增殖和成骨分化的影响并探究其可能的机制。方法原代培养人牙周膜干细胞,经过流式鉴定后选取第3代细胞进行实验。采用CCK-8试剂盒检测不同浓度(0、20、50、100 mg·L^-1)的釉基质衍生物对人牙周膜干细胞增殖的影响;实时荧光定量聚合酶链式反应(qRT-PCR)检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞成骨分化的影响;通过Trichrome染色和Von Kosa’s染色检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞胶原合成和矿化结节形成的影响;不同浓度釉基质衍生物和DDK1作用人牙周膜干细胞之后,通过Western blot和qRT-PCR检测β-连环蛋白、RunX2、CaMKⅡ及NLK表达情况。结果釉基质衍生物对人牙周膜干细胞的增殖具有明显的促进作用,并呈现剂量和时间依赖性;釉基质衍生物处理人牙周膜干细胞之后,矿化结节形成和胶原合成显著增多,骨钙素、Ⅰ型胶原、RunX2的表达明显增多;另外,釉基质衍生物处理能显著增加β-连环蛋白、RunX2、CaMKⅡ和NLK的表达,且该作用可被DDK1抑制。结论釉基质衍生物对体外培养的人牙周膜干细胞有促进增殖和成骨分化的作用,其作用可能是通过Wnt/β-连环蛋白实现的。     

Osteoprotegerin and receptor activator of nuclear factor-kappa B ligand modulation by enamel matrix derivative in human alveolar osteoblasts

BACKGROUND: Bone regeneration techniques increasingly rely on the use of exogenous molecules able to enhance tissue formation in pathologic and traumatic defects. An enamel matrix derivative (EMD) has been largely used to promote tooth ligament regeneration within periodontal pockets. Recent evidence suggests that EMD may contribute to inducing osteoblast growth and differentiation. We investigated the effects of EMD on growth and osteogenic marker modulation in human mandibular osteoblasts. METHODS: We focused our attention on cell growth by 3-(4,5-dimethyl[thiazol-2-yl]-3,5-diphery)tetradium bromide (MTT) assay, cell differentiation, mineralized nodule formation, and, in particular, the expression of receptor activator of nuclear factor-kappa B ligand (RANKL), the main osteoclast differentiation factor, and its decoy receptor, osteoprotegerin (OPG), by enzyme-linked immunosorbent assay. RESULTS: Cell growth was significantly increased by EMD. Similarly, a significantly higher quantity of OPG and a lower amount of RANKL were detectable in groups treated with 50 and 100 microg/ml at weeks 1, 2, and 3, and alkaline phosphatase activity and osteocalcin production were enhanced in cultures treated with 50 and 100 microg/ml at weeks 2 and 3. Mineralized nodules appeared bigger and more numerous in cultures treated with 50 and 100 microg/ml EMD. CONCLUSIONS: EMD was able to enhance osteoblast cell growth and the expression of markers of osteoblastic phenotype and differentiation. EMD also seemed able to create a favorable osteogenic microenvironment by reducing RANKL release and enhancing osteoblastic OPG production.     

Differentiation and cytokine synthesis of human alveolar osteoblasts compared to osteoblast-like cells (MG63) in response to titanium surfaces.

