Periodontal Ligament Fibroblast Cells Research Articles

Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

Fluorosed fibroblast attachment on fluorosed and nonfluorosed teeth after SRP and EDTA root biomodification.

Background:Fluorosis causes mineralization changes in the tooth and can lead to morphologic alterations of fibroblasts. To understand the effect of fluorosis on periodontal healing, the initial step during healing, such as fibroblast attachment to the root surface, needs to be evaluated. Hence, the objective of the present study was to study the attachment of fluorosed fibroblasts on the fluorosed and nonfluorosed root fragments.Materials and Methods:A total of 56 fluorosed and nonfluorosed, periodontally healthy and diseased tooth roots were obtained and allotted to eight groups: Fluorosed and nonfluorosed healthy controls (FH and NFH, respectively), fluorosed and nonfluorosed diseased controls (FD and NFD, respectively), fluorosed and nonfluorosed roots treated with scaling and root planing (FD + S and NFD + S, respectively), and similar groups treated with scaling and root planing and 24% ethylenediaminetetraacetic acid (EDTA) gel application for 2 min (FD + SE and NFD + SE, respectively). After the respective treatment, the root fragments were incubated in the human periodontal ligament fibroblast cells obtained and cultured from freshly extracted healthy human fluorosed premolar tooth root.Results:In the nonfluorosed roots category, greater attachment was found in the untreated nonfluorosed diseased (P = 0.036) and SRP-treated nonfluorosed diseased groups (P = 0.008) as compared to the nonfluorosed healthy group. While in the fluorosed roots category, no significant difference was observed in FL-FA (P > 0.05) within the group. However, no attachment was observed in EDTA-treated fluorosed root fragments. When fluorosed groups were compared to nonfluorosed groups, no significant changes were noted between the groups.Conclusion:SRP proves to be a standard requirement for fibroblast attachment to occur both in fluorosed and nonfluorosed roots. Although there was no significant difference in attachment between SRP and SRP + EDTA among fluorosed roots, EDTA does not seem to be a promising agent for root biomodification in fluorosed roots in a given concentration and time of treatment.

Comparative assessment of fluorosed and nonfluorosed fibroblast attachment on fluorosed and nonfluorosed teeth after scaling and root planning and ethylenediaminetetraacetic acid root biomodification

Background and Objectives: Fluorosis causes mineralization changes in the tooth and can lead to morphological alterations of fibroblasts. To evaluate the effect of fluorosis on periodontal healing, the initial step while healing such as, fibroblast attachment onto the root surface requires to be evaluated on the fluorosed and nonfluorosed tooth using nonfluorosed as well as fluorosed fibroblasts originated from the subjects influenced by high-water fluoride. Hence, the objective of the current study was to study and compare the attachment of nonfluorosed and fluorosed fibroblasts on the fluorosed and nonfluorosed root fragments. Materials and Methods: A total of 112 fluorosed and nonfluorosed, periodontally healthy and diseased tooth roots were obtained and allotted to eight groups : f0 luorosed healthy (FH) and non-FH (NFH) controls, fluorosed diseased (FD) and non-FD (NFD) controls, fluorosed and nonfluorosed teeth treated with scaling and root planning (SRP) (FD SRP and NFD SRP) and similar groups treated with SRP and ethylenediaminetetraacetic acid (EDTA) (FD SRP + EDTA and NFD SRP + EDTA) burnishing treatment with 24% EDTA gel for 2 min. After the respective treatment half of the root fragments in each group were incubated in the human periodontal ligament fibroblast cells obtained and cultured from freshly extracted FH and NFH human premolar tooth root. The nonfluorosed fibroblasts are elongated, flat cells thus they show increased attachment to root the surface. Results: When comparison was carried out between the attachment of fluorosed and nonfluorosed fibroblasts on NFD groups treated with scaling and EDTA, significant results were obtained with increased attachment seen on the group incubated with nonfluorosed fibroblasts (P = 0.029). While on comparison between the attachment of fluorosed and nonfluorosed fibroblasts on NFH group, NFD group treated with SRP and NFD group, no significant results were obtained (P > 0.05). On comparison between the attachment of fluorosed and nonfluorosed fibroblasts on FD group treated with SRP, highly significant results were obtained with increased attachment seen in the group incubated with nonfluorosed fibroblasts (P = 0.001). While the comparison of attachment of fluorosed and nonfluorosed fibroblasts on FH group, FD group treated with SRP + EDTA and FD group revealed no significant results (P > 0.05). Interpretation and Conclusion: SRP proves yet to be a standard requirement for fibroblast attachment to occur both in fluorosed and nonfluorosed teeth. Although, there is no significant difference in attachment between SRP and SRP + EDTA among fluorosed teeth, EDTA does not seem to be a promising agent for root biomodification in fluorosed teeth in given concentration and time of treatment.

