【Abstract】 Objective To investigate the feasibil ity of applying enzymatic method to prepare decellularizedporcine aorta and to evaluate its biomechanical properties, immunogenicity and cell compatibil ity. Methods 0.1% trypsin- 0.01% EDTA was appl ied to extract cells from porcine aorta under 37 continuously vibrating condition and its histology and microstructure were observed. The thickness, stress-strain curve, ultimate tension stress (UTS) and strain of failure (SOF) were compared before and after decellularization for 48, 96 and 120 hours under uniaxial tensile tests, respectively. The histological change was observed at 1, 3 and 6 weeks after the decellularized tissue was implanted subcutaneously in 3 dogs. According to the HE stains and a semi-quantitative Wakitani grading method, gross changes, category and amounts of infiltrated cells and neo-capillaries were compared between pre- and post-decellularization of porcine aortae. Endothel ial cells from canine external jugular vein were seeded onto the decellularized patches to observe the cell compatibil ity. Results Microscopy and electron microscopies examination identified that cell components was completely removed from the fresh porcine aorta and Masson’ strichrome showed that the structure of matrix (fibrins) was maintained intact at 96 hours using the decellularization method. There were no significant differences in the thickness, UTS and SOF between before and after decellularization (P gt; 0.05). However, The UTS values showed a decrease tendency and SOF showed an increase tendency. The stress-strain curve also verified a decrease tendency in mechanical intensity and an increase one in ductil ity after decellularization. After implanting the acellularized matrix subcutaneously in canine, moderately lymphocyte infiltration was seen at the 1st week and the infiltration was replaced by fibroblasts accompanied by neocapillary formation at the 6th week. A semi-quantity histological evaluation showed that there were differences in gross observation, category and the numbers of the infiltrated cells between decellularized and non-decellularized tissues（P lt; 0.05). A cell monolayer was identified by HE staining and scanning electron microscopywhen the endothel ial cells were seeded onto the inner luminal surface of the scaffold, al igned at the same direction on the whole. Conclusion The decellularized porcine aortic scaffold, prepared by trypsin-EDTA extraction under continuously vibrating condition, could meet the requirements of tissue-engineering graft in biomechanical properties, immunogenicity and cell compatibil ity.
Objective To observe the clinical efficiency of the implantation of the autologous bone marrow mononuclear cells for treatment of lower limb ischemia after the bone marrow stimulation. Methods From May to December 2005, 43 ischemic limbs in 35 patients (23 males,12 females; aged 3490 years,averaged 71.3 year) were treated. Of the 35 patients, 30 had diabetic lowerlimb ischemia with 38 lower ischemic limbs, 2 had atherosclerosis obliterans with 2 ischemic lower limbs, and 3 had thromboangiitis obliterans with 3 ischemic lower limbs. Five patients with 5 ischemic limbs were in stage Ⅰ lower limb ischemia (intermittentclaudication), 15 patients with ischemic 19 limbs were in stage Ⅱ (rest pain),9 patients with 12 ischemic limbs were in stage Ⅲa(ulceration), and 6 patients with 7 ischemic lower limbs in stage Ⅲb (gangrene); 88.4% of all the ischemic lower limbs （38/43）had a pain, 79.1%（34/43） had coldness, and 69.8%（30/43）had limb numbness. The bone marrow of each patient was stimulated by an injection of the recombinant human granulocyte-macrophage colony-stimulatory factor 300 μg/d for 2-3 days. The bone marrow 130-200 ml was drawn from the iliac spine and the mononuclear cells were obtained. Each patient received implantation of the autologous bone marrow mononuclear cells by an intramuscular injection, an arterial intraluminal injection or a combined injection of the two routes.Results The pain relief was found in 94.7% of theischemic lower limbs, and pain improvement in 97.1% . Relived numbness was found in 93.3%. The distance of the claudication was increased by all the ischemic limbs. An increase in the ankle/ brachial index (ABI)was found in 47.9%. The transcutaneous oxygen pressure (TcPO2) increased in 92.3%. The ulcer heal rate was 9.1% (1/11). Markedlyreduced ulcer wound was found in 27.3% (3/11). The amputation rate was 6.3% (3/48). Arterial angiography revealed that there was a new collateral vessel formationin 91.2%. Complications were as follows: fever and mild fatigue-developed respectively in 1 patient after the bone marrow stimulation, but relieved by themselves. Acute but mild myocardial infarction was found in 1 patient with a slight precordial pain and elevation of myocardial enzymes 1 week after transplantation of the bone marrow mononuclear cells, but recovered after medical treatment. The follow-up averaged 5 months. According to the subjective criteria, the overall efficacy was90%. ABI increased in 62.5% of the patients after operation and the value of TcPO2 was higher in 90% of the patients after this kind of therapy. Arterial angiography revealed a new collateral vessel formation in 90.5% of the 21 ischemic limbs. The foot ulcer healed in 7 and obviously improved in 3. Three of the foot ulcer patients were discharged 2-3 months after the amputation was performed on the diseased toes. Conclusion Implantation of the autologous bone marrow mononuclear cells after the bone marrow stimulation of treatment of the lower limb ischemia has advantages of less marrow aspiration, more mononuclear cell content, satisfactory shortterm effect, and relatively high safety. Itis a new method of treating the lower limb ischemia besides the autologous bone marrow and peripheral blood mononuclear cell implantation. The longterm effect of this method needs a further study.
