OBJECTIVETo investigate the integrity of multilayer of human periodontal ligament fibroblasts (HPDLF) and human gingival fibroblasts (HGF) on filter membrane of Transwell and to provide basis for the drug transcellular transport by the HPDLF and HGF in the hypothesis of delivering medicine to the periodontium and whole body through the root canal.

METHODSHPDLF and HGF derived from the primary culture were seeded on polycarbonate filter membrane of transwell respectively. After 1, 2, 3 and 4 weeks of culture, transepithelial electrical resistance (TEER) was detected and the growth of HPDLF and HGF observed by light microscope. After 2 weeks of culture, section of filter membrane where HPDLF and HGF lived was observed with light microscope and transmission electron microscope (TEM), and the permeability of the drug transport cell models was measured with fluorescein sodium.

RESULTSHPDLF and HGF converged 1 week after inoculation, and the cells connected each other tightly and completely 2 weeks later. Observation of section of filter membrane by light microscope and TEM revealed a stratified cell growth of HPDLF and HGF 2 weeks after inoculation, and TEER of HPDLF and HGF were (56.14 +/- 7.43) and (57.34 +/- 7.62) ohm.cm(-2) respectively. The values of TEER remained the same level until 4 weeks later. Two weeks after inoculation, the paracellular transport of fluorescein sodium was less than 1% after the cell models were incubated for 30 min.

CONCLUSIONSStratified cell layers of HPDLF and HGF grown on filter membrane of Transwell are analogous to periodontal membrane and gingiva 2 weeks after inoculation, the test results of permeability and TEER were consistent with the demands of development of cell models. HPDLF and HGF grown on filter membrane of Transwell could be used to study drug transcellular transport by HPDLF and HGF in vitro.

Cell Proliferation ; Cells, Cultured ; Fibroblasts ; cytology ; Filtration ; instrumentation ; Gingiva ; cytology ; Humans ; Periodontal Ligament ; cytology

OBJECTIVETo isolate and cultivate human periodontal ligament stem cells (PDLSC) and to investigate the feasibility of PDLSC in vitro differentiation into chondrogenic phenotype.

METHODSPeriodontal tissue was obtained from healthy young human teeth extracted for orthodontic purposes. PDLSCs were isolated by single-colony selection and cultivated. PDLSC of passage 3 was plated at density of 1 x 10(7) cells/cm3 and induced with chondrogenic induction medium of DMEM containing TGF-beta1 (10 microg/L), IGF-1 (50 microg/L), dexamethasone (40 microg/L) and 10% FBS. In control group, the constructs were maintained in DMEM medium + 10% FBS. After 21 days induction, the results were evaluated by histology, histochemistry, immunohistochemistry and RT-PCR.

RESULTSThe constructs in experimental group were smooth and relatively firm in texture after 3 weeks of culture. Toluidine blue staining showed the formation of distinct lacuna structure. Positive staining of type II collagen was also detected by immunohistochemistry and it was confirmed by RT-PCR. In contrast, in the control group, the constructs collapsed gradually, lacuna was barely detected in histology and type II collagen expression negative.

CONCLUSIONSPeriodontal ligament contain stem cells can be isolated and cultivated. PDLSC have the potential of chondrogenic differentiation.

Adolescent ; Adult Stem Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Child ; Chondrocytes ; cytology ; Humans ; Periodontal Ligament ; cytology

OBJECTIVETo fabricate the bio-implant supported by regenerated periodontal tissue utilizing periodontal ligament cells (PDLC)-bone marrow stromal cells (BMSC) sheet and natural root.

METHODSPremolars of 2 beagle dogs were extracted to prepare the implanted area. Autologous tooth roots were carved into cylinders. PDLC and BMSC separated from beagle dogs were cultured into cell sheets in medium. Tooth roots were wrapped by one type of cell sheets or both to fabricate bio-implant and divided into four groups, tooth roots were wrapped by PDLC sheets and BMSC sheets successively (2 samples each dog), tooth roots were wrapped by PDLC sheets alone (2 samples each dog), tooth roots were wrapped by BMSC sheet alone (2 samples each dog), tooth roots without cell sheet (1 sample each dog). The implants were implanted into the mandibles. The mandibles were dissected 12 weeks later, sliced and stained by HE and Masson dyes for histological examination.

RESULTSIn the PDLC cell sheet/root implants, histological examination revealed that new periodontal-like tissue including cementum, periodontium and alveolar bone was regenerated.In the BMSC implants, tooth ankylosis was observed.