OBJECTIVES: The aim of this study was to investigate the influence of different implant surface topographies and chemistries on the expression of differentiation/proliferation markers on MG63 cells and primary human alveolar osteoblasts. METHODS: Hydrophobic acid-etched (A) and hydrophobic coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and hydrophilic coarse-grit-blasted (modSLA) surfaces were produced. Thereby, modA and modSLA surfaces were rinsed under nitrogen protection and stored in a sealed glass tube containing isotonic NaCl solution at pH 4-6. Tissue culture plates without specimens served as controls. The behavior of MG63 cells and primary human alveolar osteoblasts (AOB) grown on all surfaces was compared through determination of alkaline phosphatase (ALP) activity, cell proliferation ((3)H-thymidin incorporation, MTT colorimetric assay) and expression of osteocalcin (OC), osteoprotegerin (OPG), transforming growth factor-beta1 (TGF-beta(1)) and vascular endothelial growth factor (VEGF), detected with commercial available test kits. RESULTS: Proliferation of MG63 and primary cells was highest on controls, followed by A surfaces, modA and SLA surfaces being almost on the same level and lowest on modSLA surfaces. modSLA surfaces exhibited highest ALP and OC production, followed by SLA, modA and A surfaces. Proliferation and OC production were comparable for MG63 cells and AOB. OPG, TGF-beta(1) and VEGF produced on primary cells showed a slightly different rank order on different surfaces compared to MG63 cells. modSLA still showed the highest production of OPG, TGF-beta(1) and VEGF, but was followed by modA, SLA and A. Statistical significance was checked by ANOVA (p<0.0035). SIGNIFICANCE: MG63 cells and primary human alveolar osteoblasts showed similar proliferation and differentiation characteristics on different titanium surfaces. Only modA surfaces showed enhanced expression of OPG, TGF-beta(1) and VEGF on MG63 cells compared to primary human alveolar osteoblasts. Overall, the lowest proliferation rates and the highest expressions of differentiation markers and growth factor productions were observed on modSLA.     

Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative

OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.     

机械刺激对牙周骨组织工程干细胞分化的影响

目前,细胞移植及其与支架材料的联合应用仍是牙周骨组织工程的主要策略之一.牙周骨组织中,牙槽骨和牙骨质等组织的硬度及空间结构有明显差异,支架材料的不同机械性能对干细胞的增殖、分化等生物学行为也具有不同诱导作用.研究显示,支架材料的基质硬度和拓扑结构等机械刺激因素参与调控包括脂肪来源间充质干细胞、牙周膜干细胞等在内的种子细...     

Subculture affects the phenotypic expression of human periodontal ligament cells and their response to fibroblast growth factor-2 and bone morphogenetic protein-7 in vitro

Background and Objective: Although periodontal ligament cells display several osteoblastic traits, their phenotypic expression is still not well established. It remains a matter of debate whether they resemble a terminally differentiated cell type or an intermediate maturation state that potentially can be directed towards a fibroblastic or an osteoblastic phenotype. Material and Methods: To explore the characteristics of periodontal ligament cells in greater detail, fourth‐passage, sixth‐passage and eighth‐passage human periodontal ligament cells were cultured for up to 3 wk. Ki‐67, alkaline phosphatase, osteocalcin, osteoprotegerin and receptor activator of nuclear factor‐κB ligand (RANKL) mRNA expression was quantified by real‐time polymerase chain reaction. Furthermore, the cellular response to fibroblast growth factor‐2 and bone morphogenetic protein‐7 was examined in first‐passage and fourth‐passage cells. Dermal fibroblasts (1BR.3.G) and osteoblast‐like cells (MG63) served as reference cell lines. Results: Proliferation decreased over time and was highest in fourth‐passage cells. The expression of differentiation parameters, osteoprotegerin and RANKL increased with culture time and was higher in fourth‐passage cells than in cells of later passages. The RANKL/osteoprotegerin ratio increased steadily until day 21. Administration of fibroblast growth factor‐2 enhanced cell numbers in both passages, whereas alkaline phosphatase and osteocalcin production remained unchanged. By contrast, exposure of periodontal ligament cells to bone morphogenetic protein‐7 resulted in a reduction of cell number in the first and fourth passages, whereas the production of alkaline phosphatase and osteocalcin was enhanced. In dermal fibroblasts, differentiation parameters did not respond to both stimuli. MG63 cells behaved similarly to periodontal ligament cells. Conclusion: These results indicate that subculture affects the phenotypic expression of human periodontal ligament cells with respect to the characteristics that these cells share with osteoblasts. Furthermore, the periodontal ligament cell phenotype can be altered by fibroblastic and osteoblastic growth factors.