Rat Periodontal Ligament Cells Research Articles

Hyaluronidase inhibits TGF-β-mediated rat periodontal ligament fibroblast expression of collagen and myofibroblast markers: An in vitro exploration of periodontal tissue remodeling

ObjectiveTo determine the effect of hyaluronic acid (HA) degradation by hyaluronidase (HYAL) in inhibiting collagen fiber production by rat periodontal ligament cells (rPDLCs). DesignPrimary rPDLCs were isolated from the euthanized rats and used for in vitro experiments. The appropriate HYAL concentration was determined through CCK-8 testing for cytotoxicity detection and Alizarin red staining for mineralization detection. RT-qPCR and western blot assays were conducted to assess the effect of HYAL, with or without TGF-β, on generation of collagen fiber constituents and expression of actin alpha 2, smooth muscle (ACTA2) of rPDLCs. ResultsNeither cell proliferation nor mineralization were significantly affected by treatment with 4 U/mL HYAL. HYAL (4 U/mL) alone downregulated type I collagen fiber (Col1a1 and Col1a2) and Acta2 mRNA expression; however, ACTA2 and COL1 protein levels were only downregulated by HYAL treatment after TGF-β induction. ConclusionsTreatment of rPDLCs with HYAL can inhibit TGF-β-induced collagen matrix formation and myofibroblast transformation.

Changes in superoxide dismutase 3 (SOD3) expression in periodontal tissue during orthodontic tooth movement of rat molars and the effect of SOD3 on in vitro hypoxia-exposed rat periodontal ligament cells.

Hypoxia during orthodontic tooth movement (OTM) induces reactive oxygen species (ROS) production in periodontal tissues. Superoxide dismutase 3 (SOD3) is an anti-inflammatory enzyme that protects cells from ROS. This study investigated the expression and function of SOD3 during rat OTM and in hypoxia-exposed rat periodontal ligament (PDL) cells. OTM of right maxillary first molars were performed in 8-week-old male Sprague-Dawley rats using closed-coil spring for 1 and 14 days (n = 6 per group). SOD3 and hypoxia-inducible factor 1-alpha (HIF-1α) protein expression was evaluated by immunohistochemistry. The effects of SOD3 on cell viability and proliferation, ROS production, and mRNA expression of Hif1-α, receptor activator of nuclear factor kappa-Β ligand (Rankl), and osteoprotegerin (Opg) in PDL cells and osteoclast differentiation were investigated under normal and hypoxic conditions. SOD3 expression in PDL tissues significantly decreased on the compression side on day 1 and on both sides on day 14 of OTM. HIF-1α levels significantly increased on the compression side on day 14. Cell viability, cell proliferation, and Opg mRNA expression decreased, whereas ROS production and Hif1-α and Rankl mRNA expression increased in the PDL cells upon SOD3 silencing. Hypoxia reduced Sod3 and Opg mRNA expression and increased ROS, Rankl mRNA expression, and osteoclast formation; SOD3 treatment attenuated these effects. SOD3 plays a role in periodontal tissue remodelling during OTM and in hypoxia-exposed PDL cells through ROS, HIF-1α, and RANKL/OPG pathways. Moreover, SOD3 treatment could attenuate the negative effects of hypoxia on the PDL cells.

Periodontal ligaments enhance neurite outgrowth in trigeminal ganglion neurons through Wnt5a production induced by mechanical stimulation.

The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force, and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca2+-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in Western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons that project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

Local delivery of simvastatin maintains tooth anchorage during mechanical tooth moving via anti-inflammation property and AMPK/MAPK/NF-kB inhibition.

Simvastatin (SMV) could increase tooth anchorage during orthodontic tooth movement (OTM). However, previous studies on its bone‐specific anabolic and anti‐inflammation properties were based on static in vitro and in vivo conditions. AMPK is a stress‐activated kinase that protects tissue against serious damage from overloading inflammation. Rat periodontal ligament cells (PDLCs) were subjected to a serial of SMV concentrations to investigate the optimization that promoted osteogenic differentiation. The PDLCs in static and/or tensile culturing conditions then received the proper concentration SMV. Related factors expression was measured by the protein array, real‐time PCR and Western blot. The 0.05UM SMV triggered osteogenic differentiation of PDLCs. The inhibition of AMPK activation through a pharmacological approach (Compound C) caused dramatic decrease in osteogenic/angiogenic gene expression and significant increase in inflammatory NF‐κB phosphorylation. In contrast, pharmacological activation of AMPK by AICAR significantly inhibited inflammatory factors expression and activated ERK1/2, P38 MAPK phosphorylation. Moreover, AMPK activation induced by SMV delivery significantly attenuated the osteoclastogenesis and decreased the expression of pro‐inflammatory TNF‐α and NF‐κB in a rodent model of OTM. The current studies suggested that SMV could intrigue intrinsic activation of AMPK in PDLCs that promote attenuate the inflammation which occurred under tensile irritation through AMPK/MAPK/NF‐kB Inhibition.

