Periodontal Ligament Cell Cultures Research Articles

Transcriptome profiles associated with human periodontal ligament differentiation

ObjectivesTissue differentiation is regulated by transcription factors. This study aimed to identify candidate transcription factors that induce periodontal ligament (PDL) cell differentiation in human pluripotent stem cells (hPSCs). MethodsHuman PDL tissues were scraped from the root surfaces of extracted teeth for orthodontic treatment and cultured using the explant culture method. We used RNA-seq to generate gene expression profiles of third-passage PDL cells and compared them with those of undifferentiated human induced pluripotent stem cells (hiPSCs) and human embryonic stem cell (hESC)-derived neural crest (NC) cells (publicly available data). ResultsPrimary cultured PDL cells exhibited a spindle-shaped fibroblast-like appearance and the gene expression of several PDL cell-specific markers. The gene expression profiles of PDL cells were relatively similar to those of hESC-derived NC cells but not those of undifferentiated hiPSCs. Thirty-seven transcription factors were identified as upregulated genes in PDL cells. Pathway analysis showed that differentially expressed genes were enriched in several functional groups and pathways, including the SMAD 2/3 nuclear pathway. ConclusionsWe identified 37 upregulated transcription genes in primary cultured PDL cells compared with hESC-derived NC cells. Regulating these genes and the SMAD signaling pathway may be promising ways to induce PDL cells from hPSC-derived NC cells.

The different irradiation parameters of carbon dioxide laser effects on periodontal ligament cells

Background/purpose: Photobiostimulation (PBS) can affect cellular functions. The objective of the present study was to evaluate the cellular changes in periodontal ligament (PDL) cells that received different carbon dioxide (CO2) laser irradiation parameters under negative pressure culture. Materials and methodsThe negative pressure-cultured PDL cells on normal medium and differentiation medium were subjected to continuous irradiation with a CO2 laser at an energy density of 5 J/cm2 or 10 J/cm2. The irradiated PDL cells were harvested at Days 1, 5 and 7, and their viability was analyzed by the Presto Blue assay and the biologic markers alkaline phosphatase (ALP), bone sialopoietin (BSP), osteopontin (OPN), osteocalcin (OC), matrix metalloproteinase-3 (MMP-3) collagen I (Col I) and cyclooxygenase-2 (COX-2) expression by reverse transcription-polymerase chain reaction (RT–PCR). ResultsThe PDL cell viability showed that the differentiation medium groups were higher than the normal culture groups. The cell morphologies were all expressed as spindle type. The inflammatory markers in the laser-irradiated groups were higher on the first day and decreased on the seventh day (P < 0.05). Osteogenesis markers were highly expressed at different time periods (P < 0.05). The Col I and OPN genes were highly expressed on the first day, and the Col I high expression lasted until the seventh day. The OC gene was highly expressed on the seventh day. The effects of PDL cultured in differential medium and normal medium were the same in the present study. ConclusionA low-dose CO2 laser continuously irradiating cultured PDL cells can induce osteogenesis and reduce cell inflammatory expression.

Regulation of gingival fibroblast phenotype by periodontal ligament cells in vitro.

ObjectivesStem cell transplantation has shown modest effects on periodontal tissue regeneration, and it is still unclear how regenerative effects utilizing this modality are mediated. A greater understanding of the basic interactions between implanted and host cells is needed to improve future strategies. The aims of this study were to investigate the effects of periodontal ligament (PDL) cells on expression of periodontal markers and alkaline phosphatase (ALP) activity of gingival fibroblasts (GF).Materials and MethodsPrimary human PDL cells were co‐cultured with primary GF cultures either by direct co‐culture with subsequent FACS sorting or indirect co‐culture using transwell cultures and PDL cell conditioned medium. Expression of periodontal markers, asporin, nestin, and periostin, was assessed by qPCR and immunofluorescence staining. Alkaline phosphatase (ALP) expression was assessed by qPCR, histochemical staining, and activity assessed by para‐nitrophenol enzymatic assay. Single cultures of PDL cells and GF were used as controls. The role of Wnt signaling on ALP activity was assessed via Dkk1‐mediated inhibition.ResultsPDL cells significantly upregulated expression of PDL markers in GF with both direct and indirect co‐culture methods when compared to controls (6.05 vs. 0.73 and 59.48 vs. 17.55 fold change of asporin expression). PDL/GF cell co‐cultures significantly increased ALP activity in GF when compared with single GF cultures. Similar results were obtained when using conditioned medium isolated from PDL cell cultures. Dkk1 caused dose‐dependent reduction in ALP activity of GF cultured in PDL cell conditioned medium.ConclusionsPDL cells stimulate expression of periodontal markers and osteogenic capacity of gingival fibroblasts via paracrine signaling which can be partially inhibited with addition of the Wnt antagonist, Dkk1.Further studies are required to identify specific secreted factors responsible for this activity.

