Proliferation Of Periodontal Ligament Fibroblasts Research Articles

The influence of platelet concentrates with and without metronidazole incorporation pre-centrifuge towards periodontal ligament fibroblast proliferation

Objective: Horizontal centrifugation was believed may improve platelet, leukocyte, and growth factors in the buffy coat. Previous study of platelet concentrates metronidazole incorporation was able to greatly inhibit bacteria. Metronidazole might help the proliferation with its ability to inhibit pro-inflammatory cytokines. This study intended to examine the influence of C-PRF and i-PRF with and without metronidazole incorporation horizontal pre-centrifugation towards periodontal ligament fibroblast proliferation. Material and Methods: 2x104 fibroblast cells on the 96 well plates were added with 1 ml of each buffy coat platelet concentrates. The samples were split into five groups: i-PRF, C-PRF, i-PRF metronidazole, C-PRF metronidazole, and control group. Then monitored after 1 day, 3 days, and 5 days using MTT assays. The data were inspected by Two Way ANOVA Test and Post Hoc Test. Results: The result indicated that cell proliferation of fibroblasts was escalated by the addition of C-PRF, i-PRF metronidazole incorporation, and C-PRF metronidazole incorporation group. While the i-PRF group showed decreasing proliferation cell count. Conclusion: This study assumed that platelet concentrates (i-PRF and C-PRF) with metronidazole incorporation can increase the cell proliferation higher than i-PRF and C-PRF only.

The effect of compression to platelet rich fibrin towards fibroblast proliferation of periodontal ligament: An in vitro study

Objective: The aim of this study was to determine the effect of compression to PRF towards the proliferation of periodontal ligament fibroblasts.
 Material and Methods: In this study, periodontal ligament fibroblasts were divided into four groups: periodontal ligament fibroblasts, periodontal ligament fibroblasts with platelet rich fibrin non-compressed, periodontal ligament fibroblasts with platelet rich fibrin compressed, periodontal ligament fibroblasts with platelet rich fibrin releasate. Each group consisted of 6 samples. The examination of fibroblast proliferation was done using MTT Assay on day 3 and 7.
 Results: Results were analyzed using ANOVA test followed by post hoc test with at least significant difference. Based on the result, non-compressed PRF showed the highest fibroblast proliferation. It also showed a significant difference between groups of periodontal ligaments, non-compressed PRF and compressed-PRF. Meanwhile, groups of non-compressed-PRF and PRF-releasate showed insignificant difference.
 Conclusion: The conclusion of this study showed that non-compressed PRF and PRF releasate is more effective to increase the periodontal ligament proliferation.

Evaluation on the efficacy of processed hydrated and dehydrated amnion chorion membrane on the proliferation of periodontal ligament fibroblasts.

The purpose of the present study was to process and assess the effect of hydrated amnion chorion membrane and dehydrated amnion chorion membrane on proliferation of periodontal ligament(PDL) fibroblast cells. The amnion chorion membrane(ACM) from placenta of 18 systemically healthy patients was obtained from the Department of Obstetrics and Gynaecology. Theywere processed as hydrated and dehydrated based on different processing methods. ThePeriodontal ligament cells were obtainedfrom periodontal ligament offreshly extracted premolars of systemically healthy patients, due to orthodontic reasons. The PDL cells werefurthercultured in laboratory and were exposed to hydrated and dehydrated amnion chorion membrane.The MTT assay was performed to assess the proliferation of PDL fibroblast cells after 24and 48 h. The hydrated and dehydrated amnion chorion membrane showed proliferation of PDL fibroblasts after 24 and 48h. The proliferation of PDL fibroblasts in hydrated (p = 0.043) and dehydrated (p = 0.050) amnion chorion membrane was statistically significant at the end of 24 and 48h respectively.On inter-groupcomparison dehydrated ACM showed significant proliferation of PDL fibroblasts after 24 (p=0.014)and 48 h (p=0.019).Withinthe limits of the present study, it can be concluded: both hydrated and dehydrated amnion chorion membrane showed proliferationof PDL fibroblast cells. However, dehydrated ACM showed significant proliferation of PDL fibroblasts.