CONCLUSIONSPDLC sheet and natural root can be used to fabricate bio-implant. PDLC sheet could promote periodontal regeneration.

Animals ; Bicuspid ; Dental Cementum ; Dogs ; Guided Tissue Regeneration, Periodontal ; Mesenchymal Stromal Cells ; Periodontal Ligament ; cytology ; Periodontium ; Regeneration ; Tooth Root

OBJECTIVETo investigate the possibility of human umbilical cord mesenchymal stem cells (hUCMSC) differentiating into human periodontal ligament cells (hPDLC) by establishing a coculture model in vitro.

METHODSIndirect coculture model of hPDLC with hUCMSC was established on Transwell system. The protein expression of bone sialoprotein(BSP), osteocalcin(OCN) and osteopontin (OPN) was examined by immunohistochemical staining. The changes of hUCMSC on molecular level were analyzed by Western blotting.

RESULTShUCMSC was in the form of polygon and rhombus after induction by hPDLC. Immunohistochemistry staining and Western blotting showed that OCN and OPN were up-regulated [OCN:(0.88 +/- 0.21)vs (1.42 +/- 0.17), OPN: (0.93 +/- 0.13) vs (1.43 +/- 0.22), P < 0.05] and BSP was down-regulated [(1.60 +/- 0.09) vs (0.75 +/- 0.20), P < 0.05] after coculture.

CONCLUSIONShUCMSC can differentiate into hPDLC under certain conditions in vitro and hUCMSC may hopefully become the stem cells in periodontal tissue engineering.

Cell Differentiation ; Cells, Cultured ; Coculture Techniques ; Humans ; Mesenchymal Stromal Cells ; cytology ; Periodontal Ligament ; cytology ; Tissue Engineering ; Umbilical Cord ; cytology

OBJECTIVETo compare the multilineage differentiation potential of human dental pulp cells (DPC) and periodontal ligament cells (PDLC) in vitro, and to identify the stem cell characteristics.

METHODSHuman DPC and PDLC were isolated by enzymatic digestion. STRO-1 expression was investigated by flow cytometry. Cells were induced to odontogenic/osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. The multilineage differentiation capacities of DPC and PDLC were evaluated by von Kossa stain, anti-osteocalcin (OCN) and anti-dentin sialoprotein (DSP) immunocytochemistry, oil red O stain, Alcian blue stain, anti-collagen type II immunocytochemistry, and real time RT-PCR.

RESULTSColony formation was observed in DPC and PDLC, with STRO-1 positive rate of (16.5% +/- 4.2%) and (11.6% +/- 1.1%) respectively. Multilineage differentiation was demonstrated in 100% of DPC samples in contrast to 83.3% of PDLC samples. OCN, dentin sialophosphoprotein (DSPP), peroxisome-proliferator-activated receptor gamma 2 (PPARgamma2), lipoprotein lipase (LPL) and collagen type II mRNA levels in DPC and PDLC were significantly upregulated after induction (P < 0.001). There were significant differences in the ratio of upregulation of OCN and PPARgamma2 mRNAs between DPC and PDLC (P < 0.001).

CONCLUSIONSDPC and PDLC contain similar proportion of mesenchymal stem cells and possess comparable multilineage differentiation capacities.

Cell Differentiation ; Cells, Cultured ; Dental Pulp ; cytology ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Periodontal Ligament ; cytology

Magnetic attachments have flux leakages, thus they will exert certain magnetic fields on the adjacent tissues when used in the patients' oral cavities. There are few research reports on the biological effects of the magnetic fields generated by magnetic attachments on human body. A cellular static magnetic field (SMF) loading system was developed in this study. By using this system, in vitro cultured human periodontal ligament cells (HPDLCs) were loaded with SMF simulating those of magnetic attachments. The cellular SMF loading system could produce constant SMF and the strength of the SMF is adjustable. The system is small and is able to exert SMF to cells cultured in different culture vessels such as culture dishes and culture plates, thus is suitable to researches in multiple biological items of cells. The results of the SMF loading experiment on HPDLCs showed that this cellular SMF loading system could effectively load cells with SMF of different strengths for different time in vitro. The development of this system has provided a useful tool for the researches on the cellular hiologioal effects of SMF.
Cells, Cultured ; Electromagnetic Fields ; adverse effects ; Humans ; Magnetics ; Periodontal Ligament ; cytology ; metabolism ; radiation effects

OBJECTIVETo obtain regulating proteins during human periodontal ligament cell (hPDLC) osteogenic differentiation and investigate its underlying molecular mechanism.

METHODSTwo-dimension difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser adsorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis was used to identify regulating proteins during hPDLC osteogenic differentiation.

RESULTSA differently expressed protein profile was obtained 7 days after osteogenic induction, and 61 protein spots with significant differences, 29 protein spots were identified by MALDI-TOF-MS, including cytoskeleton, cell membrane-bounding, nuclear regulation, matrix synthesis, and metabolic enzymes and signal transduction and other proteins. A functional class of cytoskeleton proteins showed co-regulation, these proteins were intimately involved in the reorganization of the cytoskeleton, cytokinesis and migration during PDLC differentiation.

CONCLUSIONSA database of differently displayed proteins during hPDLC osteogenic differentiation was established, which may be helpful to understand and further study molecular mechanism of PDLC osteogenic differentiation.

Cell Differentiation ; Cells, Cultured ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Osteogenesis ; Periodontal Ligament ; cytology ; Proteomics

This study aims at exploring the effects of tensile strain and loading time on the secretion of IL-1 beta of human periodontal ligament fibroblast. Five tensile strain values including 0%, 8%, 12%, 16%, 20% and three loading time including 24 h, 48 h, 72 h are set in this study. The prepared cell samples are mounted on the self-devised loading apparatus in vitro. The content of IL-1 beta in each sample was determined using double-antibody ELISA. The secretion amount of IL-1 beta per day is directly proportional to the loading time and tensile strain value in tensile strain group of 8%, 12%, 16%. The secretion amount of IL-1 beta reaches its maximum at tensile strain value of 16% in loading time groups. Loaded strain for 24 h and 48 h, the secretion amount of IL-1 beta at tensile strain value of 20% is obviously more than that at value of 0%, but the amount already begin to decrease apparently. Loaded strain for 72 h, the secretion amount of IL-1 beta decreases to a great extent that it is less than the amount at strain value of 0%. The tensile strain stimulates the secretion of IL-1 beta by human periodontal ligament fibroblast when the strain is under normal physiological extent, but the stimulation effect fades out as time goes on.
Cells, Cultured ; Fibroblasts ; secretion ; Humans ; Interleukin-1 ; secretion ; Periodontal Ligament ; cytology ; secretion ; Tensile Strength ; Time Factors

OBJECTIVETo investigate the effects of 17beta-estradiol on the expression of alkaline phosphatase (ALP) and osteoprotegerin in human periodontal ligament cells.

METHODSHuman periodontal ligament cells (hPDLC) were obtained from periodontal tissue explants of teeth extracted for orthodontic treatment ALP activity was determined by PNPP, and OPG protein and corresponding mRNA levels were quantitatively detected by ELISA and RT-PCR RESULTS: ALP activity was significantly increased at 14 days and 21 days (P <0.05). 17beta-E2 of physiological concentration promoted secretion of OPG protein and expression of OPG mRNA (P <0.05). 17beta-E2 with high-dose showed no effect on OPG protein secretion and decrease OPG mRNA expression.

CONCLUSIONS17beta-E2 may have a positive impact on periodontium through promoting expression of ALP and OPG in hPDLC.

Alkaline Phosphatase ; metabolism ; Cells, Cultured ; Estradiol ; pharmacology ; Humans ; Osteoprotegerin ; metabolism ; Periodontal Ligament ; cytology ; drug effects ; metabolism

OBJECTIVETo explore the biological effects of tetracycline on cultured human periodontal ligament fibroblasts (HPDLFs).

METHODSIncreasing concentrations of tetracycline (1, 5, 20, 100, 500, 2500 microg/ml) were added to the medium of cultured HPDLFs, respectively. After co-incubated for 2 days, cell morphology was observed under reverse microscope, meanwhile, cell proliferation activity was assayed using MTT, the total amount of protein was detected with Coumassie Bright Blue method and DNA synthesis was measured by 3H-TdR.

RESULTSOver a concentration range of 1 to 100 microg/ml, cells demonstrated a normal appearance, spindle or fusiform shaped. Moreover, at a concentration range of 20 to 100 microg/ml, tetracycline significantly enhanced the proliferating activity and biosynthesis of HPDLFs (P < 0.01). However, higher concentration (2500 microg/ml) not only changed cell morphology, but also significantly inhibited cellular activity.

CONCLUSIONThe results suggested that proper doses of tetracycline could promote proliferation and biosynthesis of HPDLFs while higher concentrations of tetracycline had cytotoxic effect.

Cells, Cultured ; Fibroblasts ; drug effects ; Humans ; Periodontal Ligament ; cytology ; drug effects ; Tetracycline ; pharmacology