An in vitro evaluation of ice apple water, Aloe vera, and propolis as a storage medium to preserve viability of human periodontal ligament fibroblasts.

A number of media that create the best possible conditions to maintain periodontal ligament (PDL) cell viability after dental avulsion have been reported. The aim of this study is to evaluate ice apple water (IAW), Aloe vera, and propolis as a storage medium to preserve the viability of human PDL fibroblasts. An in vitro comparative type of study was performed on a PDL cell culture model. PDL fibroblasts obtained from the roots of healthy premolars were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with ice apple water (IAW), 7% propolis extract (PE), 30% Aloe vera extract (AVE), positive control DMEM supplemented with fetal bovine serum, negative control (NC) without any agent, and incubated at 37°C for 1 h, 3 h, and 24 h. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay after every test period. Optical density was measured at a wavelength of 490 nm. The effects of the test storage media were evaluated by one-way analysis of variance test, followed by post hoc Tukey's multiple comparison test (P < 0.05). Seven percent PE demonstrated the highest capacity of maintaining PDL cell viability at 1 h and 24 h. IAW showed a statistically significantly lower percentage of viable cells at all three test periods as compared to 7% PE. After 3 h, 30% AVE demonstrated maximum viable cells. Within the limitations of this study, propolis at a concentration of 7% was the most effective medium for maintaining PDL cell viability.

Cyclic tensile forces enhance the angiogenic properties of HUVECs by promoting the activities of human periodontal ligament cells.

This study aimed to investigate whether human periodontal ligament (PDL) cells secrete pro-angiogenic factors that induce the vascularization of surrounding bone tissue under tensile stress. Quantitative real-time PCR and Western blotting were used to analyze the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), Angiopoietin-I (Ang-I), connective tissue growth factor （CTGF）, and macrophage colony-stimulating factor (M-CSF) in PDL cells after tensile force treatments of different durations. Enzyme-linked immunosorbent assay was used to measure the VEGF concentration in the supernatants of cell cultures. Cell viability assay, wound healing assay, and tube formation assay were performed to evaluate the angiogenic behaviors of human umbilical vein endothelial cells (HUVECs). The mRNA expression and protein expression of VEGF, bFGF, Ang-I, and M-CSF was increased in the cells that received 6 to 48hours of tensile force treatment. And, the VEGF level in the supernatant significantly increased in the human PDL cell cultures stressed for 6 to 48hours. The abilities of HUVECs to proliferate, migrate, and form tubes were enhanced in media conditioned with tensile-stressed human PDL cells. Hence, tensile force induced human PDL cells to express and release pro-angiogenic factors enhancing the proliferation, migration, and angiogenic capacity of HUVECs. Tensile stress induced human PDL cells to express and release pro-angiogenic factors, including VEGF, bFGF, Ang-I, and M-CSF, thereby enhancing the proliferation, migration, and angiogenic capacity of HUVECs.

An in-vitro mechanical strain three-dimensional culture model: periodontal ligament cell viability, apoptosis, and endoplasmic reticulum stress response.