Effects of UV Treatment on Ceria-Stabilized Zirconia/Alumina Nanocomposite (NANOZR).

Nanostructured zirconia/alumina composite (NANOZR) has been explored as a suitable material for fabricating implants for patients with metal allergy. In this study, we examined the effect of UV treatment on the NANOZR surface. The experimental group was UV-treated NANOZR and the control group was untreated NANOZR. Observation of the surface of the UV-treated materials revealed no mechanical or structural change; however, the carbon content on the material surface was reduced, and the material surface displayed superhydrophilicity. Further, the effects of the UV-induced superhydrophilic properties of NANOZR plates on the adhesion behavior of various cells were investigated. Treatment of the NANOZR surface was found to facilitate protein adsorption onto it. An in vitro evaluation using rat bone marrow cells, human vascular endothelial cells, and rat periodontal ligament cells revealed high levels of adhesion in the experimental group. In addition, it was clarified that the NANOZR surface forms active oxygen and suppresses the generation of oxidative stress. Overall, the study results suggested that UV-treated NANOZR is useful as a new ceramic implant material.

Human \u03b2-defensin 3 gene modification promotes the osteogenic differentiation of human periodontal ligament cells and bone repair in periodontitis

Efforts to control inflammation and achieve better tissue repair in the treatment of periodontitis have been ongoing for years. Human β-defensin 3, a broad-spectrum antimicrobial peptide has been proven to have a variety of biological functions in periodontitis; however, relatively few reports have addressed the effects of human periodontal ligament cells (hPDLCs) on osteogenic differentiation. In this study, we evaluated the osteogenic effects of hPDLCs with an adenoviral vector encoding human β-defensin 3 in an inflammatory microenvironment. Then human β-defensin 3 gene-modified rat periodontal ligament cells were transplanted into rats with experimental periodontitis to observe their effects on periodontal bone repair. We found that the human β-defensin 3 gene-modified hPDLCs presented with high levels of osteogenesis-related gene expression and calcium deposition. Furthermore, the p38 MAPK pathway was activated in this process. In vivo, human β-defensin 3 gene-transfected rat PDLCs promoted bone repair in SD rats with periodontitis, and the p38 mitogen-activated protein kinase (MAPK) pathway might also have been involved. These findings demonstrate that human β-defensin 3 accelerates osteogenesis and that human β-defensin 3 gene modification may offer a potential approach to promote bone repair in patients with periodontitis.

Combined effects of systemic parathyroid hormone (1-34) and locally delivered neutral self-assembling peptide hydrogel in the treatment of periodontal defects: An experimental in vivo investigation.

To evaluate in vivo combination therapy of systemic parathyroid hormone (PTH) and locally delivered neutral self-assembling peptide (SAP) hydrogel for periodontal treatment. Viability/proliferation of rat periodontal ligament cells in a neutral SAP nanofibre hydrogel (SPG-178) was evaluated using WST-1 assay. Periodontal defects were created mesially to the maxillary first molars in 40 Wistar rats. Defects were filled with 1.5% SPG-178 or left unfilled. Animals received PTH (1-34) or saline injections every 2days. Microcomputed tomography, histological, and immunohistochemical examinations were used to evaluate healing at 2 or 4 weeks postoperative. At 72hr, cells in 1.5% SPG-178 showed increased viability/proliferation compared to cells in 0.8% SPG-178 or untreated controls. In vivo, systemic PTH resulted in significantly greater bone volume in the Unfilled group at 2 weeks (p = .01) and 4 weeks (p < .0001) than in the saline control. At 4 weeks, a significantly greater bone volume was observed in the PTH/SPG-178 (p = .0003) and PTH/Unfilled (p = .004) groups than in Saline/SPG-178 group. Histologically, greater bone formation was observed in PTH/SPG-178 at 4weeks than in other groups. In the PTH/SPG-178 group, increased proportions of PCNA-, VEGF-, and Osterix-positive cells were observed in the treated sites. These findings suggest that intermittent systemic PTH and locally delivered neutral SAP hydrogel enhance periodontal healing.

