To introduce a rat model of the conversion of acute edematous pancreatitis (AEP) to necrotizing pancreatitis (ANP). One hundred and seven Sprague-Dawley rats were randomized in three experimental groups as follows: sham operation control group and AEP group and ANP group. AEP was induced by pancreatic duct ligation and exocrine stimulation, ANP was induced same as AEP，but with a large dose of dextran-110 (500mg/kg) intravenously. The serum concentration of amylase increased significantly in AEP group and ANP group. Cytosolic free Ca2+ concentration in isolated pancreatic acinar cells increased consistently after induction of ANP. Homorrhage, parenchymal necrosis and calcium deposits in acinar cells were observed in pancreas in ANP group. Ultrastructural examination showed desquamation and necrosis of the endothelium of the pancreatic capillary in ANP group. These results suggest that ischemia may induce the conversion of AEP to ANP via acinar cell Ca2+ overloading. The rat model would seem to be a suitable animal model for studying aggravating mechanism of acute pancreatitis.
Objective To study the mechanism of compound of calcium phosphate(TCP) and platelet-rich plasma(PRP) in the treatment of femoral head necrosis.Methods The left femoral heads of 48 New Zealand white rabbits were frozen by liquid nitrogen as to make themodel of femoral head necrosis.Twenty-four rabbits were randomly chosen as theexperimental group and their femoral heads were filled with TCP/PRP. The other 24 rabbits were used as the control group and their femoral heads were filled only with TCP. They were sacrificed at 2, 4,8,12 weeks after operation. The specimens were examined with X-ray and histological study.Results At 2 weeks after operation,there was no significant difference in femoral headdensity between the two groups. Four weeks after operation, femoral head density decreased in both groups, while it decreased more in the control group. At 8,12 weeks after operation, the density of the femoral heads in both groups increased, and it was higher in the experimental group. Histology examination showed thatthere was no difference between the two groups 2 weeks after operation. The head became flat at 4 weeks. Control group had more defects. At 4,8,12 weeks, more repairs were observed in the experimental group than that in the control group. The amount and maturity of osteogenesis in experimental group were much more greaterthan those in control group．Bone histomorphometry showed that the volum of thetrabecular was larger in the experimental group (36.65%±7.22%,38.29%±4.28%，39.24%±3.42%) than that of control group(P＜0.05). Conclusion TCP/PRP does not only provide osteoblasts scaffold, butalso promotes bone formation and the head repair. TCP/PRP is a good biomaterialfor the treatment of femur head necrosis.
OBJECTIVE: To study chondrogenesis of calcium alginate-chondrocytes predetermined shapes. METHODS: Chondrocytes isolated from ears of rabbit by type II collagenase digestion, and then were mixed with 1.5% solidium alginate solution. The suspension was gelled to create three spatial shapes as triangle, circle and quadrilateral by immersed into 2.5% CaCl2 for 90 minutes, and then was implanted into the subcutaneous pocket on the dorsum of the rabbit. Samples were harvested at 6 and 12 weeks after implantation. RESULTS: Gross examination of excised specimens at 6 and 12 weeks after implantation revealed the presence of new cartilage of approximately the same dimensions as the original construct. Histologic evaluation using hematoxylin and eosin stains confirmed the presence of cartilage nodules at 6 weeks after implantation. After 12 weeks, mature cartilage was observed and histologic analysis confirmed the presence of well formed cartilaginous matrix. CONCLUSION: Predetermined shapes neocartilage can be regenerated using calcium alginate as a carrier of chondrocytes in the bodies of immune animals.
Objective To investigate the preparation of a chitosanencapsulated porus calcium polyphosphate (CPP) bioceramic so as to provide a feasible approach to repair of the bone defect. Methods The chitosan microspheres were produced by chemical procedures. The CPP bioceramic was made by the following steps: annealing, ball milling, admixing, and calcinating. The chemical method was used to encapsule the calcinated bioceramic by the porus chitosan film. The physicochemical property, biomechanical property, and toxicity of the chitosanencapsulated porus CPP bioceramic were analyzed. Results The uniform holes were observed in the CPP bioceramic under a microscope. The diameter of the hole was 100300 μm. The chitosan microballoons were amber in color. The particles were uniform with a diameter of 200-400 μm, with a poor compressive strength. They could be easily ground by hand. Themaximally tolerated dose of the CPP bioceramic leaching liquor given to the Jimpy mice of both sexes was gt;24g/kg on average. The compressive strength reached 200 MPa, and the interval porosity was about 60％-80％, which could completely meet with the compressive strength of the bone substitute. Conclusion The chitosancncapsulated porous CPP bioceramic can be used as a good porous bioceramic scaffold material, which has a good biomechanical property withno acute toxicity, and so may be used as an excellent material for the bone substitute.