To develop a model to investigate a potential relationship between mechanical strain, cell responses, and endoplasmic reticulum stress in periodontal ligament (PDL) cells, primary PDL cell cultures were obtained from extracted premolars. Cells were cultured in hydrogel and subjected to 24h of static mechanical strain, resulting in 18% dimensional substrate elongation. Cell viability, caspase-3/7 activity, and mRNA levels for 28 genes, including unfolded protein response (UPR)-related and mechanically responsive genes, serving as positive controls for stress induction, were examined. Compared with unstrained cultures, no difference in caspase activity was observed; however, viability responses differed between cell lines. Multiple UPR-related genes were differentially upregulated, with marginal statistical significance, including cAMP responsive element binding protein 3 like 3 (CREB3L3) (mean fold-regulation=1.91), an adenosine monophosphate-dependent transcription factor with roles in UPR activation and the acute inflammatory response; and the pro-apoptotic UPR gene, endoplasmic reticulum to nucleus signaling 2 (ERN2) (mean fold-regulation=4.01). The observed effect on cell viability following strain with no change in caspase activity suggests that reduction in viability may be mediated via caspase-3/7-independent mechanisms. Three-dimensional mechanical strain PDL cell culture models offer a method to study the role of endoplasmic reticulum stress and UPR, and provide a framework and potential UPR targets for future investigations.

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films.

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Mesenchymal stem cell exosomes enhance periodontal ligament cell functions and promote periodontal regeneration

Mesenchymal stem cells (MSCs) are potential therapeutics for the treatment of periodontal defects. It is increasingly accepted that MSCs mediate tissue repair through secretion of trophic factors, particularly exosomes. Here, we investigated the therapeutic effects of human MSC exosome-loaded collagen sponge for regeneration of surgically created periodontal intrabony defects in an immunocompetent rat model. We observed that relative to control rats, exosome-treated rats repaired the defects more efficiently with regeneration of periodontal tissues including newly-formed bone and periodontal ligament (PDL). We also observed that concomitant with this, there was increased cellular infiltration and proliferation. We therefore postulated that MSC exosomes enhanced regeneration through increased cellular mobilisation and proliferation. Using PDL cell cultures, we demonstrated that MSC exosomes could increase PDL cell migration and proliferation through CD73-mediated adenosine receptor activation of pro-survival AKT and ERK signalling. Inhibition of AKT or ERK phosphorylation suppressed PDL cell migration and proliferation. Our findings demonstrated for the first time that MSC exosomes enhance periodontal regeneration possibly by increasing PDL migration and proliferation. This study suggests that MSC exosome is a viable ready-to-use and cell-free MSC therapeutic for the treatment of periodontal defects. Statement of significanceMesenchymal stem cell (MSC) therapies have demonstrated regenerative potential for the treatment of periodontal defects. However, translation of cellular therapies is hampered by challenges in maintaining optimal cell vitality and viability from manufacturing and storage to final delivery to patients. Although the use of MSCs for tissue repair was first predicated on their differentiation potential, the therapeutic efficacy of MSCs has increasingly been attributed to its paracrine secretion, particularly exosomes or small extracellular vesicles. In this study, MSC exosome-loaded collagen sponge enhanced periodontal regeneration in an immunocompetent rat periodontal defect model without any obvious adverse effects. These findings provide the basis for future development of MSC exosomes as a cell-free strategy for periodontal regeneration.

Inhibition of methionine gamma lyase deaminase and the growth of Porphyromonas gingivalis: A therapeutic target for halitosis/periodontitis

Background and objectivesPathogenic infections caused by Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia can result in the production of volatile sulfur compounds (VSC’s) and other toxic compounds from methionine catabolism that can lead to halitosis and periodontitis. Our aim is to block the activity of methionine gammalyase-deaminase (Mgld) of methionine catabolism to prevent halitosis/periodontitis. DesignsCloned, expressed, Mgld protein was tested for purity by SDS-PAGE and western blotting. Mgld activity was tested by UV–vis spectroscopy and DTNB assay. Effects of Mgld inhibitor propargylglycine (PGLY) was tested on P. gingivalis growth by turbidity measurements. The effects of PGLY on oral epithelial and periodontal ligament cells in culture at different concentrations and time were tested for cell viability by MTT and Live-Dead assays. Amino acid comparisons of Mgld from different oral pathogens were done using standard bioinformatics program. ResultsPropargylglycine (PGLY) inhibited purified Mgld activity completely. In vivo, PGLY is a potent inhibitor on the growth of the P. gingivalis over 24 h, grown at 25 °C and 37 °C. Correspondingly in vivo Mgld activity was also affected by PGLY. Amino acid comparisons of oral pathogens showed 100% identity on the key residues of Mgld catalysis. Mammalian oral cell lines with PGLY, showed no difference in cell death over untreated controls assessed by MTT and Live-Dead assays. ConclusionsPGLY arrest’s VSC’s production by P. gingivalis. Since initial Mgld activity is inhibited subsequent enzymatic and nonenzymatic products formed will be prevented. PGLY showed no toxicity towards cultured mammalian oral cells. Thus, PGLY can serve as a mouthwash ingredient to prevent halitosis/periodontitis.