Effect of Amelogenin Coating of a Nano-Modified Titanium Surface on Bioactivity

The interactions between implants and host tissues depend on several factors. In particular, a growing body of evidence has demonstrated that the surface texture of an implant influences the response of the surrounding cells. The purpose of this study is to develop new implant materials aiming at the regeneration of periodontal tissues as well as hard tissues by coating nano-modified titanium with amelogenin, which is one of the main proteins contained in Emdogain®. We confirmed by quartz crystal microbalance evaluation that amelogenin is easy to adsorb onto the nano-modified titanium surface as a coating. Scanning electron microscopy, scanning probe microscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy analyses confirmed that amelogenin coated the nano-modified titanium surface following alkali-treatment. In vitro evaluation using rat bone marrow and periodontal ligament cells revealed that the initial adhesion of both cell types and the induction of hard tissue differentiation such as cementum were improved by amelogenin coating. Additionally, the formation of new bone in implanted surrounding tissues was observed in in vivo evaluation using rat femurs. Together, these results suggest that this material may serve as a new implant material with the potential to play a major role in the advancement of clinical dentistry.

Effect of nano modified titanium surface on adsorption of rat periodontal ligament cells

Effect of nano modified titanium surface on adsorption of rat periodontal ligament cells

Transfer of the bone morphogenetic protein 4 gene into rat periodontal ligament by in vivo electroporation

ObjectiveRegulation of alveolar bone metabolism is required in clinical dentistry. The aim of the present study was to establish a method for gene transfer into the periodontal ligament (PDL) by in vivo electroporation with a plasmid vector and to investigate the effects of BMP-4 transfer into the PDL. DesignPlasmids containing mouse BMP-4 cDNA (pCAGGS-BMP4) were transfected into cultured rat PDL cells by in vitro electroporation, and BMP-4 production and secretion were detected by immunocytochemistry and western blotting. Next, pCAGGS-BMP4 was injected into the PDL of rats, and electroporation was performed in vivo, using original paired-needle electrodes. BMP-4 expression was examined by immunohistochemical staining 3, 7, 14, 21, and 28days after electroporation. Control groups were injected with pCAGGS by electroporation, injected with pCAGGS-BMP4 without electroporation, or subjected to neither injection nor electroporation. ResultsIn vitro-transfected rat PDL cells exhibited production and secretion of the mature-form BMP-4. After in vivo electroporation of pCAGGS-BMP4, site-specific BMP-4 expression peaked on day 3, gradually decreased until day 14, and was absent by day 21. We observed no unfavorable effects such as inflammation, degeneration, or necrosis. ConclusionsGene transfer by electroporation with plasmid DNA vectors has several advantages over other methods, including the non-viral vector, non-immunogenic effects, site-specific expression, simplicity, cost-effectiveness, and limited histological side effects. Our results indicate that the method is useful for gene therapy targeting the periodontal tissue, which regulates alveolar bone remodeling.

GDNF From Human Periodontal Ligament Cells Treated With Pro-Inflammatory Cytokines Promotes Neurocytic Differentiation of PC12 Cells.

Glial cell line-derived neurotrophic factor (GDNF) is known to mediate multiple biological activities such as promotion of cell motility and proliferation, and morphogenesis. However, little is known about its effects on periodontal ligament (PDL) cells. Recently, we reported that GDNF expression is increased in wounded rat PDL tissue and human PDL cells (HPDLCs) treated with pro-inflammatory cytokines. Here, we investigated the associated expression of GDNF and the pro-inflammatory cytokine interleukin-1 beta (IL-1β) in wounded PDL tissue, and whether HPDLCs secrete GDNF which affects neurocytic differentiation. Rat PDL cells near the wounded area showed intense immunoreactions against an anti-GDNF antibody, where immunoreactivity was also increased against an anti-IL-1β antibody. Compared with untreated cells, HPDLCs treated with IL-1β or tumor necrosis factor-alpha showed an increase in the secretion of GDNF protein. Conditioned medium of IL-1β-treated HPDLCs (IL-1β-CM) increased neurite outgrowth of PC12 rat adrenal pheochromocytoma cells. The expression levels of two neural regeneration-associated genes, growth-associated protein-43 (Gap-43), and small proline-rich repeat protein 1A (Sprr1A), were also upregulated in IL-1β-CM-treated PC12 cells. These stimulatory effects of IL-1β-CM were significantly inhibited by a neutralizing antibody against GDNF. In addition, U0126, a MEK inhibitor, inhibited GDNF-induced neurite outgrowth of PC12 cells. These findings suggest that an increase of GDNF in wounded PDL tissue might play an important role in neural regeneration probably via the MEK/ERK signaling pathway. J. Cell. Biochem. 118: 699-708, 2017. © 2016 Wiley Periodicals, Inc.