Enhanced periodontal tissue regeneration by periodontal cell implantation

Due to a lack of regenerative potential, current treatments for periodontal defects do not always provide satisfactory clinical results. Previously, the implantation of a biomaterial scaffold-cell construct has been suggested as a clinically achievable approach. In this study, it was aimed to investigate the contribution of implanted periodontal ligament (PDL) cells to periodontal tissue regeneration. Gelatin sponges were seeded with green fluorescent protein (GFP) transfected PDL or gingival fibroblasts (GF) cells, and implanted into a surgically created rat intrabony periodontal defect model. After six weeks, decalcified maxillae were used for histomorphometrical and immunohistochemical analyses. After six weeks, animals that had received the PDL cells exhibited significantly more functional bone and ligament. Furthermore, there were remarkable differences in the distribution of the transplanted cells. Periodontal ligament cells were always located directly lining the newly regenerated areas. In contrast, GF cells dispersed over the whole defect area, and did not provide a favourable effect on the regeneration of the periodontal tissues. We concluded that PDL cells transplanted into a periodontal defect survive and favour regeneration of periodontium, possibly in a paracrine manner.

Effect of Incorporation of Nanoscale Bioactive Glass and Hydroxyapatite in PCL/Chitosan Nanofibers for Bone and Periodontal Tissue Engineering

A biomimetic scaffold which can very closely mimic the extracellular matrix of the bone was fabricated by incorporating nano-bioceramic particles such as nano bioglass (nBG) and nano hydroxyapatite (nHAp) within electrospun nanofibrous scaffold. A comparative study between nHAp incorporated poly(caprolactone) (PCL)-chitosan (CS) and nBG incorporated PCL-CS nanofibrous scaffolds was carried out and their feasibility in tissue engineering was investigated. All the samples were optimized to obtain fibers of similar diameter from 100-200 nm for the ease of comparison between the samples. Protein adsorption studies showed that PCL-CS incorporated with 3 wt% nHAp and 3 wt% nBG adsorbed more proteins on their surface than other samples. Cell attachment and proliferation studies using human periodontal ligament fibroblast cells (hPLFs) and osteoblast like cells (MG-63 cell lines) showed that nBG incorporated samples are slightly superior to nHAp incorporated counterparts. Cell viability test using alamar blue assay and live/dead staining confirms that the scaffolds are cytocompatible. ALP activity confirmed the osteoblastic behavior of hPDLFs. Also the presence of nHAp and nBG enhanced the ALP activity of hPDLF on the PCH3 and PCB3 scaffolds. These studies indicate that nBG incorporated electrospun scaffolds are comparatively better candidates for orthopedic and periodontal tissue engineering applications.