Resolvin D1 modulates periodontal ligament fibroblast function.

The resolution of inflammation is an active process mediated by specialized lipid mediators called lipoxins and resolvins. Periodontal ligament fibroblasts (PDLFs) play a significant role in periodontal regeneration. The purpose of the current study was to determine the impact of resolvin D1 (RvD1) on human PDLF cell wound healing and proliferation, receptor expression (G-protein-coupled receptor 32 [GPR32] and formyl peptidereceptor2 [ALX/FPR2]), and cytokine expression and release. PDLFs were stimulated with interleukin-1β (IL-1β) (500pg/ml) with and without RvD1 (100nM). RvD1 receptor expression was determined by quantitative real-time polymerase chain reaction (qPCR), immunofluorescence microscopy, and fluorescence-activated cell sorting. Wound closure was measured by a scratch assay, and proliferation was determined by bromodeoxyuridine incorporation. Interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1, cyclooxygenase-2, matrix metalloproteinases-1, -2, and -3 (MMP-1, -2, and -3), tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2), prostaglandin E2, and osteoprotegerin (OPG) gene expression and production were measured using qPCR and Western blotting, multiplex immunoassay, and enzyme-linked immunosorbent assay. PDLF expressed GPR32 and ALX/FPR2. RvD1 reversed IL-1β-induced inhibition of wound healing and proliferation of PDLF. IL-1β also induced the production of proinflammatory cytokines and MMPs. This effect was reversed by RvD1. RvD1 reversed IL-1β-induced inhibition of TIMP-1, TIMP-2, and OPG. The data suggested that RvD1 has a pro-wound healing, proliferative, and anti-inflammatory impact on the PDLF that favors periodontal regeneration.

Effect of autogenous growth factors released from platelet concentrates on the osteogenic differentiation of periodontal ligament fibroblasts: a comparative study.

BackgroundPlatelet concentrates have been used in tissue regeneration. The purpose of this study was to examine effects of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF) and concentrated growth factor (CGF) on the osteogenic differentiation of periodontal ligament fibroblasts (PDLFs).MethodsLeukocyte- and platelet-rich fibrins, CGFs and PDLFs were obtained from New Zealand rabbits. The release of basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP-2) and transforming growth factor β1 (TGF-β1) from L-PRFs and CGFs was measured at 5 h and 1, 3, 5, 7 days, using the enzyme linked immunosorbent assay. The PDLFs were treated with exudates of L-PRF or CGF. After the treatment, cell counting kit-8 assay was performed at day 1, 3, 5 and 7. Alkaline phosphatase (ALP) assay and Western blotting were applied at day 7. Three blocking antibodies were used to neutralize the proteins of bFGF, BMP-2 and TGF-β1.ResultsLeukocyte- and platelet-rich fibrin and CGF showed different growth factor release pattern, but similar accumulated concentration of these growth factors. PDLFs proliferation was significantly promoted by both L-PRF and CGF at day 1, 3 and 7, and CGF group was superior to L-PRF group at day 1 and 3. Both L-PRF and CGF significantly enhanced PDLFs ALP activity and protein expression of osteogenic markers. The osteopontin level was higher in CGF group than in L-PRF group, but no significant differences were found between two groups for ALP activity. Three blocking antibodies significantly downregulated both L-PRF and CGF induced osteogenic markers expression.ConclusionBoth CGF and L-PRF can promote the proliferation and osteogenic differentiation of PDLFs. The bFGF, BMP-2 and TGF-β1 are involved in both L-PRF and CGF induced osteogenic differentiation of PDLFs.

