Abstract: Objective To investigate the influence of cryopreservation on cellular viability of latepregnancy fetal valved allografts in human. Methods The fetal valved allografts with gestational ages ranged from 24 to 40 weeks were sterilely procured within 6 hours after brain death. Each sample was bisected into control group and experiment group. The cellular viability of control group was directly tested and that of experiment group was examined after being storaged in liquid nitrogen for a week through a programmed frozen procedure. The light microscopy, tissue culture and Methylthiazol tetrazolium assay (MTT assay) were used to determine the cellular viability. Results Twelve latepregnancy fetal valved aortic allografts were procured. Light microscopy showed the integrity of the basic structure of the thawed aorta, the normal structure of the collagen and elastic fibers, with part of vascular endothelium lost. There were lots of cells deriving from both groups,but the cellular growing rate of the experiment group was relatively slower. At 490 nm, MTT assay valve of control group was 0.442±0.046, and that of experiment group was 0.424±0.041. The difference between two groups failed to statistically significance(t=1.617, P=0.328). Conclusion There were viable cells in latepregnancy fetal valved allografts after cryopreservation.
Abstract In order to determine the fasibility of reestablishment of circulation with cryopreserved microvenous allografts (1.0～1.4mm in diameter), 40 rabbits were divided into 2 groups. In the control group, the fresh autografts were used. In the experimental group, 20 rabbitsfemoral vein segments were treated by a two-step freezing procedure. After stored in liquid nitrogen for 48 hours, the segments were implanted into the femoral veins as allografts. The histological as well as the pathological studies were performed with light and electron microscope, and its patency was determined by angiography. The results showed that the preservation of vein was generally good. The rejective response was weak. The patency rates of 1 week and 12 weeks were 90% and 85% respectively, and there was no significant difference with that of the allogenic fresh autografts (Pgt;0.05). It was suggested that clinical use of cryoperserved allogenic microvein grafts instead of fresh autografts was possible.
Abstract： Objective To assess the effects of simultaneous antegrade/retrograde cardioplegia (SARC) on myocardial perfusion and energy metabolism in ischemic myocardium using magnetic resonance imaging (MRI). Methods After the hearts were harvested from 18 domestic pigs, left anterior descending artery, aorta, anterior ascending cardiac vein and coronary sinus were cannulated to establish the perfusion routes. 6 hearts were used to assess the effects of SARC on myocardial perfusion. Energy metabolism was observed in the other 12 pig hearts. MRI was used to monitor the distribution of contrast agent (gadoliniumdiethylenetriamine penlaacetic acid, Gd-DTPA) in the myocardium after its injection through arterial and retrograde perfusion routes. The efficacies in sustaining myocardial perfusion and energy metabolism were evaluated by using phosphorus-31 magnetic resonance spectroscopy (31P MRS) during antegrade cardioplegia (AC) and SARC respectively. Results It was found that injection of Gd-DTPA into the aorta during AC did not result in signal increase in the ischemic myocardium on MRI. During SARC, however, Gd-DTPA was found in the ischemic region as well as in the other regions, no matter if it was given into the aorta or into the coronary sinus. Moreover,31P spectra showed that occlusion of LAD during AC resulted in severe decrease of the levels of phosphocreatine (PCr) and adenosine triphosphate (ATP), while the level of inorganic phosphate (Pi) increased in LAD-support myocardium. The abnormal metabolic changes were completely abolished by use of SARC. Conclusion It is concluded that SARC can deliver cardioplegic solution to the myocardium distal to a coronary occlusion and can sustain normal energy metabolism in the jeopardized myocardium.