The Role of a Platelet Lysate-Based Compartmentalized System as a Carrier of Cells and Platelet-Origin Cytokines for Periodontal Tissue Regeneration.

Currently available clinical therapies are not capable to regenerate tissues that are lost by periodontitis. Tissue engineering can be applied as a strategy to regenerate reliably the tissues and function of damaged periodontium. A prerequisite for this regeneration is the colonization of the defect with the adequate cell populations. In this study, we proposed a bilayered system composed of (1) a platelet lysate (PL)-based construct produced by crosslinking of PL proteins with genipin (gPL) for the delivery of rat periodontal ligament cells (rat-PDLCs); combined with (2) an injectable composite consisting of calcium phosphate cement incorporated with PL-loaded poly(d, l-lactic-co-glycolic acid) microspheres. This system was expected to promote periodontal regeneration by the delivery of adequate progenitor cells and providing a stable system enriched with adequate cytokines and growth factors for the orchestration of tissue regrowth in periodontal defects. The bilayered system was tested in a three-wall intrabony defect in rats and the healing of periodontal tissue was assessed 6 weeks after surgery. Results showed that the bilayered system was able to promote the regrowth of functional periodontal tissues, both with (cells + gPL) and without the loading of PDLCs (gPL). Significant connective tissue attachment (45.0 ± 15.0% and 64.0 ± 15.0% for gPL and cells + gPL group, respectively) and new bone area (33.8 ± 21% and 21.3 ± 3% for gPL and cells + gPL group, respectively) were observed. Nevertheless, rat PDLCs delivered with gPL construct in the defect area were hardly visible 6 weeks after surgery and did not contribute for the regeneration of new periodontal tissue. Overall, our findings show that the bilayered system promotes the stabilization of PL proteins on the root surface and has a positive effect in the repair of periodontal tissues both in quality and in quantity.

Evaluation of the Viability of Rat Periodontal Ligament Cells after Storing at 0℃/2 MPa Condition up to One Week: In Vivo MTT Method

Purpose: The aim of this study was to evaluate the rat periodontal ligament cell viability under 0℃/2 MPa condition up to one week using in vivo 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. Materials and Methods: As soon as 110 upper molar teeth of rats were extracted, they were stored in Hartman’s solution under 0℃/2 MPa condition for 1, 2, 3, 4, and 7 days each. All specimens were treated with in vivo MTT assay and the value of optical density was measured by ELISA reader. These values were statistically analyzed by one-way ANOVA. Result: There was no statistical difference on MTT value between immediate and 1 day storage group. There were statistically significant differences between 1 day and 2 days storage, 2 and 3 days storage groups, respectively. Teeth of 3, 4, and 7 days storage groups showed significantly lower MTT values compared with shorter period storage groups. Conclusion: When the MTT values were substituted in standard curve, 1 day storage group at 0℃/2 MPa condition showed 68% cell viability when compared with immediate group. It dropped to 13% at 2 days, and to less than 5% at 3 days or more.

Enhanced healing of surgical periodontal defects in rats following application of a self-assembling peptide nanofibre hydrogel.

The aim of this study was to investigate the effects of a self-assembling peptide (SAP) nanofibre hydrogel on healing of surgical periodontal defects in rats. In vitro interactions between rat periodontal ligament (PDL) cells and SAP hydrogel (2.5% RADA16) were assessed by cell proliferation assays. In vivo, maxillary first molars of 45 Wistar rats were extracted and after healing, bilateral periodontal defects were surgically created mesially in second molars. Defects were treated with RADA16, Matrigel, or left unfilled. After 2 and 4 weeks, defect healing was evaluated by microcomputed tomography, histological and immunohistochemical analyses. Periodontal ligament cells grown on RADA16 showed an gradual increase in proliferation up to 72 h. At 4 weeks post surgery, the bone volume fraction and trabecular thickness of defect areas in the RADA16 group were significantly greater than those in other groups. Histologically, enhanced new bone formation was observed in the RADA16 group. At 4 weeks, PDL-like collagen bundles ran oblique to the root surface in the RADA16 group. Expression levels of PCNA-positive cells, vascular endothelial growth factor and osteopontin in the RADA16 group were significantly greater than those in other groups. Within the limitations of the study, application of the SAP hydrogel promoted healing of surgical periodontal defects by enhancing cell recruitment and possibly angiogenesis.