BioRoot RCS Extracts Modulate the Early Mechanisms of Periodontal Inflammation and Regeneration

IntroductionThe balance between periapical tissue inflammation and regeneration after the removal of necrotic/infected tissues is pivotal in determining the success of endodontic treatment. This study was designed to investigate the effect of silicate-based root canal sealer BioRoot RCS (BRCS; Septodont, Saint-Maur-des-Fossés, France) on modulating the inflammatory mechanisms and early steps of regeneration initiated by human periodontal ligament (PDL) fibroblasts. MethodsSamples of BRCS and Pulp Canal Sealer (PCS; SybronEndo, Orange, CA) were incubated in culture medium to obtain material extracts. To simulate bacterial infection and endodontic sealer use, PDL fibroblasts were stimulated with lipopolysaccharides and cultured with material extracts. The secretion of proinflammatory cytokine (interleukin 6) and growth factor (transforming growth factor beta 1) were quantified by enzyme-linked immunosorbent assay. Inflammatory cell recruitment sequence was investigated using a human inflammatory monocytic cell line (THP-1) that can be activated into macrophage-like cells. The adhesion of THP-1 to endothelial cells (human umbilical vein endothelial cells) was studied using fluorescent THP-1, their migration using Boyden chambers, and their activation into macrophage-like cells using a cell adhesion assay. The proliferation of PDL fibroblasts was quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas the migration of PDL stem cells was investigated using Boyden chambers after immunofluorescence and reverse transcription polymerase chain reaction characterization. ResultsInterleukin 6 secretion decreased with BRCS, whereas it increased with PCS. Transforming growth factor beta 1 secretion significantly increased only with BRCS. The material extracts did not affect THP-1 adhesion to human umbilical vein endothelial cells, but only BRCS inhibited their migration. Moreover, activation of THP-1 decreased with BRCS and to a lesser extent with PCS. Finally, BRCS increased PDL fibroblast proliferation without affecting PDL stem cell migration. By contrast, PCS decreased PDL fibroblast proliferation and PDL stem cell migration. ConclusionsThis work shows that the endodontic sealers modulate the PDL inflammatory and regeneration potentials in vitro. It demonstrates that BRCS has anti-inflammatory effects and the potential to promote tissue regeneration.

The Effects of human amniotic fluid on periodontal ligament fibroblast cell viability, proliferation, and cytokine/growth factor expression

Background: The importance of the amniotic fluid (AF) to the fetus is clear. However, very few studies have been published to examine the potential uses of this fluid in various areas such as tissue regeneration. AF contains epidermal growth factor, transforming growth factor-alpha, transforming growth factor beta-1, insulin-like growth factor-I, erythropoietin and granulocyte colony-stimulating factor, as well as hyaluronic acid and hyaluronic acid-stimulating factor. Previous studies suggest that AF can increase fibroblast proliferation and chemotaxis, and decrease apoptosis as well as promote wound healing. Furthermore, evidence showed that human AF inhibits hyaluronidase, elastase, and cathepsin. The current study examined the effects of human AF on periodontal ligament fibroblasts (PDLF) in terms of cell toxicity, cell proliferation, and cytokine/growth factor expression. Materials and Methods: Cytotoxicity of AF on PDLF was determined using lactate dehydrogenase assays. PDLF proliferation was determined using water-soluble tetrazolium-1 assays. Cytokine/growth factor expression was determined on AF-treated PDLF, AF alone, and PDLF alone utilizing protein arrays. Results: Human AF at 10% and below did not affect cell growth and was not toxic. AF-treated PDLF cells showed a decrease in cytokine/growth factor levels compared to the sum of cytokine/growth factor levels in AF only and cells only for 39 of the 80 proteins examined (48.8%). Of the 39 examined cytokines, 20 inflammatory cytokines, 11 cell cycle cytokines, 1 anti-inflammatory cytokine, and 7 other cytokines were decreased. Conclusion: Human AF at the examined concentrations was not toxic to PDLF cells and did not influence their proliferation. In addition, AF (10%) caused a decrease in the total protein levels of cytokines/growth factors expressed in 39 of the 80 proteins examined (48.8%). Of the 39 examined cytokines, 20 inflammatory cytokines, 11 cell cycle cytokines, 1 anti-inflammatory cytokine, and 7 other cytokines were decreased.