Objective To investigate the effect of adenovirus vector mediated transfer of human herpes simplex virus thymidine kinase (HSVtk) gene inhibits intimal hyperplasia of vein grafts. Methods Auto vein graft models of Wistar rats were established. Adenovirus vector dwelled in cervical veins which were transplanted into inferior renal abdominal aorta. The combination of HSVtk (4×109 plaque forming units) and ganciclovir (GCV) was applied to test the inhibition effect. GCV was infused 〔60 mg/(kg·d), IP, Bid〕 from day 3 to day 21 after transplantation. Vein samples were harvested and the existence of HSVtk DNA was measured by PCR and the mRNA of it was studied by in situ hybridization. Van gieson (VG) and proliferating cell nuclear antigen (PCNA) stains were carried out in paraffin sections to study the thickness of neointima and smooth muscle cells (SMCs) proliferation with a computer-assisted analysis system. The apoptosis of SMCs also was detected by TUNEL. Results The existence of HSVtk gene in veins and its transcription were demonstrated. Morphometric analysis demonstrated a reduced intima thickness in the group receiving combination therapy (HSVtk/GCV) compared with HSVtk alone 〔(17.2±3.2) μm versus (31.1±2.5) μm, P＜0.05〕. GCV per se had no effect on intimal hyperplasia after vein transplantation. The apoptosis of SMCs increased significantly and expression of PCNA decreased in HSVtk/GCV gene therapy group versus blank control group 〔(9.1±2.3)% vs (28.7±3.6)%, P＜0.05; (38.7±5.6)%vs (18.5±2.6)%, P＜0.05〕. Conclusion GCV conditions reduction of intimal hyperplasia after intraluminal delivery of HSVtk in transplanting vena veins involving SMCs apoptosis.
Objective To investigate the effectiveness of percutaneous transluminal renal artery stenting (PTRAS) in treating atherosclerotic renal artery stenosis (ARAS). Methods A total of 69 patients with severe ARAS were treated with PTRAS between January 2002 and December 2008. There were 47 males and 22 females with an average age of 66.2 years(range, 42-88 years), including 66 cases of unilateral ARAS (single functional kidney, 1 case) and 3 cases of bilateral ARAS. Renal angiography revealed that the degree of renal artery stenosis was 70%-99%. Concomitant diseases included hypertension (67 cases), atherosclerosis obl iterans (69 cases), coronary heart disease (34 cases), diabetes (44 cases), and hyperl ipidemia (36 cases). Blood pressure, serum creatinine (sCr), and patency of the renal artery were measured to assess the effectiveness of PTRAS after 12 months. Results The renal artery stent was successfully implanted in 68 patients and the technical success rate was 98.6%. One patient was converted to il io-renal bypass because of intra-operative acute renal artery occlusion. One patient died of heart failure at 6 months after PTRAS, and 1 patient was lost at 3 months after PTRAS. The other 66 patients were followed up 32 months on average (range, 13-60 months). The blood pressure decreased significantly at 1 month and gained a further decrease at 12 months after PTRAS when compared with the preoperative ones [systol ic blood pressure: (132 ± 24) mm Hg vs (163 ± 34) mm Hg, P lt; 0.05; diastol ic blood pressure: (78 ± 11) mm Hg vs (89 ± 17) mm Hg, P lt; 0.05; 1 mm Hg=0.133 kPa]. Hypertension was cured in 4 cases (6.3%), improved in 52 cases (81.2%), failure in 8 cases (12.5%), and the overall benefit rate was87.5%. The sCr level was stable after 12 months of PTRAS, showing no significant difference when compared with preoperative basel ine [(107.8 ± 35.4) μmol/L vs (104.1 ± 33.8) μmol/L, P gt; 0.05]. Renal function was improved in 9 cases (13.6%), stable in 48 cases (72.8%), deterioration in 9 cases (13.6%), and the overall benefit rate was 86.4%. Instent restenosis found in 2 patients (3.0%) at 12 months after operation. Conclusion PTRAS is a safe and effective method to treat ARAS. It can control the blood pressure and stabil ize the renal function in most ARAS patients. Long-term efficacy needs further investigation.