OBJECTIVE:To observe the effect of dexamethasone to intracellular free Ca2+ of frozen RPE cells. METHODS:The cultured human RPE cells were frozen for 30s at --70deg;C. The RPE cells were loaded with Fura-2/AM and analyzed using a digital imaging microscopy system,the effect of dexamethasone to intracellular free Ca2+ was measured at a serial concentration of 40, 60,100,150,200mu;g/ml. RESULTS:The concentration of intracellular free Ca in frozen human RPE cells was increased to 18.6%~29.8% by dexamethasone at concenlration of 40mu;g/ml~60mu;g/ml,while was decreased to 28.4%~35.2% at 150mu;g/ml~200mu;g/ml. CONCLUSIONS:Effect of dexamethasone showed two aspects of effect to frozen cultured human RPE ceils,that it was inhibitor at high concentration and stimulator at low concentration （Chin J Ocul Fundus Dis,1997,13: 86-88）
The change of Ig-forming cells in the gallbladder mucoderm were studied in the rabbit models.One hundred rabbits were randomly divided into the control group(Con,n=10),simple biliary obstruction group(BO,n=45)and biliary obstruction and infection group(BOI,n=45).The results showed that only a few Ig-forming cells presented in the gallbladder mucoderm of normal rabbit.At the 3rd,7th and 14th day,the quantities of IgG and IgA-forming cells in the mucoderm in BO group remained unchange,but increased much higher in BOI group(Plt;0.001),especially in IgG formation.This study suggests that the gallbladder of rabbit may be the important place of Ig-formation.The quantities of Ig-forming cells in bilitary tract may have a close relationship with the gallstone formation.
Objective To find out an effective technique torepair large segmental infected bony defect.Methods Calcium phosphate cement(CPC) incorporated with bone morphogenetic protein and gentamycin was embedded in the massive reconstituted bovine xenograft(MRBX), then CPC-MRBX was obtained after CPC’s solidification. In vivo test was applied to test the drug delivery capability of CPC-MRBX, in which it was implanted in the dorsal muscle pouch of 18 rabbits. The drug concentration of animal blood and surrounding soft tissue of the CPC-MRBX in the muscle pouch was measured 1, 2, 5, 10, 15, 20, 25, 30 and 35 d after operation, 2 rabbits each time. Large segmental infected femur defect in the rabbit model was created to test the repairing capability of CPC-MRBX. External fixation was done 1.5~2.0 cm above the knee, the most adjacent nail to fracture site was 0.5～0.8 cm away, and proper pressure was applied to the graft. In experimental group(n=25), the bony defect was replaced by CPC-MRBX, while in the control group(n=15) dissected bone block was re-implanted in original position. The animal was subjected to radiographic, histological examination at 4, 8, 16 and 24 weeks. The general condition was observed after the operation.Results CPC-MRBX was easily made under normal temperature and pressure. In viro drug delivery test showed that the drug concentration of the tissue remainedabove the minimal inhibitory concentration of staphylococcus 30 d after operation and no significant increase of blood drug concentration was observed. In experimental group, no adverse influence was observed. Four weeks after operation, the animal could bear load, bony callus around the graft was observed by X-ray, and abundant chondral tissues that grew into CPC-MRBX were observed by histological method. Eight weeks after operation, progressively increasing bony callus around the graft was observed, external fixation could be removed, normal function was restored, and CPC was degenerated dramatically while new bone tissues were growing. Sixteen weeks after the operation, more new bone tissues grew and CPC was degenerated furtherly while marrow tissues were taking shape. Twenty-four weeks after the operation, femur healed completely and CPC was degenerated completely. In the control group, the autograft remained unhealedon X-ray at 4 weeks, and osteomyelitis manifestation such as inflammatory cells infiltration and osteolysis was detected at 4 weeks. All the animals in the control group died before the 8th week, 4 of which showed positive hemoculture. Conclusion CPC-MRBX is readily available and can be applied to repairing large segmental infected bony defect.30 d after operation and no significant increase of blood drug concentration was observed. In experimental group, no adverse influence was observed. Four weeks after operation, the animal could bear load, bony callus around the graft was observed by X-ray, and abundant chondral tissues that grew into CPCMRBX were observed by histological method. Eight weeks after operation, progressively increasing bony callus around the graft was observed, external fixation could be removed, normal function was restored, and CPC was degenerated dramatically while new bone tissues were growing. Sixteen weeks after the operation, more new bone tissues grew and CPC was degenerated furtherly while marrow tissues were taking shape. Twenty-four weeks after the operation, femur healed completely and CPC was degenerated completely. In the control group, the autograft remained unhealedon X-ray at 4 weeks, and osteomyelitis manifestation such as inflammatory cells infiltration and osteolysis was detected at 4 weeks. All the animals in the control group died before the 8th week, 4 of which showed positive hemoculture.Conclusion CPC-MRBX is readily available and can be applied to repairing large segmental infected bony defect.