Adhesion of Ligament Periodontal Cells to Different Titanium Surfaces: An In Vitro Comparative Study

Purpose: the present study aims to evaluate, through experiments in vitro, the adhesion capacity of rats periodontal ligament cells on polishing and treated by ionic nitriding (plasma) titanium surfaces. Method: degree II titanium discs (ASTM F86), 15mm of diameter for 1,5mm of thickness, which had received different treatments from surface in 2 distinct groups (polished and cathodic cage plasma nitriding) were used. The cells were isolated from periodontal ligament of rats and cultivated in α-MEM contend antibiotic and supplemented with 10% of FBS, for 72 hours, in humid atmosphere with 5% of CO2 at 37oC. In the subculture the cells were cultivated on titanium discs in 24-well cell culture plates, with a density of 1 x 104 cells per well, including wells with no discs (control). After 24 hours of cultivation, the cells were counted in a Neubauer chamber. Results: the results had shown that the mean adhesion was greater on the control surface (0.62±0.22) than on polished (0.46±0.14) and nitriding (0.33±0.10) surfaces. Statistical significant difference was observed between the groups control and cathodic cage plasma nitriding (p=0.04), nevertheless no diference was found between polished and nitriding groups. Conclusion: the adhesion capacity of rat periodontal ligament cells on the titanium surfaces was not influenced by the different surface treatments given to the material, since none of these contributed positively in the process of cellular adhesion.

The effect of electrospun fibre alignment on the behaviour of rat periodontal ligament cells

It is envisioned that for the regeneration of highly organized structures, like tendon and ligaments, only aligned fibrous scaffolds can provide adequate topographic guidance to cells. In this study, a novel method to electrospin an aligned scaffold is presented. Electrospun fibres were deposited into a water bath and then the fibres were drawn to a rotating mandrel in a controlled manner. In this way, parallel and cross-aligned fibrous poly (lactide-co-glycolide) (PLGA) scaffolds were fabricated, which were subsequently used to study their effect on the growth behaviour of rat periodontal ligament (PDL) cells. First, the scaffolds were characterized regarding mechanical properties, scaffold stability and degradation in vitro. Then, rat PDL cells were seeded and cultured on these scaffolds for up to 7 days. Randomly oriented PLGA and solvent cast plain PLGA films served as controls. Results showed that the alignment of fibres resulted in a higher tensile stress and Young's modulus. Aligned scaffolds maintained their structural stability better compared to the controls after incubation in phosphate-buffered saline for 6 weeks. Further, cells were observed to elongate along the fibre after 3 days of culture. Proliferation and migration of PDL cells was significantly more prevalent on the aligned fibres compared to the controls. It was concluded that aligned scaffolds seem to be able to promote the organized regeneration of periodontal tissue.

Age-related differences in the expression of heat shock protein 27 by rat periodontal ligament cells in culture

To evaluate age-related differences in the expression of heat shock protein (HSP) 27 in periodontal ligament (PDL) cells at the protein and mRNA levels, we obtained PDL cells from the incisors of rats weighing approximately 150 g (young group) and 350 g (aged group) and seeded the cells in culture dishes. Over 2 weeks of cultivation, PDL cells were subjected to quantitative reverse transcription polymerase chain reaction (RT-PCR) to measure HSP27 mRNA, and HSP27 protein expression was examined by immunofluorescence. HSP27 mRNA expression in the young group increased slightly over the 2 weeks, while in the aged group it increased significantly over the same time period. The expression ratio of HSP27 mRNA in the aged group was higher than in the young group at 14 days. Furthermore, immunofluorescence signals for HSP27 were stronger in the aged group than in the young group. These data reveal that PDL cells vary their expression of HSP27 with age, and suggest that in view of such changes in HSP27 mRNA expression, the self-defense mechanism is enhanced in aged PDL cells although their overall homeostatic conditions are suppressed.

