ABSTRACT
BACKGROUND: Surface roughness of implants can directly influence cellular proliferation, differentiation, and gene expression. OBJECTIVE: To observe the early attachment of periodontal ligament cells (PDLCs) to pure titanium with different surface roughness levels, and to study the effect of surface performance on cell differentiation. DESIGN, TIME AND SETTING: Randomized controlled observation/multi-sample comparison study, which was performed at Center of Science and Technology, Gannan Medical College between January 2005 and July 2006. MATERIALS: Pure titanium stick was cut into pieces, of 10 mm diameter and 2 mm thickness, using cutting-off machine, and there were 24 sections in total. Then, the titanium sections were randomly divided into four groups: simple mechanical processing, nitric acid processing, sand blasting processing, and combination group, with 6 sections per group. METHODS: TR240 portable-type surface roughness meter was used in this study. In the simple mechanical processing group, sections were scoured by sand paper alone; in the nitric acid processing group, sections were etched with 65% HNO3 for 1 hour at 100 ℃ after scoured by sand paper; in the sand blasting processing group, sections were sandblasted by 100 μ m AI203 after scoured by sand paper; in the combination group, sections were etched with 65% HNO3 for I hour at 100～C after scoured by sand paper and sandblasted by 100 μ m Al2O3. MAIN OUTCOME MEASURES: Samples were maintained in DMEM for 30 minutes, and the third-passage cells were inoculated. Then, titanium sections were taken out at different time points of 30, 60, 120, 240 minutes, 1, 3, and 7 days. Surface roughness and early attachment of PDLCs were detected under fluorescent microscope. RESULTS: O Quantitative analysis: Surface roughness was (599.5±8.3) nm in the simple mechanical processing group, (406.5 +4.6) nm in the nitric acid processing group, (358.8±11.8) nm in the sand blasting processing group, and (8.7±2.0) nm in the combination group. On the other hand, surface roughness in the simple mechanical processing group was significantly higher Fluorescence observation exhibited that number of PDLCs attaching to pure titanium surface was increased, and the proliferation was greater with the time passing by. in addition, surface roughness of pure titanium was positively associated with number of PDLCs. CONCLUSION: The lighter the surface roughness is, the more the early attachment of PDLCs is, benefiting for cell adhesion and proliferation.
ABSTRACT
BACKGROUND: The investigation of culturing, passaging and establishing human limbal stem cells can strengthen the recognition of the stem cells and provide the enough cellular reserve for the basic and clinical research of limbal stem cell transplantation.OBJECTIVE: To explore a method of pessaging and establishing cell line of human limbal stem cells cultured in vitro.DESIGN: Randomized controlled observation.SETTING: Gannan Medical College.MATERIALS: The experiment was performed at Scientific Center of Gannan Medical College and the National Key Laboratory of Ophthalmology Hospital Affiliated to Sun Yat-Sen University from June 2003 to April 2004. Fresh human limbus corneae were isolated from two healthy donors. Procedures were performed according to the informed consent of the donors. Main reagents contained RPMI-1640 (Sigma R8755, containing L-glutamine) and 200 g/L fetal calf serum (FCS) (Gibco 16140-071). DMEM medium, chondroitin sulfatase and human epidermal growth factor (hEGF) were purchased from Sigma Co. USA; HEPES and DMSO were bought from Gibco, USA; 100% glycerinum was purchased from Yunjia Huangpu Pharmaceutical Product Limited Company, PRC; glutaraldehyde was bought from E.Merk, Germany; Alcohol, chlorhydric acid, acetone and methyl aldehyde were purchased from Beijing Chemical Agent Company, PRC; 0.25% parenzyme was bought from Shanghai Xinhua Pharmaceutical Factory, PRC.Above-mentioned reagents were analytical pure grade.METHODS: After digestion, human limbal tissues in limbal basilar part with an abundant pigment were cultured in the culture flask containing RPMI-1640 and 200 g/L FCS and in culture dish containing amniotic extracellular matrix (AECM) as the cultural supporter. Primary and passage cells were observed under light microscope and scanning electron microscope (SEM). The revival ratio of stem cell refrigeration of every generation was calculated by the trypanblau exclusion experiment.MAIN OUTCOME MEASURES: ① Observational results of limbal stem cells during the primary culture and serial subcultivation in vitro, and ② revival ratio of stem cell refrigeration.RESULTS: ①Findings of primary culture: Most limbal stem cells in the culture flask had the adherence and were arrayed uniformly sparsely to form monolayer and adhered to the bottom of culture flask under the inverted phase contrast microscope after 1-day culture. ② Findings of serial subcultivation: After human epidermal growth factor (hEGF) was added into the second passage, cells were scattered into the monolayer and adhered to grow quickly.Morphological variability of all the cells increased obviously when passage the 30th generation. The cellular volume was obviously increasing, and the round or irregular round cells gathered together. The 33rd generation human limbal stem cells still could vigorously differentiate, proliferate and grow in ACEM. ③ The revival ratio of stem cell refrigeration was 82.2%.CONCLUSION: The human limbal stem cell lines were preliminarily established by culturing and freezing the cells of 33 generations in vitro. The human limbal stem cell lines preferred to grow in the culture dish containing AECM as the cultural supporter.
