【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 investigate the feasibil ity of preparing the porous extracellular matrix (ECM) by use of some chemicals and enzymes to decellularize the porcine carotid artery. Methods The porcine carotid artery was procured, and warm ischemia time was less than 30 minunts. The porcine carotid artery was decellularized with 1% sodium dodecyl sulfate (SDS) for 60 hours to prepare common ECM; then common ECM was treated with 0.25% trypsin (for 6 hours) and 0.3 U/ mL collagenase (for 24 hours) to prepare porous ECM. The common ECM and porous ECM were stained with HE,Masson’s trichrome, and Orcein to evaluate the histological features. Then the mechanical property, cytotoxicity, and pore size of ECMs were determined. After 4 weeks of subcutaneous implantation in dogs, the histological examination was used for the study. Results Histological observation confirmed that 2 kinds of ECMs were decellularized completely and more porous structure was observed in porous ECM. Scanning electron microscope showed the pores in porous ECM were greater and the length of shorter axis in porous ECM ranged from 5 to 30 μm, the length of longer axis from 40 to 100 μm. The porosity of porous ECM (99.25%) was greater than that of common ECM (91.50%). The burst pressure of porous ECM decreased when compared with common ECM, showing significant difference [(0.154 3 ± 0.012 7) MPa vs [0.305 2 ± 0.015 7) MPa, P lt; 0.05]. There was no significant difference in suture retention strength between 2 kinds of ECMs (P gt; 0.05). The cytotoxicity test showed no obvious cytotoxicity in 2 kinds of ECMs. In vivo implantation test showed that the deeper host cells infiltration and more neo-microvessels in porous ECM were observed than in common ECM. Conclusion SDS and some enzymes can be used to prepare porous ECM as the scaffold for tissue engineered blood vessels.
Objective To evaluate effect of hypoxia condition (1% or 5% oxygen concentration) on proliferation, adhesion, migration, or viability ability of bone morrow-derived endothelial progenitor cells (EPCs). Methods The bone marrow mononuclear cells of SD rat were acquired with density gradient centrifugation method. They were cultured, induced, and differentiated to the EPCs. Then they were cultured respectively in three different oxygen concentrations (1%, 5%, or 21%). On the 3rd day and the 7th day, the effects of the different oxygen concentrations (1%, 5%, or 21%) on the EPCs’ neovascularization characteristics (including proliferation, adhesion, migration, and viability abilities) were evaluated. Results Whether cultured for the 3rd day or 7th day, the proliferation, adhesion, migration, and viability abilities of the cultured cells in the 1% and 5% oxygen concentrations were significantly better than those of the cultured cells in the 21% oxygen concentration (all P<0.05). Except for the proliferation ability of the cultured cells in the 5% oxygen concentration was significantly better than that of the cultured cells in the 1% oxygen concentration (P<0.05) on the 3rd day, and the adhesion ability on the 3rd day and the proliferation ability on the 7th day had no significantly differences, the other abilities (adhesion, migration, and viability abilities) of the cultured cells in the 1% oxygen concentration were significantly better than those of the cultured cells in the 5% oxygen concentration (allP<0.05). Conclusion Different oxygen concentration has an effect on proliferation, adhesion, migration, or viability ability of bone morrow-derived EPCs, appropriate hypoxia condition (1% or 5% oxygen concentration ) can enhance these abilities.
Objective To explore the effective surgical approaches in treating subclavian artery occlusion. Methods Between December 2005 and February 2010, 53 patients with subclavian artery occlusion were treated, including left subclavian artery occlusion (35 cases) and stenosis (5 cases), right subclavian artery occlusion (5 cases) and stenosis (4 cases), and bilateral subclavian artery occlusion (4 cases). There were 40 males and 13 females with an average age of 64 years (range, 22-77 years), including 49 cases of arteriosclerosis obl iterans and 4 cases of aortic arteritis. The disease duration was 15 days to 20 months (6.5 months on average). In 49 patients with unilateral subclavian artery occlusion, 39 cases compl icated by carotid or / and cerebral artery lesion underwent axillo-axillary bypass grafting, and 10 cases without carotid or /and cerebral artery lesion underwent carotid-subclavian bypass grafting. Ascending aorta to bisubclavian bypass graftings were performed on 4 cases with bilateral subclavian artery occlusion. After operation, patients received routine treatment with anticoagulant and antiplatelet agents. Results The operations were successfully performed in 52 cases with a successful rate of 98.11%. Thrombogenesis at anastomotic site occurred in 1 case of aortic arteritis after 48 hours. Two cases had brachial plexus crush injury and 4 had hematoma around the bilateral anastomosis after axillo-axillary bypass grafting, and all recovered with nonoperative therapy. A total of 52 patients were followed up 1-52 months (24.5 months on average). All patients survived and the symptoms of basilar and upper l imb artery ischemia disappeared. Doppler ultrasonography showed that the blood flow was patent through anastomosis and polytetrafluoroethylene graft, and the vertebral artery flow was normal. Pseudoaneurysm at anastomosis was found in 1 case after 18 months and treated by interventional embol ization. The postoperative graft patency rate was 100% at 1 year and at 2 years. Conclusion Both thoracic and extrathoracic surgical approaches are effective for treating subclavianartery occlusion. The reasonable surgical approach should be selected according to the arteriopathy and the patient’s condition. Perioperative treatment and strict intraoperative manipulation are important to guarantee the success of surgery.