Increased PELP1 expression in rat periodontal ligament tissue in response to estrogens treatment

Estrogens and their receptors are important factors involved in periodontal ligament (PDL) tissue health. As a regulator of estrogen receptors (ER), the proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) may play a role in alveolar bone formation and PDL homeostasis. The aim of the present study was to observe PELP1 expression in rat PDL tissue during estrogen levels manipulations. Twenty-one 8-week old normal female Sprague-Dawley rats were randomly divided into three equal groups: sham-operated controls, ovariectomized (OVX) group, and OVX given 17β-estradiol intraperitoneally (OVX+E2) for 16weeks. PELP1 expression was down-regulated in the OVX group and was up-regulated in the OVX+E2 group. Periodontal ligament fibroblast cells (PDLFCs) were isolated from PDL tissue, and characterized by immunohistochemical staining. Estradiol treatment of PDLFCs induced PELP1 protein level compared to untreated cells. PELP1 mRNA expression in estradiol-treated cells was relatively low at the beginning of treatment and then steadily increased from hour 4. In conclusion, results indicate that PELP1 is expressed in rat PDL tissue and PDLFCs, and that its expression is up-regulated during estrogen treatment.

Cytotoxic evaluation of two chlorine-releasing irrigating solutions on cultured human periodontal ligament fibroblasts

Objective: Endodontic irrigants solutions with anti-bacterial activity have been used in treatment of teeth with infected root canals; however, these solutions can irritate periapical tissues. The objective of this study was to evaluate the cytotoxicity of two chlorine-releasing irrigating solutions on cultured human periodontal ligament fibroblasts. Materials and Methods: Periodontal ligament fibroblasts cells were cultured from orthodontically extracted premolars. The passage number between 3-6 was taken for further experiments. 2.5% sodium hypochlorite and 0.6% sodium dichloroisocyanuarate are the irrigating solution used for testing their cytotoxicity. The viability of cells after treating with irrigating solutions was evaluated by trypan blue dye exclusion. Results: Results showed that 0.6% NaDCC solution was less cytotoxic on cultured human periodontal ligament fibroblasts compared with 2.5% NaOCl at 2 min, 30 min, and 1 hr. Conclusion: This study suggested that sodium dichloroisocyanuarate is less cytotoxic than 2.5% NaOCl, suggesting its potential for use as root canal irrigant.

Transplanted Bone Marrow-Derived Cell Migration Into Periodontal Tissues Induced by Orthodontic Mechanical Stress

Aim: Bone marrow-derived cells have abilities of cell migration and differentiation into teeth and related tissues/organs, especially into periodontal ligament fibroblast cells (Tsujigiwa T. et al. J Hard Tissue Biol 20: 147-52, 2011; Muraoka R. et al. J Hard Tissue Biol 20: 301-6, 2011). In this examination using a bone marrow transplantation model, we examined the effect of orthodontic mechanical stress to the transplanted bone marrow-derived cell migration into periodontal tissues. Methods: Bone marrow derived cells from green fluorescence protein (GFP) transgenic mice were transplanted into 8 week-old female C57BL/6 immunocompromised recipient mice (n=10), which had undergone 10 Gy of lethal whole-body irradiation. After successful transplantation (about one month period), 5 mice received orthodontic mechanical stress using the Waldo method 5 times in 5 weeks; and 5 mice were compared as control without receiving orthodontic mechanical stress (Waldo CM. J Dent Res 32: 690-1, 1953). After that, the regional tissues were removed and fixed in formalin fixative. Paraffin-embedded sections were immunohistochemically analyzed using a Dako Envision + Kit-K4006 (Dako, Glostrup, Denmark) and a primary anti-GFP-polyclonal rabbit antibody (#598; 1/500; MBL, Nagoya, Japan). For semiquantitative evaluation of immunohistochemical staining, the following procedures were performed. First immunohistochemical images with same magnification from the periodontal tissues were prepared and pixel density was counted for each image. Then typical immunohistochemically positive staining part was defined as positive area. The pixel number of positive area in the periodontal tissue was compared with the previously calculated total pixel number of the periodontal tissue and the ratio was obtained. Results and discussion: We examined the transplanted bone marrow-derived cell migration into periodontal tissues. The immunohistochemistry revealed that GFP-positive cells were detected in the periodontal tissues, both in the experimental and control specimens. The GFP-positive cells histopathologically differentiated into some cell types. The fluorescence IHC and TRAP staining techniques demonstrated these cells were detected as osteoclasts and macrophages. Furthermore, GFP-positive cells gathered adjacent blood vessels. The data suggest that GFP-positive bone marrow-derived cell migrate into periodontal tissues and differentiate periodontal tissue component-cells. In the experimental group, there were numerous GFP-positive cells appearing in the experimental periodontal tissues which received intermittent stimulation of orthodontic mechanical stress, but there were few GFP-positive cells in the control specimens. As a results of the examination group specimens and control group, the ratio of pixel number in the examination group showed 5.77 ± 3.24 % (mean ± SD); and that in the control group, 0.71±0.45 % (mean ± SD). The examination group was significantly greater than that of control group (Mann-Whitney U test: p< 0.001). Thus, these data indicated that orthodontic mechanical stress acts as a possible promoting factor of transplanted bone marrow-derived cell migration into periodontal tissues, and of differentiation to fibroblasts. Conclusions: These results suggest that orthodontic mechanical stress induces transplanted bone marrow-derived cell migration and differentiation into periodontal tissue.

