OBJECTIVETo compared the difference in contact guidance activity among microgroove surfaces with different sizes of human gingival fibroblast (HGF), with the hope of providing basis for size selection of microgroove for transmucosal part of dental implant.

METHODSBasing on the size of HGF, microgroove titanium surfaces were fabricated by photolithography with parallel grooves: 15, 30 or 60 µm in width and 5 or 10 µm in depth. The groups that used different microgroove surfaces were denoted as T15/5, T15/10, T30/5, T30/10, T60/5, and T60/10. Group T0 (the control meanwhile was a sputter of titanium on a simple planar silicon substrate). The morphology that HGF arranged along the groove was analyzed by immunofluorescence staining. Difference in contact guidance activity was quantitatively compared basing on the consistency of nucleus arrangement and deformation ratio.

RESULTSMicrogroove groups had significantly higher consistency of nucleus arrangement and deformation ratio compared to the control group, with T60/10 had the highest consistency of 0.937±0.024, and T15/5 had the lowest consistency of 0.660±0.016 and T60/10 had the highest deformation ratio of 3.555±0.205, and T15/5 had the lowest deformation ratio of 1.819±0.011.

CONCLUSIONSMicrogroove surfaces of all the different sizes show contact guidance activity on HGF, and the contact guidance activity increases with the increase of width and depth.

Cell Adhesion ; physiology ; Dental Implants ; Fibroblasts ; cytology ; physiology ; Gingiva ; cytology ; Humans ; Surface Properties ; Titanium

UNLABELLEDThe periodontal ligament fibroblast (PDLF) was cultivated artificially as the cell to be tested, and then it was loaded with mechanical stress-strain of different values and for different times. The cell and nucleus projected areas and shapes as well as the structure of cytoskeleton were tested by use of confocal laser scanning microscope and immunity fluorescence technique. Then the relationship among the stress-strain, the time, the shape and the structure of cytoskeleton of the PDLF was detected.

RESULTSIn the trial groups of 0, 8%, 12%, 16% strain values, the cell and nucleus projected areas were proportional to strain (stress) and time. The diameter, density and order of the structure of cytoskeleton increased in the strain and time dependent fashion. In the trial group of 20% strain values, the cell and nucleus projected areas decreased with the increase of time, and the structure of cytoskeleton became disorderly. It was demonstrated in this study that the shape and structure of cytoskeleton of PDLF underwent regular changes when the PDLF was loaded with the mechanical stress-strain.

Cells, Cultured ; Cytoskeleton ; physiology ; Fibroblasts ; cytology ; physiology ; Humans ; Periodontal Ligament ; cytology ; Radioimmunoassay ; Stress, Mechanical ; Tensile Strength ; physiology

To investigate the effects of human mesenchymal stem cells (MSC) and human fibroblastoid cell line (HFCL) as feeder layer on expansion of umbilical cord blood CD34(+) cells in vitro, (60)Co gamma-ray irradiated MSC and HFCL were used as feeder layer to expand cord blood CD34(+) cells in culture. The efficiencies of MSC and HFCL on expansion of CD34(+) cells in culture with or without cytokines were compared. The results showed that no matter whether cytokines (rhFL, rhSCF, rhTPO) were added, the proliferation of nucleated cells after expansion for 12 days in HFCL group was statistically higher than that in MSC group, i.e. with cytokines (9797 +/- 361)% vs (7061 +/- 418)%; without cytokines (5305 +/- 354)% vs (1992 +/- 247)%, when the cell numbers at day 0 was accounted as 100%), P < 0.01. The proliferation of propagated CD34(+) cells between MSC group and HFCL without addition of cytokines was not statistically different (820 +/- 191)% vs (825 +/- 305)%, P > 0.05. However, in the presence of cytokines, the propagating rate of MSC group was lower than that of HFCL group (939 +/- 212)% vs (1617 +/- 222)%, P < 0.01. MSC was better than HFCL in maintaining the LTC-IC of UCB CD34(+) cells, i.e. the number of CFU-GM colonies in the fifth week was (129.95 +/- 8.73) /10(5) seeded cells vs (89.81 +/- 10.29) colonies/10(5) cells, P < 0.05; with addition of cytokines, the effect was more obvious, i.e. the number of CFU-GM colonies in the fifth week (192.93 +/- 4.95)/10(5) seeded cells vs (90.47 +/- 14.28) colonies/10(5) seeded cells, P < 0.01. MSC mixed with a certain proportion of HFCL facilitated maintaining the LTC-IC of UCB CD34(+) cells. When the proportion was 4:1, the number of CFU-GM colonies was the highest (186.89 +/- 11.11)/10(5) seeded cells, which was higher than that of both 3:2 group [(138.92 +/- 14.84) colonies/10(5) seeded cells] and MSC only group, i.e. (64.63 +/- 6.11) colonies/10(5) seeded cells, both P < 0.01. It is concluded that HFCL is better than MSC in maintaining the expansion of CD34(+) cells and cytokines can enhance this effect, while MSC are stronger than HFCL in maintaining the LTC-IC of UCB CD34(+) cells in vitro. MSC with addition of a certain proportion of HFCL can significantly enhance the efficiency of CD34(+) cell expansion.
Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; physiology ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Fibroblasts ; cytology ; physiology ; Humans ; Mesenchymal Stromal Cells ; cytology ; physiology

Epithelial-mesenchymal transition (EMT) is a process by which fully differentiated epithelial cells undergo a phenotypic conversion and assume a mesenchymal cell phenotype, including elongated morphology, enhanced migratory and invasiveness capacity, and greatly increased production of extracellular matrix (ECM) components. The EMTs associated with wound healing, tissue regeneration, and organ fibrosis are termed as type 2 EMT. Over the past two decades, emerging evidence suggested that injured epithelial cells, via type 2 EMT, may serve as important sources of fibroblasts and contribute to organ fibrosis, such as kidney, liver, lung and eyes. There is perhaps no doubt that adult epithelial cells can undergo EMT in vitro in response to transforming growth factor (TGF)-β1 and other inflammatory or pro-fibrotic stimuli. However, whether type 2 EMT really occurs in vivo, whethers it is actually a source of functional and activated interstitial fibroblasts and whether it contributes to tissue fibrosis have already been the subjects of heated debate. In this review, we will describe the main features of EMT, the major findings of type 2 EMT in vitro, the evidences for and against type 2 EMT in vivo and discuss the heterogeneity and pitfalls of the techniques used to detect EMT during fibrotic diseases. We suggest that in order to ascertain the existence of type 2 EMT in vivo, different proper phenotype markers of epithelial and mesenchymal cells should be jointly used and cell lineage tracking techniques should be standardized and avoid false positives. Finally, we believe that if EMT really occurs and contributes to tissue fibrosis, efforts should be made to block or reverse EMT to attenuate fibrotic process.
Animals ; Epithelial-Mesenchymal Transition ; physiology ; Fibroblasts ; cytology ; metabolism ; Fibrosis ; metabolism ; pathology ; Humans

To evaluate the feasibility of using human embryonic fibroblast(HEF) as feeder layer in the culture of human embryonic stem(ES) cells in vitro, we investigated the morphology, the sensitivity to 0.25% trypsin, the growth curve and cell cycle of HEF with DMEM(low glucose) +10% FBS used as culture medium, and then we compared HEF with mouse embryonic fibroblast (MEF). The results showed that both HEF and MEF are adherent cells in vitro, and HEF has longer life span and better growth ability than MEF. In room temperature, HEF is more sensitive to 0.25% trypsin. Our research suggested that HEF can be used as feeder layer in culture of ES cells. HEF has longer service life than MEF and is worthy to be studied further.
Animals ; Cell Cycle ; Cell Differentiation ; Cell Division ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fibroblasts ; cytology ; physiology ; Humans ; Mice

The effect of alveolar macrophages (AM) harvested from Wistar rats by lung lavage on proliferation of human embryo pulmonary fibroblasts in culture was investigated. It was observed that supernatants of AM decreased the uptake of (3)H TdR by the pulmonary fibroblasts. The AM activated with opsonized zymosan (OPZ) showed a stronger inhibitory effect on fibroblast proliferation compared with inactivated AM. Following pretreatment with indomethacin, the inhibitory effect of AM was abolished and reversed to stimulatory effect on pulmonary fibroblast proliferation. The PGE content in AM supernatant was measured with radioimmunoassay. It was observed that the inhibitory effect of AM was highly correlated to prostaglandin (PGE) content in the supernatant of AM. The results suggest that AM has both inhibitory and stimulatory effects on the proliferation of pulmonary fibroblast; the inhibitory effect is primary under normal conditions. This inhibitory action is mainly due to PGE secreted from AM. It is, therefore, suggested that AM plays an important role in suppressing pulmonary fibrosis under normal conditions.
Animals ; Cell Division ; physiology ; Cells, Cultured ; Female ; Fibroblasts ; cytology ; Humans ; Lung ; cytology ; embryology ; Macrophages, Alveolar ; physiology ; Male ; Prostaglandins E ; analysis ; physiology ; Pulmonary Fibrosis ; pathology ; Rats

AIMTo explore the effects of tRNA on the growth of mammalian cells.