Evaluating the effectiveness of rehydrating solutions in preserving periodontal ligament cells vitality: An in vitro study

Background and Purpose of the Study: Two of the most critical factors affecting the prognosis of an avulsed tooth after replantation are extraoral dry time and the storage medium in which the tooth was placed before treatment could be rendered. The ability of a storage medium to support cell viability of the periodontal ligament (PDL) can be an important factor in the prognosis, and thus, the aim of this study was to evaluate the efficacy of commonly available storage media. Materials and Methods: Cultured PDL cells of sixty freshly extracted human teeth were randomly divided into four groups of 15 each, Group I: Electral solution, Group II: Ringer's lactate, Group III: Oral rehydration salt liquid (ORS-L), and Group IV: Coconut water. Samples in each group were further divided into three subgroups depending on the time duration they were placed in the storage medium, i.e., 1, 4, and 8 h. Further these samples were subjected to Collagenase assay, the cells were stained with trypan blue and viable PDL cells were counted under light microscope. Results: Statistical analysis showed that Group II demonstrated significantly (P Conclusion: Ringer's lactate maintained highest PDL cell viability followed by coconut water, electoral solution, and ORS-L at various time intervals. More studies with large samples are required to prove the efficacy of Ringer's lactate as viable storage media.

Impact of nicotine on the interplay between human periodontal ligament cells and CD4+ T cells.

Periodontitis is a common infectious disease associated with destruction of periodontal ligaments and alveolar bones. CD4(+) T cell-mediated immune response is involved in the progression of periodontitis. Tobacco consumption increases the risk of periodontal disease. However, the impact of nicotine on the interaction between human periodontal ligament (PDL) cells and CD4(+) T cells remains unrevealed. Our study aims to investigate the effect of nicotine on PDL cells and the cocultured CD4(+) T cells. The PDL cell cultures were established by explants from healthy individuals, exposed to nicotine or α-bungarotoxin (α-BTX), and incubated solely or in combination with CD4(+) T cells. Afterwards, cell viability, secreted cytokines, and matrix metalloproteinases (MMPs) were evaluated. In monoculture of PDL cells, nicotine dramatically repressed cell viability and increased apoptosis. Meanwhile, α-BTX largely reversed the nicotine-induced apoptosis and increased viability of PDL cells. Compared with the monoculture, MMP-1, MMP-3, interleukin (IL)-1β, IL-6, IL-17, and IL-21 in supernatant of cocultures were markedly elevated after treatment with nicotine. Moreover, α-BTX significantly attenuated nicotine-triggered production of these components either in mono- or co-cultures. In addition, PDL cell-derived CXCL12 following nicotine treatment recruited CD4(+) T cells. Above all, nicotine deteriorated periodontitis partially by promoting PDL cell-CD4(+) T cell-mediated inflammatory response and matrix degradation.

The preservative effect of Thai propolis extract on the viability of human periodontal ligament cells.

