To investigate the effect of hepatocyte growth factor (HGF) on prol iferation of cultured human eccrine sweat gland epithel ial cells (hESGc) and the involvement of phosphorylation of ERK1/2. Methods hESGc were cultured in keratinocyte serum free medium (KSFM) and the first generation of hESGc was harvested. The expression of C-met was detected by immunocytochemistry. MTT assay was used to detect the effect of HGF on the prol iferation of hESGc. The cells were divided into blank group, control group and experimental group. The culture density was 2 × 103 cells/hole in control group and experimental group. Two hundred μL KSFM with HGF in different levels was added to every hole. hESGcwere cultured in KSFM with HGF at different levels (2, 20, 40 and 80 ng/mL) in experimental group, in KSFM without HGF incontrol group, and in KSFM without HGF and no hESGc in blank group. The cell prol iferation was observed in xperimental group 2 and 4 days later. Western blot was used to detect the expression of phosphorylated ERK1/2 at 40 ng/mL HGF after 0, 5, 30, 90 and 120 minutes. Results The results were positive for anti-C-met staining in the cytoplasm. HGF (40 ng/mL and 80 ng/mL) significantly improved the prol iferation of hESGc (P lt; 0.05). When cultured in the KSFM with 40 ng/mL HGF, the cell prol iferation rate and the absorbance were 74.2%, 0.239 3 ± 0.070 9 at 2 days and 74.8%, 0.287 8 ± 0.074 3 at 4 days; showing significant differences when compared with control group (P lt; 0.05). When cultured in KSFM with 80 ng/mL HGF, the cell prol iferation rate and the absorbance were 54.5%, 0.212 3 ± 0.059 2 at 2 days and 40.3%, 0.231 0 ± 0.056 7 at 4 days; showing significant differences when compared with control group (P lt; 0.05). The expression of p-ERK1/2 reached to the maximum after stimulation of 40 ng/mL HGF for 5 minutes, and relative integral absorbance (RIA) was 0.593 2 ± 0.192 2, increased 8.1 times compared with instant stimulation (P lt; 0.01). Conclusion HGF could induce the prol iferation of hESGc and activate the phosphorylation of ERK1/2 protein.
In order to study the biological characteristics of tenocyte and fibroblast, the former was obtained from rabbit’s tendon, and the latter from rabbits’s skin. Both cells were cultured according Heuderson’s method. The cell morphology, strapping and expanding time, and the type of collagen fiber synthesized in culture were observed. The results showed that the strapping and expanding time of fibroblast was faster than that of tenocyte. The cellular arrangement of fibroblast was irregular, but that in tenocyte was regular. Type I and III collagen of fibers were found in cultured fibroblost while only type I collagen fibers were found in culture of tenocyte. The tenocyte and fibroblast could be identified individually by strapping and expanding time, arrangement of cells and type of collagen fiber synthesized.
The heart valve prosthesis must have excellent hydrodynamic performance which is usually tested in vitro, not in vivo. This paper comprehensively introduced the principles and methods of hydrodynamic performance in vitro testing, helping clinicians to understand valve performance parameters, evaluate valve applicability, and reduce clinical risk of the valve prosthesis. In vitro testing not only serves as the "gold standard" for valve prosthesis assessment, but also provides detailed data for design and optimization of the prosthesis. ISO 5840 defines the items and methods for valve in vitro testing, which consists of three parts: (1) pulsatile flow testing, which reproduces the pulsating flow of the valve prosthesis after implantation in the human body; (2) steady flow testing, which assesses valve forward flow resistance; (3) durability testing, which evaluates the durability of the valve prosthesis and determines the expected failure mode. In addition, the paper presented the differences between atrioventricular and aortic valve testing, the method of mitral valve testing, the differences between transcatheter and surgical valve testing, and the method of valve flow visualization.
To study the effect of autogeneic PRP on prol iferation and osteogenetic differentiation of human adipose-derived stem cells (ADSCs) in vitro. Methods ADSCs were isolated from adipose tissue obtained from donor undergoing l iposuction and were cultured, and growth condition of the cells was observed by inverted microscope. ADSCs at passage 3 were cultured in adipogenic or chondrogenic medium and underwent identification, immunofluorescence staining observations for CD29 and CD44 were performed. ADSCs at passage 3 were divided into 2 groups: PRP group cultured by osteogenic induction culture medium containing 10 mL/L PRP, and control group cultured by osteogenic induction culture medium without PRP. Then growth condition of the cells was observed by inverted microscope. MTT method was used to observe cell prol iferation activity 1, 2, 3, 4 and 5 days after culture. ALP activity detection was conducted 7, 14, 21 and 28 days after culture. ALP staining was performed on PRP group 7 and 14 days after culture. Al izarin red staining was performed on PRP group 14 days after culture to detect the formation of calcium nodule. Results Under the inverted microscope, most ADSCs at passage 3 were spindle-shaped and the doubl ing time was about 35 hours. Adipogenic and chondrogenic differentiation were confirmed, and the cells were positive for CD29 and CD44 immunofluorescence staining. MTT method revealed the absorbance value of PRP group at 1, 2, 3, 4 and 5 days was 0.137 ± 0.015, 0.219 ± 0.023, 0.367 ± 0.031, 0.586 ± 0.039 and 0.948 ± 0.046, respectively, and in the control group, it was 0.081 ± 0.009, 0.115 ± 0.012, 0.162 ± 0.017, 0.242 ± 0.025 and 0.356 ± 0.032, respectively, suggesting there were significant differences between two groups (P lt; 0.01). At 7 days after osteogenic induction, PRP group was positive for ALP staining, grey-black cell plasm and black precipitate were evident; the positive cells increased