Effect of Stretching Stress on Gene Transcription Related to Early-phase Differentiation in Rat Periodontal Ligament Cells

Mechanical stress such as occlusal and orthodontic loading has been suggested to induce a homeostatic and regenerative response in periodontal ligament (PDL), but the underlying mechanism remains to be clarified. The purpose of this study was to investigate expression of mRNAs encoding proteins involved in osteogenesis and homeostasis by PDL cells following application of tensile stress and characterize the relationship between such expression and the regenerative and homeostatic functions of the PDL. PDL cells were obtained from rats and stretched by 9% or 18% at a frequency of 6 cycles/min for 12 hr to 5 days in a FX-4000T&trade; culture system. After stretching, expression of mRNAs encoding collagen type I (Col-I), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4), heat shock protein 70 (HSP70) and basic fibroblast growth factor (bFGF) was investigated. The highest levels of Col-I, ALP and BMP-2 mRNA expression occurred at 12 hr, while those of BMP-4 and HSP70 occurred at 1 day and 5 days, respectively. Expression levels of Col-I, ALP, BMP-2, BMP-4 and HSP70 increased magnitude-dependently with stretching force in the stretching groups. In contrast, expression of bFGF mRNA showed statistically significant reduction in both stretching groups, with the largest reduction seen in the 9% stretching group (p<0.01). These results suggest that stretching of PDL cells provokes significant increases in expression of factors promoting osteogenic differentiation and HSP70, which protects PDL cells undergoing mechanical stress and contributes to maintenance of PDL homeostasis. However, expression of bFGF was restrained. Reduced expression of bFGF mRNA suggested that there was an optimum magnitude of stretching force for increasing expression.

The efficacy of programmed cryo-preservation under pressure in rat periodontal ligament cells

The purpose of this study was to evaluate the viability of peri odontal ligament cells in rat teeth usingslow cryo-preservation method under pressure by means of MTT assay and WST-1 assay. Eighteen teeth ofSprague-Dawley white female rats of 4 week-old were used for each group. Both sides of the first and second maxillary molars were extracted as atraumatically as possible underTiletamine anesthesia. The experimental groups were group 1 (Immediate control), group 2 (Cold preserva-tion at 4℃for 1 week), group 3 (Slow freezing), group 4 (Slow f reezing under pressure of 3 MPa). F-medi-um and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups,thawing was performed in 37℃water bath, then MTT assay and WST- 1 assay were processed. One wayANOVA and Tukey method were performed at the 95% level of confi dence. The values of optical densityobtained by MTT assay and WST-1 were divided by the values of e osin staining for tissue volume stan-dardization. In both MTT and WST-1 assay, group 4 showed significantly higher viability of periodontal ligament cellsthan group 2 and 3 (p < 0.05), but showed lower viability than immediate control group. By the results of this study, slow cryo-preservation method under pressure suggests the possibility forlong term cryo-preservation of the teeth.

Behavior of rat periodontal ligament cells on fibroblast growth factor‐2‐immobilized titanium surfaces treated by plasma modification

The aim of this study was to investigate the behavior of rat periodontal ligament (PDL) cells cultured on fibroblast growth factor-2 (FGF-2)-immobilized titanium surfaces treated by oxygen (O2) plasma. We used cell disks (15 mm in diameter), and 35-mm culture dishes sputter-coated with titanium. These were treated with oxygen plasma and dipped in FGF-2 solution. Immobilized FGF-2 was visualized with a confocal laser-scanning microscope, and its weight was calculated to be approximately 22.6 ng/cm(2) using a quartz crystal microbalance-dissipation apparatus. The PDL cells were obtained from rat incisors. Cells from fourth subculture were seeded onto the FGF-2-immobilized titanium surface. Proliferation ratio, alkaline phosphatase (ALP) activity, and expressions of type I collagen and vascular endothelial growth factor (VEGF) mRNAs were evaluated. Proliferation ratio and expressions of type I collagen and VEGF mRNAs were significantly higher, whereas ALP activity was significantly lower in FGF-2-immobilized cells than in control group (p < 0.05). These findings suggest that oxygen plasma modification can immobilize FGF-2 onto a titanium surface. Immobilized FGF-2, although inferior to culture medium with FGF-2, influenced the proliferation of PDL cells and might have promoted collagen and vascular synthesis.