BACKGROUND:The low output of seed cells and long cycle of traditional ceils culture methods in tendon animal models(rabbits and chicken) restrict the researches of tendon tissue engineering study.OBJECTIVE:To establish an ideal culture protocol of tail tendon in SD rats,to get more seed cells within less time for subsequent engineered tendon construction research.DESIGN,TIME AND SETTING:Controlled observation was performed in the National Key Laboratory of Biotherapy,Department of Stem Cells and Tissue Engineering,Sichuan University between February and November in 2006.MATERIALS:Rat tail tendon cells were harvested from 2 SD rats,aged 7-10 days;human prepuce fibroblasts were offered by National Key Laboratory of Biotherapy,Department of Stem Cells and Tissue Engineering,Sichuan University.METHODS:Tail tendon of SD rats was draw off and cut into pieces,which were then cultured in 10% fetal bovine serum+DFculture medium for getting primary tendoncyte by using suspension tissue culture method. The third generation cells were processed into immuocytochemistry stain with collagen type Ⅰ and Ⅲ,while human prepuce fibroblasts served as controls.Absorbance of stain result was measured by image-pro plus 5.02 for statistical analysis.MAIN OUTCOME MEASURES:Immuocytochemistry stain and absorbance measurement of SD rat tail tendon cells.RESULTS:The second generation of SD rat tail tendon cells were positive for type Ⅰ collagen stain,and negative for type Ⅲ collagen stain;human fibroblast were positive for both Ⅰ and Ⅲ collagen. In the rat tail tendon cells and human flbroblasts,the absorbance value of type Ⅰ collagen expression was dramatically higher than of type Ⅲ collagen(P＜0.05). There was no significant differences addressing the absorbance of type Ⅲ collagen expression between type Ⅰ and Ⅲ collagen of SD rat tail tendon cells and blank control group (P＞0.05).CONCLUSION:Cells cultured from SD rat tail tendon have biological characteristic of tendon cells. Tissue piece suspensionculture can obtain a quantity of primary or subcuitured cells of rat tail tendon within a short time.

BACKGROUND:Accumulative evidence supports that vitreous cryopreservation can improve the cel survival rate. OBJECTIVE:To investigate the effect of vitreous cryopreservation on the tenocytes co-cultured with the porous polydimethylsiloxane (PDMS) scaffold. METHODS:Tenocytes were co-cultured with the porous PDMS scaffold for 9-14 days, and then preserved and resuscitated in the 10%dimethyl sulfoxide (DMSO), 21%DMSO and VS55, respectively. One hour later, the survival rate of post-resuscitated tenocytes versus pre-resusciated tenocytes was analyzed by live/dead double color fluorescent staining and flow cytometry. RESULTS AND CONCLUSION:Live/dead double color fluorescent staining revealed that tenocytes in the 10%DMSO group appeared to be irregular and double stained, and a large number of cel s shedding from the scaffold. The VS55 and 21%DMSO groups showed some spindle and hemispherical cel s single stained for green fluorescence and few double stained irregular cel s. Additional y, the cel density in the two groups was significantly lower than that in the control group. Flow cytometry results found that there were homogenous cel s in the control group;the number of cel s in the 10%DMSO group was too low to undergo flow cytometry;smal cel particles were visible in the VS55 group;in the 21%DMSO group, the cel volume was similar with the control group, and smal particles also existed. The survival rate in the VS55 group (64.9%) was significantly lower than that in the 21%DMSO group (76.2%;P<0.05). Conversely, the survived cel s were rare in the 10%DMSO group. To conclude, 21%DMSO vitreous cryopreservation improves the cel survival rate and is beneficial for tenocyte adherence to the scaffold.

OBJECTIVETo elucidate the significance of detecting cytokeratin gene (CK-20 mRNA) expression in metastatic lymph nodes of colon carcinoma patients.

METHODSThe expression of CK-20 mRNA was detected in 342 lymph nodes by reverse transcription-polymerase chain reaction (RT-PCR) in 49 colon carcinoma patients. Its incidence was compared with that by routine pathologic examination.

RESULTSThe positive rates of detecting metastasis in the lymph nodes were 21.9% by RT-PCR and 11.1% by routine pathologic examination.

CONCLUSIONThe detection of cancer metastasis in the lymph nodes in colon carcinoma is almost doubled (21.9% vs 11.1%) by CK-20 mRNA, which may provide a guiding significance in staging, treatment planning and prognosis in the prognostic estimation of colon carcinoma patients.