ObjectiveTo explore the effects of cryopreservation on the cell survival rate, cell viability, early apoptosis, migration ability, and tendon-related marker expression of tendon-derived stem cells (TDSCs) in rat patellar tendons.MethodsThe patellar tendon tissues were harvested from 12 4-month-old male Sprague Dawley rats; 12 patellar tendon tissues from 6 rats were cryopreserved (the experimental group), and the other 12 patellar tendon tissues were not treated (the control group). The patellar tendons were digested with 0.3% type I collagenase to obtain nucleated cells. The survival rate of nucleated cells was detected by trypan blue exclusion assay, and colony-forming ability by crystal violet staining. TDSCs were isolated and cultured to passage 3 (P3). The cell viability of TDSCs was detected by Alamar Blue method, the early apoptosis by Annexin V-FITC/PI assay, the cell migration ability by Transwell method, and the mRNA expressions of tendon-related markers [collagen type I (Col1α1), scleraxis (Scx), and tenomodulin (Tnmd)] by real-time quantitative PCR.ResultsThe survival rate of nucleated cells was 91.00%±3.63% in the control group, and was 61.65%±4.76% in the experimental group, showing significant difference (t=12.010, P=0.000). The formation of the primary nucleated cell clones was observed in 2 groups. At 12 days, the number of colonies forming of the experimental group [(8.41±0.33)/1 000 nucleated cells] was significantly lower than that of the control group [(15.19±0.47)/1 000 nucleated cells] (t=28.910, P=0.000). The percentage of TDSCs in the active nucleated cells in the experimental group (1.37%±0.09%) was significantly lower than that in the control group (1.67%±0.10%) (t=5.508, P=0.003). The growth trend of TDSCs (P3) in the 2 groups was consistent within 14 days. There was no significant difference in absorbance (A) value between 2 groups at each time point (P>0.05). The early apoptotic rate of TDSCs was 1.67%±0.06% in the experimental group and was 1.63%±0.06% in the control group, showing no significant difference (t=0.707, P=0.519). Under microscope, TDSCs adhered to the lower chamber of the Transwell chamber; the number of cells was 445.00±9.70 in the experimental group and was 451.50±12.66 in the control group, showing no significant difference (t=0.998, P=0.342). The relative mRNA expressions of Col1α1, Scx, and Tnmd were 3.498±0.065, 0.062±0.002, and (4.211±0.211)×10–5 in the experimental group and were 3.499±0.113, 0.062±0.001, and (4.341±0.274)×10–5 in the con-trol group, showing no significant difference (t=0.013, P=0.991; t=0.042, P=0.969; t=0.653, P=0.549).ConclusionThe survival rate of nucleated cells in cryopreserved rat tendon tissues is lower, but a large number of active TDSCs, and its cell viability, early apoptosis rate, migration ability in vitro, and cell tenogenic differentiation ability are remained.
Objective To investigate the effect of tissue engineered bone with cryopreservation on healing of bone defects and to explore feasibility of cryopreservation for tissue engineered bone. Methods Tissue engineeredbones were constructed with osteoblasts being seeded onto bio-derived materials made from freshhuman bones,and they were preserved at 4℃ and －196℃ for 3 months and 6 monthsrespectively.They were applied to repair segmental bone defects of rabbit’s radius while the tissue engineered bone without cryopreservation and bio-derived materials were brought into control groups.The experiment was divided into groups A3,A6,B3,B6,C and D(group A3:tissue engineered bones were preserved at 4℃ for 3 months; group A6:tissue engineered bones were preserved at 4℃ for 6 months;group B3:tissue engineered bones were preserved at －196℃ for 3 months; group B6:tissue engineered bones were preserved at －196℃ for 6 months; group C: tissueengineered bones without cryopreservation; group D: bio-derived materials). Macroscopical and histologial examination were done at the 2nd,4th,6th,12th weeks, X-ray examination was done at the 6th,12th weeks and biomechanics were determined at 12th weeks after operation respectively. Results Macroscopical observation showed no significant differences among group A3, A6, B3, B6 and C, but less new bone formation and more obvious boundary in group D were observed. Histological observation showed more collagen and new bone around the edge of implant of group A3, A6, B3, B6 and C than group D, and histological evaluation showed significant differences between group D and other groups(P＜0.05). Radiographic observation showed no absorbability of the implant cortex and less new bone formation in group D, but the unity between implant and host bone, medullary cavity reopened, disappearance of fracture line and fine bone modelling were observed in other groups at 12 weeks after operation. Biomechanics between group D and other groups showed significant differences(P＜0.05). Conclusion Cryopreservation (4℃ and -196℃) were capable of preserving tissue engineered bone for long time, and tissue engineered bone withcryopreservation has significant effect on healing of bone defects. The methods f it clinical application.