Correction to: human periodontal ligament fibroblasts stimulated by nanocrystalline hydroxyapatite paste or enamel matrix derivative. An in vitro assessment of PDL attachment, migration, and proliferation.

In the original published version of this article, the middle name for author Mirko H. H. Schmidt is missing.

Topical application of bFGF on acid-conditioned and non-conditioned dentin: effect on cell proliferation and gene expression in cells relevant for periodontal regeneration.

Periodontal regeneration is still a challenge in terms of predictability and magnitude of effect. In this study we assess the biological effects of combining chemical root conditioning and biological mediators on three relevant cell types for periodontal regeneration.Material and Methods:Bovine dentin slices were conditioned with 25% citric acid followed by topical application of basic fibroblast growth factor (bFGF, 10 and 50 ng). We used ELISA to assess the dynamics of bFGF release from the dentin surface and RT-qPCR to study the expression of Runx2, Col1a1, Bglap and fibronectin by periodontal ligament (PDL) fibroblasts, cementoblasts and bone marrow stromal cells (BMSC) grown onto these dentin slices. We also assessed the effects of topical application of bFGF on cell proliferation by quantification of genomic DNA.Results:Acid conditioning significantly increased the release of bFGF from dentin slices. Overall, bFGF application significantly (p<0.05) increased cell proliferation, except for BMSC grown on non-conditioned dentin slices. Dentin substrate discretely increased expression of Col1a1 in all cell types. Expression of Runx2, Col1a1 and Fn was either unaffected or inhibited by bFGF application in all cell types. We could not detect expression of the target genes on BMSC grown onto conditioned dentin.Conclusion:Acid conditioning of dentin improves the release of topically-applied bFGF. Topical application of bFGF had a stimulatory effect on proliferation of PDL fibroblasts, cementoblasts and BMSC, but did not affect expression of Runx2, Col1a1, Bglap and fibronectin by these cells.

Physical and Biological Properties of PCL/CS/nHA Composite Membranes for Guided Tissue Regeneration

With the aim to explore a barrier membrane with stable degradation rate and excellent biological properties for guided tissue regeneration (GTR), a series of PCL/ CS/nHA (100/0,95/5,90/10,85/15,80/20,w/w) composite membranes were composed via freeze drying. PCL/CS/nHA samples were divided into five groups by adjusting the PCL to CS ratio, and polyvinylpyrrolidone was used as porogen. The fabricated membranes were evaluated by mechanical properties, material degradation rate, morphological characterization and biological properties. Through these experiment, we can proved that for a PCL to CS ratio of 95/5 optimal conditions were as follows: porosity 31.25±2.35, water absorption 101.7, degradation 21.3%, tensile strength 19.2MPa, and elongation at break 218.6%. Moreover, in vitro cytologic research revealed that PCL/CS/nHA (95/5) composite membrane could efficiently increased the infiltration of periodontal ligament fibroblasts (PLFs). Finally, cell proliferation assay showed that PCL/CS/nHA (95/5) composite membrane could promote the differentiation and proliferation of PLFs. These results suggested that PCL/CS/nHA ((95/5) composite membrane could serve as a promising barrier membrane for guided tissue regeneration due to appropriate biodegradability, preferable mechanical properties and outstanding biocompatibility.

Role of Periostin in Adhesion and Migration of Bone Remodeling Cells.

Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology.

In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts

BackgroundFindings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts.MethodsHuman alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined.ResultsMore than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens.ConclusionsThe present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.

Interleukin‐17A Stimulates Migration of Periodontal Ligament Fibroblasts Via p38 MAPK/NF‐κB ‐Dependent MMP‐1 Expression

Interleukin-17 (IL-17) is a cytokine secreted predominantly by Th17 cells. Although IL-17 is primarily associated with the induction of tissue inflammation, the other biological functions of IL-17, including its wound-healing functions, have yet to be thoroughly explored. Fibroblast proliferation and migration play essential roles in periodontal wound-healing responses. In this study, we report that IL-17A can increase the migration and expression of matrix metalloproteinase (MMP)-1 in human periodontal ligament (PDL) fibroblasts but has no effect on PDL fibroblast proliferation. IL-17A-induced MMP-1 expression led to cell migration, which was attenuated by pre-treatment with IL-17 receptor neutralizing antibody and small interfering RNA (siRNA) for MMP-1. The IL-17A-induced cell migration was also attenuated by its tissue inhibitor of matrix metalloproteinase (TIMP)-1. In addition, a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) inhibited IL-17A-induced increase of the migration and MMP-1 upregulation of PDL fibroblasts. The involvement of p38 MAPK in IL-17A-induced MMP-1 expression and cell migration was further confirmed by transfection of p38α siRNA. A nuclear factor kappaB (NF-κB) inhibitor (pyrrolidine dithiocarbamate) also suppressed the cell migration and MMP-1 expression enhanced by IL-17A. Moreover, transfection with p38α siRNA inhibited IL-17A-induced NF-κB nuclear translocation as well as NF-κB binding activity. Our results suggest that IL-17A enhances the migration of PDL fibroblasts by increasing MMP-1 expression through the IL-17 receptor, p38 MAPK, and NF-κB signal transduction pathways.

Resolvin D1 protects periodontal ligament

Resolution agonists are endogenous mediators that drive inflammation to homeostasis. We earlier demonstrated in vivo activity of resolvins and lipoxins on regenerative periodontal wound healing. The goal of this study was to determine the impact of resolvin D1 (RvD1) on the function of human periodontal ligament (PDL) fibroblasts, which are critical for wound healing during regeneration of the soft and hard tissues around teeth. Primary cells were cultured from biopsies obtained from three individuals free of periodontal diseases. Peripheral blood mononuclear cells were isolated by density gradient centrifugation from whole blood of healthy volunteers. PGE2, leukotriene B4 (LTB4), and lipoxin A4 (LXA4) in culture supernatants were measured by ELISA. The direct impact of RvD1 on PDL fibroblast proliferation was measured and wound closure was analyzed in vitro using a fibroblast culture "scratch assay." PDL fibroblast function in response to RvD1 was further characterized by basic FGF production by ELISA. IL-1β and TNF-α enhanced the production of PGE2. Treatment of PDL cells and monocytes with 0.1-10 ng/ml RvD1 (0.27-27 M) reduced cytokine induced production of PGE2 and upregulated LXA4 production by both PDL cells and monocytes. RvD1 significantly enhanced PDL fibroblast proliferation and wound closure as well as basic FGF release. The results demonstrate that anti-inflammatory and proresolution actions of RvD1 with upregulation of arachidonic acid-derived endogenous resolution pathways (LXA4) and suggest resolution pathway synergy establishing a novel mechanism for the proresolution activity of the ω-3 docosahexaenoic acid-derived resolution agonist RvD1.

Human periodontal ligament fibroblast response to rhPDGF-BB application on periodontally diseased root surfaces-in vitro

The aim of the present study was to evaluate the influence of recombinant Platelet Derived Growth Factor-BB (rhPDGF-BB) on the cellular proliferation, morphology and adherence of human periodontal ligament fibroblasts (PLFs) to the root surfaces of periodontally diseased and healthy human teeth roots at two different time periods. Primary cell cultures of PLFs were obtained from clinically healthy premolar or mandibular third molar teeth. 11 scaffolds were prepared from healthy teeth for group-1 (Healthy Substrates), 33 scaffolds were prepared from periodontally diseased teeth, which were further divided in Group-2 – Periodontally diseased substrates, Group-3 – Scaled and Root planed (SRP) substrates, Group-4 – SRP + rhPDGF-BB (50 ng/ml). Groups were further subdivided into two groups (n = 5 scaffolds per subgroup) and PLFs were incubated on the scaffolds for three and seven days, topographical assessment was done on the remaining substrate. Cell morphology and counting was assessed under a scanning electron microscope at 350× on day three and seven and statistically compared with the Mann–Whitney U test and the Kruskal–Wallis test. On day three, Group 1 showed least number of cells attached, whereas maximum number of cells were attached on Group 3 (SRP only) substrates. For day 7, Group 1 and Group 4 showed increase in the number of cells from day 3 to 7, while number of cells attached/substrate reduced drastically for Group 2 and 3 substrates. Group 3 and 4 showed better adhesion and proliferation of PLFs as compared to Group 1 and 2. Group-1 and Group-4 showed predominantly spindle cells with flat appearance, Group-3 showed stellate cells and Group-2 showed predominantly distorted spindle shaped cells. The results of this in-vitro study indicates that rhPDGF-BB plays a significant role as an adjunct to periodontal therapy in influencing maturity, attachment and proliferation of PLFs.