Adult ; Aged ; Colonic Neoplasms ; diagnosis ; genetics ; pathology ; Female ; Gene Expression ; Genetic Markers ; genetics ; Humans ; Intermediate Filament Proteins ; genetics ; Keratin-20 ; Lymphatic Metastasis ; diagnosis ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; RNA, Messenger ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

It is crucial to improve the orientation and growth of cells on substrates in tissue engineering. In this study, we investigated the effects of micropatterned surfaces coated with type I collagen (CNI) on the orientation and growth of SD rat tenocytes. Using the technique of microcontact printing and microfluidic channels, we prepared micropatterned microgrooves with a 10 microm width and 4 microm depth on silicone membrane substrates. The microgrooves were coated with CNI at concentrations 0.25, 0.5, 0.75, 1.0, and 1.25 mg/ml, respectively. The rat tenocytes at 1 x 10(5)/ml were seeded onto the CNI-coated substrates and the control substrates (without CNI coating), and then cultured in a humidified 37 degrees C/5% CO2 incubator for 48 hours. Cell proliferation was measured by MTT method. After 1, 12, 24, 48 hrs of incubation, the tenocytes' alignment and morphology were observed by means of inverted phase microscope, scanning electron microscope and fluorescent microscope. The results showed there was obvious orientation of tenocytes in CNI-modified grooves, and most of the tenocytes spread along the grooves. The tenocyte orientation became more obvious with the increasing CNI concentration over a range from 0.25 to 1.25 mg/ml. This method could find important application in the construct of engineered tendons which need precise spatial organization of cells.
Animals ; Animals, Newborn ; Cell Culture Techniques ; methods ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coated Materials, Biocompatible ; chemistry ; pharmacology ; Collagen Type I ; pharmacology ; Guided Tissue Regeneration ; methods ; Rats ; Rats, Sprague-Dawley ; Surface Properties ; Tendons ; cytology ; Tissue Engineering ; methods

In search of a practical method for the cryopreservation of tissue engineered tendon (TET) by vitrification, we adopted 3 kinds of different cryoprotective agents (CPA)(21% DMSO, DP6 and VS55) in studying the freeze-stored effect of different CPA. The cellular morphology and post-thaw viability of the TET were examined by scanning electron microscopy (SEM), flow cytometry, and confocal laser microscopy (CLM). The results showed that there existed statistically significant difference in respect to the post-thaw viability between 21% DMSO and DP6, VS55; The cells specially adhered to the surface of scaffold both before or after cryopreservation by use of 21% DMSO. It was suggested that 21% DMSO as a CPA for TET cryopreservation was better than DP6 and VS55 in the current study.
Animals ; Cell Differentiation ; Cells, Cultured ; Cryopreservation ; Cryoprotective Agents ; analysis ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Tendons ; cytology ; transplantation ; Tissue Engineering ; Tissue Preservation ; methods ; Tissue Scaffolds ; Vitrification

OBJECTIVETo detect occult metastases in lymph nodes from patients with colorectal carcinoma and its clinical significance.

METHODSThe metastases in 260 lymph nodes from 39 histologically verified colorectal cancer patients were studied by both hematoxylin-eosin (HE) staining and cytokeratin-20 (CK20) specific RT-PCR. Ten normal lymph nodes were served as negative controls, and HT29 colon cancer cell line and 5 colorectal cancer specimens as positive controls.

RESULTSTen normal lymph nodes were CK20-negative, HT29 cells and 5 tumor specimens were all CK20-positive. All 29 lymph nodes from 16 patients which confirmed metastases by HE staining exhibited CK20 positive expression; an additional 28 lymph nodes from 5 patients with no histologically detectable metastases expressed CK20 mRNA, i.e. presence of metastases. The difference of the positivity was significant (11.1% vs 21.9%, P < 0.01). According to the HE staining, the cases of Dukes' A, B, C and D were 3, 20, 12 and 4, respectively. In the 20 patients of Dukes' B stage, 5 of them had CK20-positive lymph nodes.

CONCLUSIONCK20-specific RT-PCR is a highly sensitive, specific and simple method for detecting occult metastases in lymph nodes. The detection of CK20 mRNA expression in lymph nodes is recommended to precisely determine tumor stage and postoperative adjuvant therapy for patients with colorectal cancer, and further studies should be done in future to confirm the findings.

Adult ; Aged ; Colorectal Neoplasms ; genetics ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; HT29 Cells ; Humans ; Intermediate Filament Proteins ; genetics ; Keratin-20 ; Lymph Node Excision ; Lymph Nodes ; metabolism ; pathology ; Lymphatic Metastasis ; genetics ; pathology ; Male ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction