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

Animals ; Cellular Reprogramming ; Dentate Gyrus ; cytology ; Fibroblasts ; cytology ; Mice ; Neural Stem Cells ; cytology ; transplantation ; Swine

A line of fibroblast-type cells derived from embryos of a domestic rabbit has been cultivated continuously for over 3 years by serial passages up to the level of the moth passage. The cell line was tentatively named rabbit embryo fibroblast (REF). The establishment of primary culture, serial passages, growth rate and cytology are described in this communication. In addition some of the results of experiments on the detection of Mycoplasma contamination, on storage of the frozen cells and on its susceptibility to vaccinia virus infection are included.
Animal ; Cell Line ; Embryo/cytology* ; Fibroblasts* ; Rabbits ; Tissue Culture*

OBJECTIVETo acquire lots of cell to culture during the primary cell culture.

METHODWe take the split-thickness skin slide technique to acquire the dissociated fibroblast cell in two big-ear rats.

RESULTSThe cell number is above 10(6) from 1 cm x 2 cm split-thickness skin slide and the technique is simple, economic, effectve.

CONCLUSIONWe think this way is better than other methods, and should be adopted in the primary cell culture, especially in fibroblast transplantation by injection.

Animals ; Cell Culture Techniques ; methods ; Fibroblasts ; cytology ; transplantation ; Rabbits

OBJECTIVETo explore the way of stably inducing canine bone marrow mesenchymal stem cells (BMSCs) to differentiate into fibroblasts and myofibroblasts in vitro, and provide seed cells for fabricating tissue engineering heart valves (TEHV).

METHODSAdult canine BMSCs were separated by a gradient centrifugation on Percoll (density 1.073 g/ml), then the cells were incubated in low-glucose Dulbecco Eagle's minimum essential medium (LG-DMEM) with 10% bovine calf serum. Cell phenotype were identified by immunohistochemistry staining. The second and third generation of BMSCs were committedly induced by conditioning culture medium, which were detected by immunohistochemistry staining. The induced-BMSCs were freezed, preserved and resuscitated after 7 d to observe the cell growth, proliferation and function.

RESULTSBMSCs deriving from the bone marrow mononuclear cells separated by a Percoll gradient were positive expression of alpha-smooth muscle antibody, vimentin and negative expression of CD34, laminin. About (50 +/- 3)% induced-BMSCs were positive expression of laminin. Approximately (85 +/- 3)% freezed induced-BMSCs could be resuscitated. And the growth, proliferation and function were well.

CONCLUSIONBMSCs could be committedly induced to differentiate into fibroblasts and myofibroblasts in vitro. It is suitable to be the seed cells.

Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Dogs ; Fibroblasts ; cytology ; Mesenchymal Stromal Cells ; cytology ; Monocytes ; cytology ; Myoblasts ; cytology

OBJECTIVETo explore the feasibility of constructing tissue-engineered cartilage with human dermal fibroblasts (HDFs) in vitro.

METHODSPorcine articular chondrocytes and HDFs were isolated and in vitro expanded respectively. Then they were mixed at the ratio of 1:1 (chondrocytes: fibroblasts) . The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml as co-culture group. Chondrocytes and HDFs at the same ultimate concentration were seeded respectively onto the scaffold as chondrocyte group ( positive control group) and fibroblast group ( negative control group). The specimens were collected after in vitro culture for 8 weeks. Gross observation, histology and immunohistochemistry were used to evaluate the results.

RESULTSIn chondrocyte group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture. The new formed cartilage-like tissue had typical histological structure and extracellular matrix staining similar to normal cartilage. In co-culture group the constructs shrunk slightly at 8 weeks, cartilage-like tissue formed and GAG could be detected for strong expression by Safranin O staining. Furthermore, using the specific identification, a few HDFs derived cells were found to form lacuna structure at the peripheral area of cartilage-like tissue. In fibroblast group, the constructs deformed and shrunk gradually without mature cartilage lacuna in histology.