Objective To explore the middle-term outcome of autologous bone marrow mononuclear cells transplantation in the treatment of lower l imb ischemia. Methods From March 2003 to June 2005, 65 patients with lower l imb ischemia were treated by autologous bone marrow mononuclear cells transplantation. Of the patients, there were 50 males and 15 females, with a mean age of 66.5 years (range 36-89 years), including 4 cases of simple arteriosclerotic occlusion,5 cases of thromboangiitis obl iterans and 56 cases diabetic lower l imb ischemia. A total of 400 mL bone-marrow blood were extracted from the posterior superior il iac crest. And then the mononuclear cells were isolated from the bone-marrow blood in the laboratory. The amount of transplantation bone marrow mononuclear cells was (0.60-1.80) × 109 (mean 1.05 × 109). Twelve patients received cell transplantation from two to four times and the other patients one time. According to the improvement of cl inical finding, the outcome was evaluated. Results All the patients were followed up for 8-56 months (mean 21.5 months). There were 8 deaths, and the mortal ity was 12.3%; 5 were due to myocardial infarction and heart failure and 3 were due to cerebral infarction. The general effective rate was 70.8% (46/65) and the recurrent rate was 10.7% (7/65). Of them, the response to treatment lasted over 12 months in 42 cases, accounting for 91.3% (42/46); over 24 months in 24 cases, accounting for 52.2% (24/46); and over 37 months in 12 cases, accounting for 26.1% (12/46). The effective rates were 100% in 12 patients who received 2-4 times transplantation and 64.2% in 53 patients who received 1 time transplantation, showing statistically significant difference between them (P lt; 0.001). Conclusion The middle-term outcome of autologous bone marrow mononuclear cells transplantation show that it is a feasible and simple method for treatment of lower l imb ischemia.
Objective To investigate the effect and safety of autologous bone marrow-mononuclear cell (BM-MNC) transplantation on ischemic limb of patients with thromboangiitis obliterans (TAO). Methods Thirteen patients with TAO underwent transplantation of autologous BM-MNC into ischemic muscles of 17 lower limbs. A series of subjective indexes (improvement of pain and cold sensation) and objective indexes including increase of ankle brachial index (ABI), transcutaneous oxygen pressure (TcPO2), and improvement of foot skin ulcer were used to evaluate the effects. Results The outcomes were evaluated after 2 months of transplantation. The pain relief and improvement of cold feeling were in 15 limbs and 16 limbs, respectively. Before transplantation and 2 months after transplantation, ABI was 0.37±0.06 and 0.50±0.17, respectively (Plt;0.05), and TcPO2 of the ischemic legs were (24.59±3.36) mm Hg (1 mm Hg=0.133 kPa) and (35.00±10.44) mm Hg, respectively (Plt;0.05). ABI increased in 9 limbs. TcPO2 elevated in 14 limbs. Skin ulcer improved in 7 limbs. Thirteen patients were followed up from 4 to 18 months (average 8 months), the patients’ symptoms improved in 13 limbs. ABI was 0.45±0.14, which wasn’t different from those before transplantation and 2 months after transplantation (Pgt;0.05). TcPO2 was (33.24±10.43) mm Hg, which was different from those before transplantation and 2 months after transplantation (Plt;0.05) and was elevated in 12 limbs. Skin ulcer healing was in 5 limbs. The ischemic symptoms in 2 patients were not relieved. There was no mortality and high level amputation. The following complications, such as proliferative retinopathy, malignant tumor, myocardial infarction, stroke or hemangioma, were not found in all patients.Conclusion In patients with TAO, intramuscular transplantation of autologous BMMNC is a safe and effective method, and may improve symptoms and accelerate the healing of skin ulcer.