BFGF-induced human periodontal ligament fibroblasts proliferation through T-type voltage-dependent calcium channels

Objective.To test the hypothesis that T-type voltage-dependent calcium channels (T-CaCNs) are involved in basic fibroblast growth factor (bFGF)-induced proliferation of human periodontal ligament fibroblasts (HPLFs). Materials and methods.This study examined the expression of the T-type calcium channel sub-units CaV 3.1, CaV 3.2 and CaV 3.3 in HPLFs by real-time PCR. Mibefradil, a T-CaCNs antagonist, was used to block the effect of T-CaCNs and the proliferation of HPLFs was evaluated by the water-soluble tetrazolium (WST) assay. The level of intracellular calcium was measured by laser confocal microscopy. Results.Expression of the three subunits of T-CaCNs in HPLFs was detected, which was strongly up-regulated upon stimulation by bFGF. The proliferation of HPLFs induced by bFGF was decreased significantly by treatment with Mibefradil. This effect was associated with the decreased expression of T-CaCNs and a decreased level of intracellular calcium. Conclusions.Expression of the T-CaCNs affected the proliferation of HPLFs that was induced by bFGF, indicating that T-CaCNs could be important in mediating periodontal ligament (PDL) remodeling.

Tensile force inhibits the proliferation of human periodontal ligament fibroblasts through Ras‐p38 MAPK up‐regulation

The capacity of human periodontal ligament fibroblasts (PLF) to proliferate in response to mechanical force plays a critical role in orthodontic tooth movement. Extensive research has not fully revealed the mechanisms by which the PLF respond to mechanical force. The responses to force differ according to the origin of cells and the type of stress applied. In this study, we examined the proliferative response of PLF to tensile force. We also explored cellular mechanisms involved in the mechanosignal transduction of tensile force. Application to the force to PLF with 1.5% elongation for 1 h inhibited cell proliferation. This was accompanied by reductions in several cyclins and cyclin-dependent kinases (CDKs) involved in the G(1)/S transition; however, p21 knockdown prevented these events. Pharmacological inhibitor of p38 MAPK suppressed the force-mediated growth inhibition as well as the decrease in p21 expression. Ras inhibitor almost completely blocked the tension-mediated increases in p-p38 MAPK and p-p21, and the attendant increase in PLF proliferation. These findings suggest that tension force activates Ras-p38 MAPK pathways in PLF, which up-regulate p21 and arrest cell cycle progression at the G(1) phase.

Human periodontal ligament fibroblasts stimulated by nanocrystalline hydroxyapatite paste or enamel matrix derivative. An in vitro assessment of PDL attachment, migration, and proliferation

