Isolation and in vitro culture of neonatal rats liver cells / 中国组织工程研究

BACKGROUND: Neonatal rat liver cells are moderately differentiated cells with the characterization and function of both liver progenitors and hepatocytes, thus, it is an ideal cell source for the study of the hepatocyte characterization. OBJECTIVE: To explore the isolation and in vitro culture of neonatal rat liver cells.DESIGN, TIME AND SETTING: An in vitro cytology trial was carried out at the Institute of General Surgery, General Hospital of Chinese PLA from March to August 2008.MATERIALS: Neonatal SD rats with 3 months old were used, irrespective of genders.METHODS: Liver cells from neonatal rat were isolated by tissue piece-cold trypsin digestion combining with multi-step low centrifuge, and cultured onto the plate in HepatoZYME-SFM supplemented with 10% fetal serum. The growth and function of the cultured liver cells was evaluated by contrast microscopy, MTT assay, PAS staining and urine enzyme test. MAIN OUTCOME MEASURES: Cell morphology and viability, content of glycogen, as well as urea level in the supernatant.RESULTS: Totally 1.0×10~6-2×10~6 cells per whole liver could be obtained with viability above 90%. The cells displayed round or orbicular-ovate shapes with big nuclei, and cell body was smaller than mature cells. More than 95% purity achieved after removal erythrocyte, nonparenchymal cells and dead cells with multi-step low-speed centrifugalization. The viability of cells were gradually increased at the beginning of culture, noticeably alleviated at the 3 days, and reached a peak at the 11 days, and then gradually decreased. The difference between day 1 and days 11, as well as days 15 and days 11 had significance (P=0). Liver cells cultured in HepatoZYME-SFM attached and kept hepatocyte-specific morphology. PAS staining showed the cultured liver cells at day 7 were strongly positive and then the positive cells decreased gradually, until 15 days, only few positive cells could be seen. Urea level in the supernatant remained stable at the initial time and dramatically decreased after 7-day culture. CONCLUSION: The tissue piece-cold trypsin digestion and HepatoZYME-SFM is a simple and efficient isolation and culture system for neonatal rat liver cells.

Study on Evaluation Method of Scaffold for the Use of Liver Tissue Engineering / 医疗卫生装备

Obiective To set up a method of scaffold evaluation using human cell line as seed cells and screen appropriate scaffold for live tissue engineering, Methods HepG2 cells were plated onto biodegradable polymer scaffolds: PLGA, 3% chitosan (3%CS) and 2% silk fibroin (2%SF), and cultured in vitro. The growth, distribution and function of HepG2 cells in the scaffolds were evaluated using MTT assay, H.E. staining, and urea assay kit. Results HepG2 cells plated on the three scaffolds maintained a proliferative state. In contrast, the cells on the 2%SF proliferated strongly, while the cells on the PLGA and chitin proliferated poorly. Histological examination showed that HepG2 cells distributed evenly on the 2%SF scaffold with a high amount, while few cells could be found on the PLGA and ehitin at day 7. Cell function assay showed that HepG2 cells on the 2%SF and PLGA exhibited slower decrease of urea synthesis compared to HepG2 cells on the chitosan. Conclusion The three scaffolds have good biocompatibility. In contrast, 2%SF scaffold is more appropriate for liver tissue engineering. This method may be used for scale screening of scaffolds for liver tissue engineering.

Preliminary report of composite external fixator in the treatment of the severely proximal tibial fractures / 中华骨科杂志

Objective To explore the clinical outcome of self-designed composite external fixator in the treatment of severely comminuted or open proximal tibial fractures. Methods From July 2000 to January 2003, 22 cases of severely comminuted or open proximal tibial fractures were treated by self-designed composite external fixators, which involved 15 males and 7 females with an average age of 43 years (range, 20-80 years). This technique consisted of indirect reduction and limited open cannulated screw or K-wires fixation in case of necessity. Results All the patients had achieved fracture union at the 5th month averagely after operation. The period of follow-up was from 6-21 months(mean, 11 months). The mean duration of external fixation was 4.5 months. 4 cases received bone autograft from the iliac crest, primary union occurred in 20 cases, 2 cases with delayed union healed after bone-grafting. No infection occurred in 6 cases with open fracture. The other 16 cases also had no infection or skin necrosis. Only 4 cases with pin track infection healed by dressing change. 19 cases had knee flexion beyond 90?(containing 3 cases with second-stage knee release), 2 was 60? and 1 was 45?. 1 case took NSAIDs intermittently because of traumatic arthritis. According to Merchant grade, 14 were evaluated as excellent, 3 as good, 3 as fair and 2 as poor. The rate of excellent and good results was 77.3%. Conclusion The composite external fixator in treating the severely comminuted or open proximal tibial fracture has the following advantages, such as less-invasion, stable fixation, early-mobilization, high union rate and fewer complication.

Trans-articular movable external fixation of severely comminuted and open tibial Pilon fractures / 中华骨科杂志

Objective To investigate the results of comminuted or open tibi al Pilon fractures treated with a joint bridging movable external fixator. Methods From January 2000 to May 2002, fifteen patients with either severely comminuted or open Pilon fractures were treated with self-designed joint bridging movable external fixators. In some patients, the external fixation was combined with li mited open reduction and Kirschner wire internal fixation. Results All patients were followed up. The average period of following up was 10 months (range 6 to 2 2 months). The fractures were externally fixed for averagely 3 months. The inter val between fracture and bony union was averagely 4.5 months. There was no nonun ion occurred. Two delayed unions were further treated with bone grafting and one of them was stabilized with a plate. All the 9 open fractures were infection fr ee, so were the closed injuries. There was no skin sloughs found in all cases bu t minor pin tract infection was found in two cases. Range of motion at last foll ow-up examination was excellent in five patients, good in seven, fair in two, an d poor in one. One patient required ankle arthrodesis for posttraumatic arthriti s. Conclusion The results of the severely comminuted or open tibial Pilon fractu res treated with joint bridging movable external fixators were fairly satisfying . The fixator can firmly stabilize the fracture while ankle movement can be resu med early. The operation is less-invasive, and is with higher union rate and few er complications.