1-(3-Aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1-ones: A novel group of tumour-selective cytotoxins

Two series of 1-(3-aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1-ones, designed as novel cytotoxins, were synthesized. The compounds had low CC50 values in the micromolar range against HL-60 promyelocytic leukemic cells and HSC-2, HSC-3 and HSC-4 oral squamous cell carcinomas. The CC50 values of these compounds were higher towards non-malignant HGF (gingival fibroblasts), HPC (pulp cells), and HPLF (periodontal ligament fibroblasts) cells, which reveals the tumour-selectivity of these enones. A representative compound 4c caused cleavage of PARP1 in HSC-2 cells but not in HGF cells, which may be a contributing factor to the tumour-selectivity.

Strontium‐Substituted Calcium Deficient Hydroxyapatite Nanoparticles: Synthesis, Characterization, and Antibacterial Properties

Strontium‐substituted apatites have provoked increased interest in recent years for their beneficial effects on osteoporotic bone treatment and replacement. In this study, rod‐ and acicular‐shaped, strontium‐substituted calcium deficient hydroxyapatite (CDHA) nanoparticles with (Ca + Sr)/P ratio of 1.61 were synthesized via accelerated microwave processing. The X‐ray powder diffraction analysis indicates the synthesized nanoparticles as apatite phase with diffraction patterns similar to those of hydroxyapatite. The hydrodynamic diameter of the particles were observed to be ~200–500 nm and found to increase with strontium substitution along with an increase in the negative zeta potential by dynamic light scattering method, suggesting the particles to be agglomerates in water. The morphology of the nanoparticles was studied using transmission electron microscopy (TEM), where, pure CDHA showed globular and strontium substituted CDHAs showed rod and acicular shape for 5% and 10% Sr substitution, respectively. The average size of the particles in TEM was measured to be 33 nm × 5 nm, 40 nm × 6 nm, and 55 nm × 8 nm (L × W) for pure and strontium‐substituted CDHAs, respectively. Inductively coupled plasma spectroscopy, energy dispersive X‐ray analysis, and Fourier transform infrared spectroscopy further confirm the substitution of strontium and deficiency of calcium in the synthesized nanoparticles. Thermal stability and in vitro solubility of CDHA nanoparticles were observed to increase with strontium substitution. The MTT [3‐(4, 5‐Dimethylthiazole‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide] assay indicate that the substituted nanoparticles are non‐toxic to human periodontal ligament fibroblast (HPDLF) cells. Cell uptake study by fluorescence microscopy using rhodamine‐123 and actin/DAPI stained HPDLF cells show cellular localization of the nCDHA, nSr5CDHA, nSr10CDHA nanoparticles without any adverse effects. The strontium‐substituted CDHAs showed significant antimicrobial activity against Escherichia coli and Staphylococcus aureus bacteria by colony count method. The 10% Sr substituted CDHA show the maximum microbial reduction of around 56% for E. coli and 35% for S. aureus with 1 × 105 cells/mL of respective bacterial culture.

Sol–gel based fabrication and characterization of new bioactive glass–ceramic composites for dental applications

Abstract The fabrication of a new composite glass–ceramic with potential application in dental restoration was investigated. The developed material aims to modify the surface of dental ceramics creating bioactive surfaces able to improve material–cell interaction enhancing the bonding of the marginal gap between restoration and tooth. The application of the sol–gel method led to a microporous homogeneous glass–ceramic which can be applied as coating on commercial dental ceramic substrates. The microstructural, thermal, mechanical and biological properties of the fabricated coatings were studied and compared to the respective results of a previously investigated glass–ceramic composite. The material–cell interaction on these two sol–gel dental composites was studied in detail. The attachment and proliferation of both periodontal ligament and gingival fibroblast cells confirmed the bioactive behavior of the new materials and their ability to be potentially applied in dental restorations for soft tissue regeneration and sealing of the marginal gap.

Attachment and Proliferation of Cultured Human Periodontal Ligament Cells on 75:25 Poly (DL-Lactide-&lt;i&gt;co-&lt;/i&gt;ε-Caprolactone) Electrospun Resorbable Membrane

75:25 poly (DL-lactide-co-ε-caprolactone) electrospun resorbable membranes were prepared by electrospinning and film casting techniques. Primary cultures of human periodontal ligament (HPDL) fibroblasts cells were seeded on the electrospun membranes, film-cast membranes, and control glass cover slips. The number of cell attached was determined at 1, 3, 24 and 72 h after cell seeding. Cell proliferation on the membranes was determined by MTT assay at 1.5 h and 1, 3, 5 and 7 days after cell seeding. The morphology of cells was also determined by SEM. The results indicated that the cell attached number and cell proliferation on the electrospun membranes were significantly higher than that of the film-cast membranes at every time point. From an SEM study, HPDL cells could healthy attach on both, the electrospun membranes and the film-cast membranes. HPDL cells were firmly attached to the membrane matrix in the electrospun membrane group. In the film-cast group, HPDL were loosely attached to the membrane surfaces. In conclusion, an electrospun membrane had better ability to promote HPDL cell attachment and proliferation than did film-cast membranes.

Hard Tissue Formation by Human Periodontal Ligament Fibroblast Cells Treated with an Emdogain^|^reg;-Derived Oligopeptide in vitro

Hard Tissue Formation by Human Periodontal Ligament Fibroblast Cells Treated with an Emdogain^|^reg;-Derived Oligopeptide in vitro

In vitro analysis of the cytotoxicity and the antimicrobial effect of four endodontic sealers

IntroductionThe aim of this study was to investigate in vitro the cytotoxicity and antibacterial properties of four different endodontic sealers using human periodontal ligament fibroblast cell proliferation and visual analysis of growth inhibition.MethodsA silicone (GuttaFlow), silicate (EndoSequence BC), zinc oxide eugenol (Pulp Canal Sealer EWT) and epoxy resin (AH Plus Jet) based sealer were incubated with PDL fibroblasts (104 cells/ml, n = 6) up to 96 h. Cell proliferation (RFU) was determined by means of the Alamar Blue assay. Cell growth and morphology was visualized by means of fluorescent dyes. Possible antibacterial properties of the different sealers were visualized by means of SEM (Enterococcus faecalis; Parvimonas micra).ResultsFibroblast proliferation depended on sealer and cultivation time. After 72 and 96 h GuttaFlow and EndoSequence BC showed relatively non-cytotoxic reactions, while Pulp Canal Sealer EWT and AH Plus Jet caused a significant decrease of cell proliferation (p < 0.001). Visualization of cell growth and morphology with various fluorescent dyes supplemented the results. No antibacterial effect of EndoSequence BC to P. micra was found, whereas GuttaFlow showed a weak, Pulp Canal Sealer EWT and AH Plus Jet extensive growth inhibition. Also, no antibacterial effect of GuttaFlow, EndoSequence BC or AH Plus Jet to E. faecalis could be detected.ConclusionsThese in vitro findings reveal that GuttaFlow and EndoSequence BC can be considered as biocompatible sealing materials. However, prior to their clinical employment, studies regarding their sealing properties also need to be considered.