【Abstract】 Objective To investigate the effect of verapamil on apoptosis, calcium and expressions of bcl-2 and c-myc of pancreatic cells in ischemia-reperfusion rat model. Methods Wistar rats were randomly divided into three groups: control group （n=10）; ischemia-reperfusion group （n=10）; verapamil treatment group （n=10）. The anterior mesenteric artery and the celiac artery of rats in both ischemia-reperfusion group and verapamil treatment group were occluded for 15 min followed by 12-hour reperfusion. Verapamil （1 mg／kg） was injected via caudal vein to the rats in verapamil treatment group 15 min before occlusion and 1 hour after the initiation of reperfusion, respectively; and ischemia-reperfusion group was given the same volume of salient twice intravenously. Pancreatic tissues were collected from the dead rats after twelve hours since the reperfusion. The pathologic characters of pancreatic tissue were observed under light microscope; The level of calcium in the tissue was measured by atomic absorption spectrometer; TUNEL was used to detect apoptosis of pancreatic cells; and the expressions of c-myc and bcl-2 in the cells were also analyzed by immunohistochemistry technique and flow cytometry. Results The pathologic change in verapamil treatment group was less conspicuous than that of ischemia-reperfusion group. Both the calcium level and the number of apoptotic cells in verapamil treatment group were less than those of ischemia-reperfusion group 〔（411.1±55.8） μg/g dry weight vs （470.9±31.9） μg/g dry weight, P＜0.05 and （9.5±2.9）% vs （18.4±3.1）% 〕, P＜0.05. After taking verapamil, the number of apoptotic cells decreased, whereas the expressions of bcl-2 and c-myc increased. The fluorescent indexes of bcl-2 and c-myc in verapamil treatment group were significantly higher than those of ischemia-reperfusion group (1.72±0.11 vs 1.41±0.07, P＜0.05; 1.76±0.19 vs 1.55±0.13, P＜0.05. Conclusion Ischemia-reperfusion injury can induce apoptosis of pancreatic cells. Verapamil could protect the injured pancreatic tissue by reducing the level of calcium, stimulating the expressions of bcl-2 and c-myc and inhibiting apoptosis of pancreatic cells.
Objective To search the most suitable concentration of calcium in the medium for the basement membrane reconstruction in tissue engineering skin in vitro. Methods Composite chitosan tissue engineering skin was prepared according to previous studies. Four groups were included according to the concentrationof calcium (1.00, 1.45, 1.65 and 1.95 mmol/L respectively). After 7 days and 15 days of culture, the histological manifestation of basement membrane in tissue engineering skin was observed by hematoxylin amp; eosin staining and PAS staining, and collagen Ⅳ of basement membrane was detected immunohistochemicallyatthe dermalepidermal junction. Results This tissue engineering skin shared some histological features of normal skin, including a welldifferentiated stratifiedepidermis and a dense dermis. The epithelium in the group of 1.95 mmol/L calcium differentiated better than those in other groups. PAS staining showing a regularly red dying strap domain at the dermal-epidermal junction. Collagen Ⅳ was positively stained immunohistochemically at the dermalepidermal junction inthe tissue engineering skin. Conclusion The above results suggest that the medium with 1.95 mmol/L calcium should be suitable for the growth of composite chitosan tissue engineering skin and the reconstruction of basement membrane.
PURPOSE:To approach the establishment of t be optimal method for determining the intracellular free Ca2+ concentration[Ca2+]i of dissociated newborn rabbit retina cells by using fluorescent indicator-Fura-2/AM. METHODS:Trypsin was employed to prepare the retlna cell suspensions which were then loaded with Fura-2/AM followed by fluorescence determination. RESULTS:The cellular viability rate of retina cell suspensiotls prepared by 0.05% trypsin 10 minutes at 37deg;C was over 90%. Loading the retina cell suspensions with Fura-2/AM 40 minutes at 37deg;C and then measurlng the fluorescent intensity of the suspensions within 30 minutes were proved to be the optimum. CONCLUSIONS:The resting [Ca2+]i of retina cell suspension was (223plusmn;27)nmol/L whlch was within the expected range of [Ca2+]i level. 25mmoI/L and S0mmol/L K+ increased the [Ca2+Ji 59% and 148% respectively. These results indicate that the preparation of retina cell suspensions and the method of [Ca2+Ji determination are reliable and feasible. （Chin J Ocul Fundus Dis,1996,12: 108-110 ）