Tooth avulsion causes an injury to the periodontal ligament (PDL). The success of tooth replantation depends on the quantity and quality of PDL cells. The aim of this study was to examine the preservative and proliferative effects of Thai propolis extract, previously shown to exert anti-inflammatory and antioxidant activities, on human PDL cells. Ninety-six premolars were left to air dry for 30 min and stored in Hank's balanced salt solution (HBSS), milk, or various concentrations of propolis extract from 0.25 to 10 mg ml-1 for 3 h. PDL cells were isolated by collagenase and trypsin digestion, and their viability was determined by a trypan blue dye exclusion assay. PDL tissues were also scraped off the root surface and cultured to determine cell growth and morphology. The alamarBlue® and BrdU assays were performed to determine the cytotoxic and proliferative effects of the extract on cultured PDL cells, respectively. A non-toxic dose of 2.5 mg ml-1 of propolis extract yielded the greatest percentage of cell viability (78.84 ± 3.34%), which was significantly higher than those of the other concentrations (P < 0.001). Nevertheless, this percentage was not significantly different from that of HBSS (80.14 ± 2.44%; P = 1.00), but was significantly higher than that of milk (71.27 ± 2.79%; P < 0.001). The cells grown from PDL explants looked like fibroblasts. However, 2.5 mg ml-1 of the extract did not induce PDL cell proliferation. Thai propolis extract at 2.5 mg ml-1 appears to be the most effective dose for preserving the viability of PDL cells, and this was comparable to HBSS.

Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains

It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them.

Effects of Slow Programmable Cryopreservation on Preserving Viability of the Cultured Periodontal Ligament Cells from Human Impacted Third Molar

Purpose: This study was conducted to determine cell viability and differentiation capability of human periodontal ligament (PDL) cells and to elucidate the effects of cryopreservation on the activity of human third molar PDL cells by comparing PDL cells with and without cryopreservation. Materials and Methods: Human PDL fibroblasts obtained from immature third molars were cultured and divided into two groups. The experimental group was cryopreserved with a slow freezing rate of 0.5℃/min from 4℃ to –35℃ followed by plunging in liquid nitrogen at –196℃ and cultured after fast thawing. The control group was cultured without cryopreservation. Cell viability, growth capacity and morphology were evaluated in both groups. Bivariate statistics were used to compare 2 groups and linear mixed model analysis was used to investigate the growth trends difference over time. Result: Cell viability and growth capacity were not significantly different between the 2 groups (P>0.05). Cultured cell of both groups showed fibroblast-like in appearance, and there were no significant differences in morphology between 2 groups. The mixed model analysis revealed no significant difference of growth capacity between 2 groups over time (β=–0.0009; P=0.138). Conclusion: This study demonstrates that cryopreservation under control does not affect the biological properties of PDL cells, supporting the feasibility of autotransplantation of cryopreserved impacted third molars.

Residents' journal review

Residents' journal review

Curcumin in Combination with Piperine Suppresses Osteoclastogenesis In Vitro

IntroductionThe dietary pigment curcumin is a natural polyphenol extracted from the Curcuma longa rhizomes native to South Asia. The antioxidative, antimicrobial, and anti-inflammatory activities besides its unknown side effects suggest that curcumin could be a promising antiresorptive agent to prevent replacement resorption in replanted teeth after traumatic avulsion. Piperine, an alkaloid present in black pepper, seems to enhance the bioavailability and activity of curcumin. Therefore, this study evaluated the biocompatibility of curcumin and piperine in cultures of periodontal ligament cells as well as their effects in an in vitro osteoclastogenesis model of RAW 264.7 macrophages. MethodsThe cytotoxicity in human periodontal ligament fibroblasts, human osteogenic sarcoma cells (SAOS-2), and murine osteoclastic precursors (RAW 264.7) was analyzed by using cell number determination and proliferation assays. The ability of curcumin and its conjugate to suppress the receptor activator of nuclear factor kappa B ligand–induced osteoclastogenesis was assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity as well as real-time polymerase chain reaction. ResultsCurcumin at concentrations ≥ 10 μmol/L was cytotoxic in all cell types tested, whereas piperine showed only slight cytotoxicity at 30 μmol/L in RAW and SAOS cultures. Although curcumin caused already significant effects, the combination with piperine completely suppressed the osteoclastogenesis by decreasing the TRAP activity and inhibiting the expression of the specific osteoclast markers TRAP, cathepsin K, and calcitonin receptor. ConclusionsWe demonstrated that curcumin combined with piperine suppressed the osteoclastogenesis in vitro without causing cytotoxic effects in periodontal ligament cells. These findings suggest its potential therapeutic application for the prevention and treatment of replacement resorption in replanted avulsed teeth.