Abstract： Objective To investigate the mechanism of protein kinase C(PKC) in immature myocardial ischemic preconditioning in order to further its clinical applicability. Methods Langendorff perfusion heart models of 24 rabbits were set up and they were randomly divided into 4 groups: ischemic reperfusion group (I/R group), myocardial ischemic preconditioning group (MIP group), chelerythrine group (CLT group) and protein kinase C group (PKC group). The emodynamics, biochemistry and myocardial ultrastructure were observed. Results The heart function recovery and myocardial water content in the MIP and the PKC groups were better than those of the I/R and the CLT groups (Plt;0.01). The adenosine triphosphate (ATP) content, superoxide dismutase activity, mitochondrial Ca2+-ATPase activity and synthesizing ATP activity of mitochondria in the MIP and the PKC groups were significantly higher than those of the I/R and the CLT groups (Plt;0.01). The dehydrogenase and creatine kinase leakage, malondialdehyde content, myocardial cell Ca2+ content and mitochondrial Ca2+ content in the MIP and the PKC groups were significantly lower than those of the I/R and the CLT groups (Plt;0.01). The myocardial ultrastructure injuries in the MIP and the PKC groups were less than that of the I/R and the CLT groups. Conclusion Myocardial ischemic preconditioning plays an important role in protecting immature myocardium, which is probably realized by the activation of PKC.
ObjectiveTo analyze clinical experience and outcomes of bileaflet preservation in mitral valve replace-ment (MVR) for patients with severe mitral regurgitation (MR). MethodsWe retrospectively analyzed clinical data of 17 patients with severe MR who underwent MVR with bileaflet preservation in the Department of Cardiovascular surgery of Guangdong General Hospital from June 2011 to January 2013. There were 14 males and 3 females with mean age of 63.41±11.82 years (range, 38 to 82 years). There were 13 patients with atrial fibrillation. Preoperatively, 5 patients were in New York Heart Association (NYHA) functional class Ⅲ, and 12 patients were in NYHA class Ⅳ. There were 7 patients with ischemic MR, 9 patients with degenerative MR, and 1 patient with rheumatic MR. ResultsMVR with bileaflet preservation was performed for all the patients. Concomitant coronary artery bypass grafting was performed for 4 patients. Eleven patients received bioprosthetic MVR, and 6 patients received mechanical MVR. There was no in-hospital death, postoperative low cardiac output syndrome or left ventricular rupture. All the 17 patients were followed up for a mean duration of 16.44±5.02 months (range, 2 to 25 months). During follow-up, 1 patient died of severe paravalvular leak 2 months after surgery. All the other patients had good mitral valve function. None of the patients had anticoagulation or prosthetic valve related complication. Patient's heart function was significantly improved. Eleven patients were in NYHA functional class Ⅰ, 4 patients were in NYHA class Ⅱ, and 1 patient was in NYHA class Ⅲ. Cardiothoracic ratio, left atrial dimension, left ventricular end-diastolic dimension and left ventricular end-systole dimension postoperatively and during follow-up were significantly smaller than preoperative values. Postoperative left ventricular ejection fraction (LVEF) was significantly lower than preoperative LVEF(50.94%±8.78% vs. 55.31%±10.44%, P=0.04), but LVEF during follow-up was not statistically different from preoperative LVEF(55.31%±10.44% vs. 56.13%±9.67%, P=0.73), and LVEF during follow-up was significantly higher than postoperative LVEF(56.13%±9.67% vs. 50.94%±8.78%, P=0.02). There was no statistical difference between postoperative mitral pressure half-time (PHT)and PHT during follow-up (95.06±19.00 ms vs. 94.56±19.19 ms, P=0.91). ConclusionMVR with bileaflet preservation is a safe and effective surgical technique for patients with severe MR, and can significantly improve postoperative left ventricular remodeling and function.