Objective To design a novel stentless porcine aortic bioprosthesis and test the feasibility and its function in vitro after the valve was implanted by a modified method. Methods Six stentless porcine aortic bioprosthesis were divided into two groups according to different implantation, single layer suture group: new improvement stentless porcine aortic bioprosthesis sutured with single layer was implanted; double layer suture group: stentless porcine aortic bioprosthesis developmented by our laboratory used double layer suture was implanted. Each group contained three scales: 23 mm ,25 mm and 27 mm. Analogue ex vivo aortic valve replacement was performed , the feasibility of the new implantation was detected. Effective orifice area, transvalvular pressure gradient and regurgitation ratio were recorded at the cardiac output of 2.0 L/min, 3.5 L/min, 5.0 L/min and 7.0 L/min under the guideline of International Organization for tandardization （ISO）5840. Results The average aortic valve implantation time used for single layer suture and tradition double layer suture were 50 min and 70 min respectively. The transvalvular pressure gradient in the single layer suture group were significantly lower than those in double layer suture group under the flow of 5.0 L/min in 23 mm valve and 27 mm valve （13.51±0.51 mm Hg vs. 14.44±0.99 mm Hg, 7.36±0.19 mm Hg vs. 7.53±0.28 mm Hg；P＜0.01）;and the effective orifice area in the single layer suture group were larger than those in double layer suture group in the same case(1.87±0.06 cm2 vs. 1.76±0.08 cm2, 2.26±0.07 cm2 vs. 2.16±0.05 cm2；P＜0.01). There was no statistically difference in other parameters between both groups. Conclusion The novel design of new improvement stentless porcine aortic bioprosthesis used single layer suture has good hemodynamic characteristics as the nature structure . The modified suture method decrease the implantation time.Nemerical data of the evaluation in vitro show that the difference between single layer suture group and double layer suture group in effective orifice area,transvalvular pressure gradient and regurgitation ratio haveno statistical significance. This experiment is the foundation of the animal and clinical experiment in the future.
Objective To investigate the effect of hepatocyte-l ike cells induced by CD34+ cells in vitro on the repair of the injured hepatic tissues of mice in vivo. Methods Mononuclear cells were isolated from umbil ical blood by density gradient centrifugation and enriched CD34+ cells were obtained. The cells were (1 × 105 cells/mL) cultured in serumfreemedium containing stem cell factor (SCF), hepatocyte growth factor (HGF), EGF, oncostatin M (OSM), bFGF (the concentration were 50, 20, 20, 10, 10 ng/mL respectively) in vitro for 10 days. Forty-eight 6-week-old female ICR mice werechosen to prepare l iver injury model by injecting carbon tetrachloride and 2-acetylamionoflu-orene. The mice were randomly divided into two groups (n=24 per group): the experimental group, the cultured cells were injected into the mice through the tail vein; the control group, the equivalent serum-free medium was injected. Six mice from each group were killed at 7, 14, 21, and 28 days after operation to receive HE staining, PCR gel electrophoresis, immunohistochemistry staining, and hepatic function detection. Results HE staining: the morphology of injured hepatic tissues in the control group recovered to normal 28 days after operation, while in the experimental group, it recovered to normal 14 days after operation. PCR gel electrophoresis and immunohistochemistry staining: the cells expressing human serum albumin were detected in the hepatic tissue of the experimental group at each time point after operation; while in the control group, no such cells were detected within 28 days after operation. Hepatic function detection: the activity of alanine aminitransperase in the control group recovered to normal 14 days after operation; the mean activity of aspartate aminotransferase of two groups failed to recover within 28 days. Conclusion The hepatocyte-l ike cells induced by CD34+ cells in vitro can promote the morphological and functional recovery of the injured hepatic tissue in mice. Moreover, it can be transformed into human-derived hepatic cells in l iver-injured mice.
Objective To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa（SIS） and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. Methods The enzymetreatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth musclecells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co cultured for a further observation. At 5,7 and 9 days of the co culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. Results The primary bladder smooth muscle cells that had been harvested by the enzyme treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitrofor 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith the enzyme treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
In order to investigate the compatibility and growth between the tendon cell or fibroblast of rabbit and artificial materials, the combined-culture of the two cells with the carbon fiber, terylene and chitin was observed respectively. Results showed as following: in vitro, the compatibility of carbon fiber with these two cells was well, cell-adhesion ability was good as well. Few cells grew on terylene. Chitin inhibited the growth of either cells. No matter the tendon cell or the fibroblast, the amount of cells adhering on the carbon fiber was far more than that on terylene or chitin. When the three materials were interlaced together, the collagen fibers produced by the cells were arranged in direction parallel to the carbon fibers. As the time elapsed, the cells on the carbon fiber distributed evenly and enveloped the material in network-like fashion, this suggested that carbon fiber was a good material for producing living artificial tendon and ligament.
The purpose of this study was to find some solutions to the problem of tendon cell proliferation control. Under the condition of in vitro culture, several materials including IGF-1 receptor antibody and mRNA antisense oligonucleotide were added to the culture medium to block the IGF-1-Receptor system. The effect of the material on the tendon cell proliferation was judged by cell count after incubation of 48 hours. The results showed that both IGF-1 Receptor antibody (IGF-1R alpha) and IGF-1 Receptor mRNA antisense oligonucleotide had negative effect on tendon cell proliferation (P lt; 0.01 and P lt; 0.05). These findings lead us to think that the above two materials could be used in the experiment of tendon adhesion preventing and living ready-made tendon producing.
At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.