Biocompatible alginate/nano bioactive glass ceramic composite scaffolds for periodontal tissue regeneration.

Periodontal regeneration is of utmost importance in the field of dentistry which essentially reconstitutes and replaces the lost tooth supporting structures. For this purpose, nano bioactive glass ceramic particle (nBGC) incorporated alginate composite scaffold was fabricated and characterized using SEM, EDAX, AFM, FTIR, XRD and other methods. The swelling ability, in vitro degradation, biomineralization and cytocompatibility of the scaffold were also evaluated. The results indicated reduced swelling and degradation and enhanced biomineralization and protein adsorption. In addition, the human periodontal ligament fibroblast (hPDLF) and osteosarcoma (MG-63) cells were viable, adhered and proliferated well on the alginate/bioglass composite scaffolds in comparison to the control alginate scaffolds. The presence of nBGC enhanced the alkaline phosphatase (ALP) activity of the hPDLF cells cultured on the composite scaffolds. Thus results suggest that these biocompatible composite scaffolds can be useful for periodontal tissue regeneration.

Evaluation of biomimetic scaffold of gelatin–hydroxyapatite crosslink as a novel scaffold for tissue engineering: Biocompatibility evaluation with human PDL fibroblasts, human mesenchymal stromal cells, and primary bone cells

Biomimetic gelatin (gel)-hydroxyapatite (HA) composites have been prepared for studying hard tissue engineering scaffolds. However, the biocompatibility test of this form of material using these three cell types, which are periodontal ligament (PDL) fibroblast cells, human mesenchymal stromal cells (HMSc) and primary cells from human hip bone (HBc) has never been evaluated. The objective of this article is to prepare and evaluate the biocompatibility of gel-HA crosslinked scaffold for tissue engineering. Two different scaffolds were prepared: preparation (1), 2.5% gel/2.5% HA; preparation (2), 2.5% gel/5% HA. Three cell types including PDL, HMSc, and HBc were used. Assessment of biocompatibility and osteoblastic cellular responses was evaluated using a three-dimensional cell culture method and scanning electron microscopy (SEM). From SEM, it was observed that scaffold (1) exhibits stable porous formation with well-blended and dispersed HA powder. All three cell types were able to proliferate in both scaffolds. The HMSc and HBc got attached to the scaffolds to a significantly higher degree and subsequently proliferated more than PDL. The alkaline phosphatase (ALP) activities of HMSc and HBc were stronger when cultured in scaffold (S1) than (S2). It was seen that the two scaffold preparations show good biocompatibility with all three cell types tested. The better cellular responses with scaffold (S1) than (S2) might be due to the different structural and morphological characteristics, that is, scaffold (S1) retained more small-sized apatite crystals and a better developed pore configuration than scaffold (S2). Based on these findings, the biomimetically synthesized composite scaffolds have the potential to be used in hard tissue regeneration and tissue engineering fields.

The Roles of Angiotensin II in Stretched Periodontal Ligament Cells

The loading caused by occlusion and mastication plays an important role in maintaining periodontal ligament (PDL) tissues. We hypothesized that a loading magnitude would be involved in the production of biological factors that function in the maintenance of PDL tissues. Here, we identified up-regulated gene expressions of transforming growth factor-β1 (TGF-β1), alkaline phosphatase (ALP), and angiotensinogen in human PDL fibroblastic cells (HPLFs) that were exposed to 8% stretch loading. Immunolocalization of angiotensin I/II (Ang I/II), which was converted from angiotensinogen, was detected in rat PDL tissues. HPLFs that were stimulated by Ang II also increased their gene expressions of TGF-β1 and ALP. Furthermore, the antagonist for Ang II type 2 receptor, rather than for type 1, significantly inhibited gene expressions induced by the stretch loading. Analysis of these data suggests that Ang II mediates the loading signal in stretched HPLFs to induce expressions of TGF-β1 and ALP.

Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer

Because periodontal ligament (PDL) cells are reported to contain progenitor or stem cell populations, they are considered a beneficial cell source for clinical periodontal regeneration. Both bone morphogenetic protein 4 (BMP4) and human telomerase reverse transcriptase (hTERT) have essential roles in the modulation of stem cell properties. In this study we report for the first time that the combined ectopic expression of BMP4 and hTERT significantly enhanced the multipotent differentiation efficiency and capacity of human PDL fibroblasts (PFs), as shown by osteogenic, adipogenic and neurogenic differentiation in vitro, and cementum/PDL-like tissue regeneration in vivo. These findings may be attributed, at least in part, to the original upregulation of important stem cell markers, such as scleraxis, Stro-1 and CD146, and the extremely lowered threshold for BMP concentration to activate BMP signaling by enhanced basal phosphorylation levels of Smad 1/5/8. In addition, the significantly reduced expression levels of CD146 and CD90 with the presence of Noggin confirms the direct effect of BMP4 on the stem cell-like phenotype of genetically modified PF cells (BT-PFs). Furthermore, BT-PFs exhibited a high neural differentiation capacity (>75%). After transplantation into NOD/SCID mice, genetically modified-PFs generated cementum/PDL-like structures on the surface of the carrier. The multipotency of these modified cells potentially provides an attractive source of stem cells for therapeutic purposes and regenerative medicine.

Human PDL Fibroblasts Proliferation in Scaffolds on Bioactive Glass Modified Ceramics

Scaffolds are commonly used as cell’s vehicles in order to promote cell development. The aim of this study was to investigate the proliferation of human periodontal ligament fibroblasts (PDLF) in composite ceramic scaffolds. Chitin (CHN), chitosan (CHS) and chitosangelatin (CH-G) scaffolds were fabricated on the surface of ceramic disks, coated with a mixture of a bioactive glass modified ceramic. Cell proliferation was evaluated with the MTS method. All composite ceramic scaffolds were successfully loaded with PDLF cells. However, only in CH-G composite ceramicscaffolds cell population progressively increased until day 7 and presented a sudden drop at day 10 before being stabilized for the rest of the testing period. In the case of CHN and CHS the cell number was progressively decreasing. The successful observed cellular response in CH-G composite ceramic scaffolds could support the development of a protocol for tissue engineering on the materials used.

Synergic induction of human periodontal ligament fibroblast cell death by nitric oxide and N-methyl-D-aspartic acid receptor antagonist

PurposeNitric oxide (NO) has been known as an important regulator of osteoblasts and periodontal ligament cell activity. This study was performed to investigate the relationship between NO-mediated cell death of human periodontal ligament fibroblasts (PDLFs) and N-methyl-D-aspartic acid (NMDA) receptor antagonist (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801).MethodsHuman PDLFs were treated with various concentrations (0 to 4 mM) of sodium nitroprusside (SNP) with or without 200 µM MK801 in culture media for 16 hours and the cell medium was then removed and replaced by fresh medium containing MTS reagent for cell proliferation assay. Western blot analysis was performed to investigate the effects of SNP on the expression of Bax, cytochrome c, and caspase-3 proteins. The differences for each value among the sample groups were compared using analysis of variance with 95% confidence intervals.ResultsIn the case of SNP treatment, as a NO donor, cell viability was significantly decreased in a concentration-dependent manner. In addition, a synergistic effect was shown when both SNP and NMDA receptor antagonist was added to the medium. SNP treated PDLFs exhibited a round shape in culture conditions and were dramatically reduced in cell number. SNP treatment also increased levels of apoptotic marker protein, such as Bax and cytochrome c, and reduced caspase-3 in PDLFs. Mitogen-activated protein kinase signaling was activated by treatment of SNP and NMDA receptor antagonist.ConclusionsThese results suggest that excessive production of NO may induce apoptosis and that NMDA receptor may modulate NO-induced apoptosis in PDLFs.