Objective To investigate the cl inical effect of vacuum seal ing drainage (VSD) on late-stage large skin avulsion injury with infection. Methods From May 2007 to August 2008, 9 patients with large-area skin avulsion injury and infection were treated. There were 1 male and 8 females aged 9-52 years old (median 27 years old). All patients suffered from closed skin avulsion injury involving the lower back, buttock, and part of the thigh. The injury area varied from 30 cm × 25 cm to92 cm × 38 cm. The time between injury and hospital admission was 15-23 days. The skin avulsion injury was compl icated with pelvis fracture, urethral injury, anal injury, sacrum exposure, and l imb fractures. The interval between hospital admission and operation was 3-23 hours. Free spl it-thickness skin graft was performed after the focus debridement and three VSD treatments (40-60 kPa). Results After three VSD treatments, no patient had general pyemia and severe local tissue necrosis or infection, the tissue edema in the skin avulsion area was alleviated obviously, and all the wound cavities were closed. All the wounds in the graft site healed after 28-45 days of treatment (average 39 days), and all the donor sites healed. Nine patients were followed up for 4-14 months (average 10 months). The appearance of the reparative area was good, and there was no occurrence of joint dysfunction in the injured area due to scar contracture. Conclusion VSD is effective in treating late-stage large skin avulsion injury with infection.
Objective To observe the changes in the number and function of bone marrow-derived endothel ial progenitor cells (EPCs) after bone-marrow stimulation, and to investigate the possible mechanism of improving ischemicl imb disease after bone-marrow stimulation through autologue bone-marrow stem cell implantation. Methods Twelvemale Lewis rats, weighing 200-250 g, were classified into the bone marrow stimulation group (n=6) and the control group(n=6). In the stimulation group, the bone marrow of each rat was stimulated by injection of recombinant human granulocytemacrophage colony-stimulatory factor. Mononuclear cells were harvested from bone marrow and cultured in EBM-2 medium. After 7-day culture, EPCs were stained by 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labbled acetylated low density l ipoprotein/fluorescein isothiocyanate-ulex europaeus agglutinin 1, and the double positive cells were counted by the fluorescent microscope. The adhesive abil ity of EPCs was determined by counting the number of re-cultured EPCs. The unilateral ischemia hindl imb model was made with 12 Lewis rats. Three days later, EPCs were transplanted into the ischemic tissues. According to different sources of EPCs, the 12 rats were divided into 2 groups: the stimulation group (n=6) and the control group (n=6). At 3 weeks after EPCs transplantation, the quantity of the collateral vascular was observed by digital subtraction angiography (DSA). Results After 7-day culture, the number of EPCs in the stimulation and control groups was (145.2 ± 37.0)/HP and (95.2 ± 39.4)/HP, respectively, and there was significant difference between the two groups (P lt; 0.05). Meanwhile, the number of adhesive EPCs in the stimulation and control groups was (21.8 ± 4.3)/HP and (15.0 ± 5.2)/HP, respectively, and the difference between the two groups was significant (P lt; 0.05). At 3 weeks after the EPCs implantation, the number of the collateral vascular was significantly larger in the stimulation group (4.2 ± 1.2) compared with the control group (2.7 ± 0.8), (P lt; 0.05). Conclusion Bone marrow stimulation increases the number of EPCs and improves the function concurrently, which may be the reason why autologue bone-marrow stem cell implantation improves the curative effect of ischemic l imb diseases after bone-marrow stimulation.
【Abstract】ObjectiveSome studies have demonstrated that recombinant adenoviruses are efficient vectors for gene transfer to the venous wall and AdCMV.tk encoded thymidine kinase can be used to reduce restenosis. In this study AdCMV.tk was apply to human vein smooth muscle cells (SMC) and organ cultured saphenous veins to study its effects on proliferation of SMCs and reduction of intimal hyperplasia. MethodsThe adenovirus vector transferred tk gene and mark gene lacZ to the SMC of human saphenous veins and organ cultured vein segments. Various concentrations ganciclovir (GCV) were contained in culture media. The efficiency of gene transfer was studied by using Xgal staining. The proliferation of SMC was monitored by the method of trypan blue exclusion. The bystander effect was observed by mixed cell culture. After vein segments treated by AdCMV.tk+GCV and cultured for 14 days, HE and VG staining were carried out and intimal thickness was analysis by computer image system. ResultsAdenovirus vector could infect saphenous vein SMC efficiently both in cultured SMCs and organ cultured vein segments. Gene expression sustained 14 d at least. The inhibition of SMCs proliferation in vitro was a positive correlation in GCV concentrations and the levels of tk expression. The proliferation of SMCs transfectered lacZ wasn’t restrained by GCV (P＜0.05). In mixed cell experiment there was at least 55% reduction in total cell number when as few as 10% of the cells express tk. Assessment of this “suicide gene strategy” in saphenous vein organ culture model demonstrated that veins treated with AdCMV.tk+GCV had a significant reduction at 14 days in the intimal thickness compared to control group (P＜0.01). ConclusionThe results suggest that adenovirusmediated gene transfer of tk along with GCV administration may be a useful strategy to treat the proliferation of intimal hyperplasia of transplanting saphenous veins. Bystander effects are amplified by AdCMV.tk/GCV gene therapy system.