We determined the effects of soluble or coated nanocrystalline hydroxyapatite paste (nano-HA) and enamel matrix derivative (EMD) on proliferation, adhesion, and migration of periodontal ligament fibroblasts (PDLs). Cultured PDLs were stimulated with nano-HA paste or EMD in a soluble form or were coated to the surface of cell culture dishes. Proliferation of PDLs on coated nano-HA and EMD was quantified by various methods including bromodeoxyuridine (BrdU) incorporation and Western blot. Cell migration was investigated in a modified Boyden chamber. The surface integrin profile of PDLs was determined using an integrin-specific ELISA, and integrin-specific signaling was measured by immunoblotting of phosphorylated focal adhesion kinase (FAK). Coated nano-HA stimulated PDL proliferation to a larger extent as compared with coated EMD. PDL migration towards a nano-HA or EMD gradient was more efficiently mediated by soluble EMD as compared with nano-HA but vice versa, adhesion of PDLs to compound-coated dishes was more effectively mediated by nano-HA as compared with EMD. Mechanistically, majorly integrin α5β1-mediated adhesion of PDL and both coated compounds mediated a significant increase in FAK activation though to a different extent. Current findings offer two different modes of action for EMD and nano-HA paste. EMD efficiently acts as a chemoattractant in its soluble form, while nano-HA paste effectively serves as a synthetic extracellular matrix component in its coated form. Our findings suggest that EMD and nano-HA paste display different molecular characteristics and apply alternative routes to mediate their beneficial effects on periodontal tissues.

Gene expression pattern during osteogenic differentiation of human periodontal ligament cells in vitro

PurposePeriodontal ligament (PDL) cell differentiation into osteoblasts is important in bone formation. Bone formation is a complex biological process and involves several tightly regulated gene expression patterns of bone-related proteins. The expression patterns of bone related proteins are regulated in a temporal manner both in vivo and in vitro. The aim of this study was to observe the gene expression profile in PDL cell proliferation, differentiation, and mineralization in vitro.MethodsPDL cells were grown until confluence, which were then designated as day 0, and nodule formation was induced by the addition of 50 µg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone to the medium. The dishes were stained with Alizarin Red S on days 1, 7, 14, and 21. Real-time polymerase chain reaction was performed for the detection of various genes on days 0, 1, 7, 14, and 21.ResultsOn day 0 with a confluent monolayer, in the active proliferative stage, c-myc gene expression was observed at its maximal level. On day 7 with a multilayer, alkaline phosphatase, bone morphogenetic protein (BMP)-2, and BMP-4 gene expression had increased and this was followed by maximal expression of osteocalcin on day 14 with the initiation of nodule mineralization. In relationship to apoptosis, c-fos gene expression peaked on day 21 and was characterized by the post-mineralization stage. Here, various genes were regulated in a temporal manner during PDL fibroblast proliferation, extracellular matrix maturation, and mineralization. The gene expression pattern was similar.ConclusionsWe can speculate that the gene expression pattern occurs during PDL cell proliferation, differentiation, and mineralization. On the basis of these results, it might be possible to understand the various factors that influence PDL cell proliferation, extracellular matrix maturation, and mineralization with regard to gene expression patterns.

Functionalized scaffolds of shorter self-assembling peptides containing MMP-2 cleavable motif promote fibroblast proliferation and significantly accelerate 3-D cell migration independent of scaffold stiffness

The functionalization of self-assembling peptide scaffolds with biologically active motifs presents many possibilities for the design of synthetic biological materials for 3D cell culture, reparative, regenerative medicine and tissue engineering. We here report the development of short self-assembling peptide scaffolds designed especially for cell migration. We designed self-assembling peptide scaffolds VEVK9 (Ac-VEVKVEVKV-CONH2) and VEVK12 (Ac-VEVKVEVKVEVK-CONH2) and modified variants of these peptides directly coupled to short biologically active motifs. These motifs include two-unit RGD binding units (PRGDSGYRGDS), cell adhesion sequences of laminin (YIGSR and IKVAV) and a Matrix metalloproteinase-2 (MMP-2) cleavable sequence (PVGLIG). We show here that the peptide scaffolds functionalized with these cell adhesion motifs significantly promoted proliferation of periodontal ligament fibroblasts. Moreover, when combined with the MMP-2 cleavable motifs, the peptide scaffolds significantly accelerated the fibroblasts' migration independent of scaffold stiffness. Our results suggest that the interaction between integrin receptors of fibroblasts and cell adhesion motifs of the scaffolds stimulated MMP-2 production from the fibroblasts to accelerate proteolytic cell migration in the similar manner as natural extracellular matrix. These peptide scaffolds may be useful in tissue engineering and regenerative medicine including periodontal tissue reconstruction.