Preparing single cell suspension of rat diaphragm tissues: Mechanical grinding versus mechanical-enzymatic digestion / 中国组织工程研究

BACKGROUND: Flow cytometry is currently the advanced cell analysis technique with single cell suspension as a research basis, but there is no report on the preparation method of single cell suspension of diaphragm tissue. OBJECTIVE: To explore the feasibility of preparing single cell suspension of rat diaphragm tissue by mechanical grinding method and mechanical-enzymatic digestion method and to compare cell number and viability of the cells obtained using different methods. METHODS: The fresh diaphragm tissues of Sprague-Dawley rats were harvested. Based on the mechanical method, trypsin, collagenase I, collagenase II, collagenase IV and their different combinations were used to digest and prepare the single cell suspension of diaphragm tissues. Cell morphology was observed; cell number and viability were determined by trypan blue staining. The living cells, inactivated cells, and cell aggregates were counted, and cell survival rate and concentration of the single cell suspension were calculated. Statistical analyses were performed thereafter. RESULTS AND CONCLUSION: (1) The single cell suspension with better cell dispersion, more complete morphology, clearer boundary, fewer impurities and cell debris, and cleaner background were obtained by mechanical-enzymatic digestion compared with mechanical grinding method. (2) The single cell suspension prepared by simple mechanical grinding method has low number of living cells, high number of inactivated cells, low survival rate and many cell aggregates. (3) The number of living cells and concentration of single cell suspension obtained by same-volume addition of trypsin, collagenase I and collagenase IV mixed enzymes based on the mechanical grinding method were the highest, with (1.0-2.0)×106 cells per 0.1 g diaphragm tissue. There were significant differences between mechanical-enzymatic digestion and mechanical grinding method in terms of living cells, inactivated cells, cell aggregates, cell survival rate and suspension concentration (P < 0.05). Moreover, the single cell suspension prepared by the same-volume addition of trypsin and collagenase IV had higher suspension concentration, higher cell survival rate, and less inactivated cells and cell aggregates. To conclude, the single cell suspension of diaphragm tissues could be prepared successful by both mechanical and mechanical-enzyme digestion methods. Mechanical-enzyme digestion is superior to simple mechanical grinding method, with the best single cell suspension after same-volume addition of trypsin, collagenase I and collagenase IV. This is the preferred method for preparation of single cell suspension of the diaphragm tissue.

Psychological influence of mini-invasive Carisolv III gel for caries removal in primary teeth / 医学研究生学报

Objective　Mini-invasive Carisolv is an aid to treat dental caries for patients with dental phobia. The article was to investigate the level of pain in caries removal using mini-invasive Carisolv III gel and mechanical methods with four psychological indicators.　Methods　We collected 120 primary molar tooth caries of 60 children treated in our hospital. Two primary molar tooth caries of each child were respectively treated with Carisolv III gel (Group A) and mechanical method (Group B) for caries removal. Psychological indicators including the visual analog scale (VAS), the Frank1 behavior rating scale (Frank1), the Kuttner law (Kuttner), and the Houpt behavior rating scale (Houpt) were used to assess the level of pain, degree of cooperation, pain tolerance and comfort. The clinical efficiency after six months and treatment time were compared between the two groups.　Results　There was no statistically significant difference before treatment between the two groups using the four psychological indicators (P>0.05) , while significant differences were found during and after the treatment between the two groups (P0.05). In the mechanical group, there were statistically significant differences before and during treatment or before and after treatment using the four psychological indicators (P<0.05). The treatment time in Carisolv III gel group was longer than in mechanical group (P=0.001). There was no statistical difference between the two groups in filling examination after six months (P=0.082).　Conclusion　Carisolv III gel for caries removal can effectively avoid pain, improve comfort and decrease fear in children, which can be promoted in clinical application.

Comparison of MMP-9 Expression in Osteoclasts Obtained by Different Methods / 昆明医科大学学报

Objective To investigate the differences of protein and mRNA expression of matrix metalloproteinase 9 (MMP-9) in osteoclasts (OC) and osteoclast-like cells (OLC) obtained by mechanical and inducement methods. Methods Mechanical separation method was used to separate mature osteoclasts from long bones of SD rats aged one-day;and inducement culture method was applied to induce OLC by using RANKL (100 ng/mL) and M-CSF (100 ng/mL) . The protein expression of MMP-9 was measured by immunocytochemistry and mRNA expression of MMP-9 was assayed by in situ hybridization. Results The integral optical density (IOD) and average optical density (AOD) of positive cells in the visual field were higher in the 12-d group of inducement method as compared with the 3 d-group of mechanical method. Conclusions It is suggested that the protein and mRNA expression of MMP-9 in OLC obtained by 12 d inducement method is much high than in OC obtained by 3 d mechanical method. OLC obtained by inducement method can be applied in the study of osteoporosis.

The efficacy of chemo-mechanical removal of dentin carious lesion / 대한치과보존학회지

Mechanical removals in decayed teeth have been performed using drill and sharp hand instruments. These methods have some disadvantages such as pain, local anesthesia and overextended cavities. Therefore chemo-mechanical excavation of dentin carious lesions has been introduced. The purpose of this study was to evaluate the efficacy of traditional mechanical methods using burs and chemo-mechanical methods (Carisolv) of caries dentin. Mechanical caries removal was carried with low speed round bur. Chemo-mechanical caries excavation was performed with Carisolv (Medi-team), using the Carisolv hand instruments. The mean time to remove caries with two different methods was evaluated and the data analyzed with SPSS software (ver 11.5) by t-test (p 0.05). 4. The remaining carious dentin was detected after the chemo-mechanical removal of the carious dentin, and no smear layer were seen after the mechanical and chemo-mechanical removal of the carious dentin.