METHODSL929, NIH3T3, MCF-7 and PC12 cells were seeded in 96 well culture plate individually, and incubated at 37 degrees C in 5% CO2 for 4 h, the tRNAs from different species were added to the culture media individually. After certain time of incubation, the viability of the cells was evaluated by the MTT methods. Sub-confluent L929 cells were incubated with 200 microg/ml ytRNA for different times, then the cells were pooled and analyzed with flow cytometry assay.

RESULTStRNA specifically inhibited the growth of L929 cells in a dose-dependent manner. The sizes of tRNA-treated cells showed larger sizes and longer processes than those of untreated cells. Flow cytometric analysis further showed that most of tRNA-treated cells were arrested in S phase of the cell cycle.

CONCLUSIONThe cell growth inhibitory effects of tRNAs were caused mainly by their degraded fragments. The results suggested that tRNA or its degraded fragments might play important roles in regulation of cell proliferation.

Animals ; Cell Cycle Checkpoints ; physiology ; Cell Line ; Cell Proliferation ; Fibroblasts ; cytology ; Flow Cytometry ; Mice ; RNA, Transfer ; physiology

Animals ; Bone Regeneration ; physiology ; Cell Differentiation ; Dental Enamel Proteins ; pharmacology ; Dental Pulp ; cytology ; Fibroblasts ; cytology ; Gingiva ; cytology ; Guided Tissue Regeneration, Periodontal ; methods ; Humans ; Induced Pluripotent Stem Cells ; cytology ; physiology ; Mouth Mucosa ; cytology ; Periodontal Ligament ; cytology ; Tissue Engineering ; methods

One of the differences between fetal and adult skin healing is the ability of fetal wounds heal without contraction and scar formation. Extracellular matrix (ECM) provides a substratum for cells adhesion, migration, and proliferation and can directly influence the form and function of cells. As motility is essential for many important biological events, including wound healing, inflammatory response, embryonic development, and tumor metastasis, this study was designed to compare the motilities cultured dermal fetal and neonatal fibroblasts in the extracellular matrix. The motility of cultured fetal and neonatal fibroblasts was compared using a video-microscopy system that was developed in combination with a self-designed CO2 mini-incubator. To determine migration speed, cells were viewed with a 4X phase-contrast lens and video recorded. Images were captured using a color CCD camera and saved in 8-bit full-color mode. We found that cultured fetal fibroblasts move faster than neonatal fibroblast on type I collagen (fetal fibroblast, 15.1 micrometer/hr; neonatal fibroblast, 13.7 micrometer/hr), and in fibronectin (fetal fibroblast, 13.2 micrometer/hr; neonatal fibroblast, 13.0 micrometer/hr) and hyaluronic acid (fetal fibroblast, 11 micrometer/hr; neonatal fibroblast, 9.8 micrometer/hr).
Cell Movement ; Cells, Cultured ; Comparative Study ; Extracellular Matrix/*physiology ; Fetus/physiology ; Fibroblasts/*physiology ; Human ; Infant, Newborn ; Skin/cytology/*embryology ; *Skin Physiology

UNLABELLEDObtaining human blastocysts is a prerequisite for cell replacement therapy using embryonic stem cells. We established an interspecies somatic cell nuclear transfer (iSCNT) technique for producing blastocysts without sacrificing human oocytes. Human foetal fibroblasts were used as donor cells injected into the enucleated bovine oocytes in nuclear transfer, whereas bovine foetal fibroblasts were used to produce intraspecies embryos. We also examined the fate of human and bovine mitochondrial DNA (mtDNA) during preimplantation development after nuclear transfer by PCR. PCR analysis for the detection of human and bovine mtDNA was done at the 2,8-morula, and blastocyst stages of the embryos.

RESULT2.8% interspecies embryos developed to blastocysts after cultured in an SOF medium, while blastocyst rate of intraspecies embryos were 10.1%. Both human and bovine mtDNAs existed until the morula stage, whereas only the bovine mtDNA was found at the blastocyst stage. These results indicated that interspecies cloning without using human oocytes could generate human blastocysts. Because of the incoordination between bovine mtDNA and human nuclear gene, developmental rate of interspecies embryos was significantly lower than intraspecie. Whether the embryonic stem cell could be used for cell replacement therapy need further research.

Animals ; Blastocyst ; cytology ; physiology ; Cattle ; Cloning, Organism ; DNA, Mitochondrial ; genetics ; Embryonic Development ; physiology ; Female ; Fibroblasts ; physiology ; Humans ; Nuclear Transfer Techniques ; Oocytes ; physiology ; Species Specificity