Isolation, characterization and investigation of differentiation potential of human periodontal ligament cells and dental follicle progenitor cells and their response to BMP-7 in vitro.

The aim of this study was to assess the factors, mechanisms and the differences between periodontal ligament (PDL) cells and denta l follicle (DF) progenitor cells towards the osteoblastic/cementoblastic differentiation and to investigate the effects of BMP-7 on developmental (DF) and mature tissue-derived (PDL) cells, respectively. Primary cell culture of PDL cells and DF progenitor cells was performed. Osteogenic differentiation was evaluated using von Kossa, Alizarin Red S and immuno-histo-chemistry staining of osteocalcin. Gene expression pattern was evaluated via real-time PCR. A series of CD surface marks were tested using flow cytometry and fluorescence-activated cell-sorting analysis was performed. Real-time RT-PCR demonstrated similar gene expression pattern of PDL cells and DF progenitor cells: the expression of OPN and OCN significantly was elevated when incubated with osteogenic components, Runx2 was unaffected, and Osteorix was hardly expressed whether in basic medium or induction medium. In addition, BMP-7 induced osteoblast/cementoblast differentiation of PDLSCs and DF progenitor cells in a dose- and time-dependent manner, as reflected by enhanced Runx2 and (OCN) mRNA transcript expression. BMP-7 triggers PDL cells and DF progenitor cells to differentiate towards an osteoblast/cementoblast phenotype.

Human oral cells' response to different endodontic restorative materials: an in vitro study.

IntroductionThe aim of this study was to compare the biological interaction of human osteoblasts and cells of the human periodontal ligament (PDL) with different endodontic restorative material as Mineral Trioxide Aggregate (MTA), Biodentine, amalgam and composite over a time period of 20 days.Materials and methodsHuman PDL cells and osteoblasts were harvested, cultured and according to standardized protocols. The cell populations were characterized with the corresponding surface markers following standardized procedures. The specimens were produced with special regard to constant dimensions and volume in the different groups. Cell attachment and proliferation were evaluated morphologically after Richardson staining and cell count was performed after 1d, 8d, 13d and 20d. All experiments were performed in triplets. The results were statistically analyzed using the ANOVA- and Tukey-test (p < 0.05).ResultsMorphological analysis proved good proliferation and cell attachment in both cements. A remarkable result was the organized spreading and parallel alignment of the PDL cells in contact with MTA and especially Biodentine (cells maturing in a second cell layer crossway to the first one). From 8d onward Biodentine showed the highest quantity of PDL cells (p < 0.05). Biodentine and MTA resulted in a significantly higher cell density in osteoblast and PDL cell culture. The other groups showed a lower PDL cell density from 8d and a lower osteoblast cell density from 13d when compared to control and cement samples (p < 0.05).ConclusionsMTA and Biodentine showed a good biocompatibility in contact with the human osteoblasts and cells of the periodontal ligament. Regarding cell survival and proliferation particularly of PDL cells Biodentine showed good results and can be considered as a well-tolerated bioactive endodontic material.Electronic supplementary materialThe online version of this article (doi:10.1186/s13005-014-0055-4) contains supplementary material, which is available to authorized users.

In vivo investigation of the osteogenic potential of human periodontal ligament cell sheet cultured on amniotic membrane

The ultimate aim of this study is to develop a novel culture sheet for bone regeneration using periodontal ligament (PDL) cells cultured on an amniotic membrane (AM). The AM is a thin membrane that covers the outermost layer of the placenta, and is composed of parenchyma of a constant thickness. It can mostly be aseptically collected from the placenta, and is easily obtained without any ethical or technical problem because it is usually discarded after delivery. It has been used for various surgeries, attracting attention for its usefulness and effectiveness as a culture substrate as well as graft material. We have focused on such usefulness of AM, and prepared various cultured PDL cells sheet using the membrane as a substrate (ref. 1, 2). In addition, we have previously reported the possibility of PDL cells sheet on AM having osteogenic potential in vitro. In this study, we report new findings from an investigation of in vivo cell kinetics of PDL cells sheet on AM.

ヒト (歯根膜・歯髄) 由来細胞の分離培養方法

ヒト (歯根膜・歯髄) 由来細胞の分離培養方法