CONCLUSIONThe 3D-co-culture system can effectively induce the differentiation of HDFs to chondrocytes. The tissue-engineered cartilage can be constructed in vitro with the 3D-co-culture system.

Animals ; Cartilage ; cytology ; Cells, Cultured ; Chondrocytes ; cytology ; Coculture Techniques ; Dermis ; cytology ; Fibroblasts ; cytology ; Humans ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo investigate the methods and conditions for the isolation, purification and culture of human spermatogonial stem cells (SSCs) on the feeder layer cells of human embryonic fibroblasts (hEFs).

METHODSSSCs isolated and purified from normal human fetal testicular tissues by sequential two-step enzyme digestion and Percoll uncontinuous density gradient centrifugation were cultured on the feeder layer cells of hEFs isolated from 5-9 weeks old human embryos. The surface markers SSEA-1 and OCT4 of the SSCs were detected by immunohistochemistry; the alkaline phosphatase (AKP) activity of the SSC clones measured; and the expressions of the SSC-related genes determined by RT-PCR.

RESULTSSSCs survived, proliferated and formed colonies on the feeder layers, and the colonies were highly positive for SSEA-1 and OCT4, with strong AKP activity and high expressions of the SSC-related genes.

CONCLUSIONThe feeder layer of hEFs supports the growth of human spermatogonial stem cells.

Cell Culture Techniques ; methods ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fibroblasts ; cytology ; Humans ; Male ; Spermatogonia ; cytology ; Stem Cells ; cytology

OBJECTIVETo investigate the influence of human telomerase reverse transcriptase (hTERT) gene transfection on the proliferation of human embryonic fibroblasts (hEF).

METHODShEFs were cultured in vitro. Sense recombinant eukaryotic plasmid (pIRES2-EGFP-hTERT) and pIRES2-EGFP vacant vector were transfected into hEF respectively with Lipofectin reagent, and were named as hEF-hTERT and hEF-EGFP. The hTERT, Id1, PCNA and I, III type collagen expression in these cells were detected by Western blot. Then the cell cycle and growth curve were measured and plotted with flow cytometry and MTT method, respectively.

RESULTS1. The expression of hTERT, Id1, PCNA, type I and III collagen in hEF-hTERT were much higher than that in hEF and hEF-EGFP. 2. As shown in the growth curve, the OD value of hEF-hTERT at 4 to 6 days after culture was obviously higher than that of hEF and hEF-EGFP (P < 0.05), while no difference existed between hEF and hEF-EGFP from 1 to 6 days after culture (P > 0.05). 3. The cell number in G0/G1 phase in hEF-hTERT was less than that in hEF and hEF-EGFP. The cell number of hEF-hTERT in S and G2/M phase and its proliferation index (57.47%) increased when compared with that in hEF-EGFP (13.13%) and hEF (17.38%), but there was no difference between hEF and hEF-EGFP.

CONCLUSIONExogenous hTERT gene transfection could promote the proliferative capacity of hEF.

Cell Proliferation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Fibroblasts ; cytology ; Humans ; Telomerase ; genetics ; Transfection

OBJECTIVETo investigate the role of apoptosis in the remodeling of temporomandibular joint (TMJ) under pressure.

METHODSSynovial fibroblasts obtained from rat temporomandibular joint were subjected to different hydrostatic pressure for 12 h. Changes of ultrastructure were observed by transmission electron microscope.

RESULTSAt 30 kPa, the ultrastructure of synovial fibroblasts showed no obvious changes. At 60 kPa, the chromatin was condensated, the baryon took on crescent and the mitochondria seemed varicose. At 90 kPa, the apoptosis-like body was wrapped by membrane and embedded in the high density chromatin.

CONCLUSIONSApoptosis-like change took place in ultrastructure of synovial fibroblasts under hydrostatic pressure, and the degree of the change was related to the hydrostatic pressure exerted.

Animals ; Apoptosis ; Fibroblasts ; ultrastructure ; Hydrostatic Pressure ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; Temporomandibular Joint ; cytology

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