Objective To investigate the effects on myocardial perfusion of simultaneous antegrade/retrograde cardiopiegia （SARC） through a single coronary artery and coronary sinus （CS）. Methods SARC was conducted in isolated pig hearts through CS in conjunction with the left anterior descending （LAD）, the left circumflex （LCX）, or the right coronary artery （RCA） respectively. After injecting magnetic resonance （MR） contrast agent （gadolinium diethyienetriamine pentaacetic acid, Gd-DTPA） into arterial or venous route, the distribution of Gd-DTPA with magnetic resonance imaging（MRI） was monitored and the effluent from the venting coronary arteries to assess the efficacy of SARC for myocardial perfusion was measured. Results Injection of Gd-DTPA into a perfusing artery during SARC resulted in increased signal intensity not only in the territory of the perfusing artery but also in the areas normally served by the other two venting coronary arteries （including the right ventrieuiar free wall）. With Gd DTPA given into the CS during SARC, the myocardium in the territories of the two venting coronary arteries was lightened. Signal intensity of the myocardium in the perfusing artery territory and right ventricuiar free wall remained unchanged. Moreover, a significant amount of effluent was collected from the venting coronary arteries during SARC： the LAD 10.5-17.7 ml/min; LCX 9.7-15.2 ml/min, and RCA 4.7-7.8 ml/min. Conclusion SARC through a single coronary artery and CS can provide homogeneous perfusion to the entire heart and is sufficient to prevent ischemic injury in the myocardium normally supported by the venting coronary arteries.
OBJECTIVE To search an optimal method for improving viability of cryopreserved articular cartilage. METHODS Articular cartilage which was sampled from the rabbits were randomly divided into 5 groups. Fresh cartilage was group I, other groups were frozen. Before frozen, other cartilage was exposured in 10% DMSO at 4 degrees C for 30 minutes(group II), 1 hour(group III), 2 hours (group IV), 4 hours(group V), then were stored in liquid nitrogen for 1 week. Viabilities of the chondrocytes were detected by Typan-blue staining, electron transmission microscope, and determination of incorporation 3H-TdR after the temperature returned to normal. RESULTS 1. The cells were injuried at different extent after the cartilage was frozen. In group I, survival rate of cells was 96% and incorporation of 3H-TdR was (4,953.13 +/- 583.27)%, statistic difference was significant between group I and other groups(P lt; 0.01). The microstructure of group I was normal while other groups all had damage of the organella, 2. Structures and functions of cells in group IV were best among frozen groups. Organella were less damaged than group II, III, V, survival rate of cells was 56% and incorporation of 3H-TdR was (1,139.88 +/- 146.39)%, statistic difference was significant between group IV and group II, III, V(P lt; 0.01). CONCLUSION If cartilage are exposured in 10% DMSO at 4 degrees C for 2 hours before frozen, optimal cryopreservation can be achieved.
Objective To elucidate the protective effect of leukocyte depletion on the myocardium during the settings of myocardial reperfusion injury. Methods Twenty patients undergoing cardiopulmonary bypass with continuous infusion of blood cardioplegia were randomized into two groups:the control group (n=10) with no leukocyte depletion filter used, and the experimental group (n=10) with the use of leukocyte depletion filter on the bypass circuit. The blood cells count before and after the filtration were measure...