ABSTRACT
BACKGROUND: The complex prescription of lujiao prescription is made up of antle, malytea scurfpea fruit, korean epimedium herb, dogwood, glossy privet fruit, and Chinese eaglewood wood. It is characterized by its positive myodynamia as well as promoting diuresis and expanding vessels. OBJECTIVE: To observe the effect of lujiao prescription in reversing left ventricular remodeling in rats with pressure overload. DESIGN: A randomized controlled trial. SETTING: Department of Integrated Traditional Chinese Medicine and Western Medicine, Nanjing General Hospital of Nanjing Military Area Command of Chinese PLA; Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine; Postdoctor Institute of Hebei Medical University.MATERIALS: The experiment was conducted at the Laboratory of Shanghai University of Traditional Chinese Medicine from January to December 1998. Totally 153 male Wistar rats were provided by the Experimental Animal Center of Shanghai Medical College. There were five experiments with 36 rats selected for each experiment except 9 rats for the fourth one.METHODS: Totally 36 Wistar male rats selected for each experiment were randomly divided into sham operation group, model group and lujiao prescription group. Sham-operation group: The rats' abdominal aortas were separated, but not narrowed by silver clip. After four weeks, the rats were treated with twice-distilled water (15 mL/mg, ig), once a day for 4 consec utive weeks. Model group: Four weeks after modeling, the rats were treated with twice-distilled water (15 mL/mg, ig) once a day for 4 consecutive weeks. Lujiao prescription group: Four weeks after rat modeling, the ratswere given orally liquid decoction of lujiao prescription (15 mL/mg, ig),once a day for 4 weeks. Observations on left ventricular mass index (LVMI): Collagen type Ⅰ and Ⅲ was measured by immunohistochemistry. mRNA expression of collagen type Ⅰ and Ⅲ was measured by RT-PCR. Ang Ⅱ, ANF and ALD were measured by radioimmunoassay. MAIN OUTCOME MEASURES: LVMI, left ventricular myocardium collagen type Ⅰ and Ⅲ, mRNA expression of collagen type Ⅰ and Ⅲ, and changes of plasm, myocardium Ang Ⅱ, plasm ANF and serum aldosterone.RESULTS: Totally 153 rats entered the final analysis. During the detection of mRNA expression of collagen type Ⅰ , 3 rats in sham-operation group died and 1 in lujiao prescription group died. The other 29 rats en tered the final analysis. During the detection of mRNA expression of collagen type Ⅲ, 3 rats in sham-operation group died, 3 in lujiao prescription group died, and 6 in model group died. The other 26 rats entered the final analysis. All rats were lost because of operation injury. ① LVMI: It was lower in sham-operation group and lujiao prescription group than in model group [(2.24±0.12)/1 000, (2.60±0.44) /1 000, (3.15±0.47)/1 000,t=2.959-6.499, P < 0.05]. ② Changes of absorbency of collagen type Ⅰ :It was lower in sham-operation group and lujiao prescription group than in model group [(0.56±0.23, 0.57±0.19, 2.79±2.00), t=0.661-3.964,P < 0.01]. ③ Changes of absorbency of collagen type Ⅲ: It was lower insham-operation group and lujiao prescription group than in model group [(0.48±0.10,0.55±0.09,0.84±0.27), t=2.898-3.560, P < 0.01]. ④ mRNA expression of collagen type Ⅰ: It was lower in sham-operation group and lujiao prescription group than in model group [58.0±36.0)%, (79.1±18.6)%,(139.0±29.2)%, t=3.027, P < 0.05]. ⑤ Changes of myocardium Aug Ⅱ: It was lower in sham-operation group and lujiao prescription group than in model group [(2.49±0.57, 2.64±0.57,3.96±0.77) pg/mL, t=4.773-5.315,P < 0.001]. ⑥ Changes of serum aldosterone: It was lower in sham-operation group and lujiao prescription group than in model group [(501.6±94.6,476.6±85.7, 647.8±72.2) ng/mL, t=4.256-5.292, P < 0.001].CONCLUSION: Indexes of integrating absorbency of myocardial collagen type Ⅰ and type Ⅲ, mRNA expression of collagen type Ⅰ , myocardial Aug Ⅱ and serum aldosterone are all similar to the changes LVMI. Lujiao prescription can reverse left ventricular remodeling and myocardial fibrosis.