组织工程血管的实验研究
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摘要
目的:研究脱细胞血管支架的快速高效的制备方法,对其进行力学性能及生物相容性的评价,用CD34抗体修饰脱细胞血管支架后,植入动物体内募集循环中的内皮祖细胞参与再内皮化,找到一种更接近现实应用的组织工程血管。方法:新鲜的羊颈动脉、前肢动脉经-80℃冷冻、37℃水浴复温,反复冻融3次,在5000大气压(atm)4℃条件下超高压处理20min,0.125%十二烷基硫酸钠溶液(SDS)中,37℃振摇(100r/min)12h,彻底去除细胞后,进行苏木精-伊红染色、Masson三染色、扫描电镜及生物力学测定研究其组织结构的变化,荧光染色和苯酚-氯仿提取法分析细胞DNA残留量,对脱细胞的效果进行评价,通过接触细胞毒性实验、材料浸提液细胞毒性实验及皮下埋植毒性实验,对制备的脱细胞血管支架进行生物相容性分析。用光交联的方法,以交联剂SANPAH为介导,CD34抗体修饰脱细胞血管支架。20只新西兰大白兔右下腹做一带蒂皮瓣,皮瓣供血动脉为股动脉的分支血管,血管移植部位选取同侧股动脉,切取一段,造成1cm长的全段缺损,实验组选用CD34抗体修饰脱细胞血管支架,对照组用未经交联的脱细胞血管支架,各10根,显微外科端端吻合替代缺损的兔股动脉。术后通过对皮瓣色泽质地的观察,了解皮瓣的血供情况,间接反映吻合血管的通畅性。术后行彩色多普勒,数字减影血管造影检查和病理切片观察移植血管的通畅情况及细胞粘附情况。结果:苏木精-伊红染色及Masson三染色显示脱细胞血管支架无细胞成分残留,保持较完整的细胞外基质成分;电镜扫描血管表面以胶原为主,适合细胞粘附;荧光染色脱细胞支架材料未见阳性核染色;电泳检测分析显示脱细胞血管支架DNA含量显著降低;力学检测证实脱细胞血管支架保持了正常血管的力学特征;接触细胞毒性实验与材料浸提液细胞毒性实验测定脱细胞支架与细胞有良好的粘附性,细胞毒性为0-1级;大鼠皮下埋植8周后基本未见炎性细胞,说明脱细胞血管支架在动物体内无排斥,与周围组织具有良好的相容性。实验组皮瓣术后血运较好,对照组皮瓣术后肿胀坏死。移植术后4周,行彩色多普勒和数字减影血管造影检查,实验组血管7例通畅,血管无明显狭窄,通畅率为70%,闭塞3例可见吻合口附壁血栓形成;对照组术后1周无1例通畅,均为血栓形成。实验组术后4周处死动物,解剖移植血管,病理切片行苏木精-伊红染色,显示实验组通畅的脱细胞血管支架表面形成连续融合单层细胞,管腔无明显狭窄,对照组术后1周脱细胞血管支架腔内血栓形成,腔内表面缺乏细胞覆盖。结论:反复冻融、超高压及小剂量SDS处理能在较短时间内彻底脱除血管的细胞成分,对血管的力学性能影响小,用这种结合物理和化学的方法脱细胞处理的血管支架无细胞毒性,具有优良的生物相容性。用光交联的方法,以交联剂SANPAH为介导,可将CD34抗体牢固的交联在血管内腔壁上。经交联CD34抗体的脱细胞血管支架对比未经交联的脱细胞血管支架无论在早期抗凝及通畅率上均有优势。经过修饰的血管材料在体内募集循环中的内皮祖细胞参与再内皮化是完全有可能的,这种方法简化了组织工程血管体外构建的过程,为组织工程血管的研究又向前推进一步。
Objective To study the method and process of obtaining decellularized vascular scaffolds and evaluate its mechanical characterization and biocompatibility.To evaluate the effects of the decellularized vascular scaffolds coated with anti-CD34 antibodies capturing endothelial progenitor cells from the circulation to participate recellularization.Methods Fresh caprine carotids and brachial arteries were treated with repeated frozen/thawing,cold isostatic pressing(5000 MPa,4℃)and 0.125% sodium dodecyl sulfate(SDS) separately for preparation of decellularized vascular scaffolds.Biological characteristics were compared with the raw caprine carotids using hematoxylin and eosin staining,Masson staining,scanning electron microscopy and mechanical test.Using fluorescence staining and DNA remains test to assess the cell extracting results.Biocompatibility were detected using cell adhesion test,MTT assay,and subcutaneously embedding test.After reacted with photochemical crosslinker SANPAH,anti-rabbit CD34 antibody was coated onto the decellularized vascular scaffolds using ultraviolet ray.20 New Zealand white rabbits were performed right lower-abdominal pedicled skin flap which supplyed by branches of femoral artery.Whole defect 1cm in femoral artery were made and end-to-end anastomosis repaired by 10 crosslinked and 10 non-crosslinked scaffolds in experimental group and control group separately.The patency of the pedicle was observed through color and appearance of the flap postoperatively.After 4 weeks of transplantation,patency rate and cell seeding were detected by Color Doppler,DSA and pathological test. Results Hematoxylin and eosin staining and Masson staining revealed that no nucleus was detected in the decellularized vascular scaffolds.Scanning electron microscopy demonstrated that a lot of collagen fibers were preserved which was beneficial for cell adhesion.Fluorescence staining and DNA remains test showed that the cells were removed completely.Mechanical test revealed that the decellularized vascular scaffolds reserved mechanical properties of the raw caprine arteries.Cell adhesion test and MTT assay confirmed that cytotoxicity was grade 0-1,and the decellularized vascular scaffolds had a good compatibility to endothelial cells.After subcutaneously embedding for 8 weeks,negligible moderately lymphocyte infiltration was observed. Flaps of experimental group had good blood supply after transplantation while swelling and necrosis could be found in the control group.Doppler and DSA showed that the 7 of 10 crosslinked scaffolds remained patent for 4 weeks and also no stenosis had been developed,but bare scaffolds were obstructed in the control group 1 week. Hematoxylin and eosin staining revealed that the inner layers of crosslinked scaffolds were partly covered with endothelial cells in one month.In contrast,no cell coverage was observed in the bare scaffolds but thrombosis.Conclusion The cells were removed completely after multistep processes and no debris and DNA residue.The decellularized vascular scaffolds show negligible cell toxicity but favorable bio compatibility.The mechanical characterization of the decellularized vascular scaffolds is well-preserved by the combination of repeated frozen/thawing,ultrahigh pressure treatment and chemical detergent.Anti-CD34 antibody was firmly coated onto the decellularized vascular scaffolds using ultraviolet ray by crosslinker SANPAH.Crosslinked scaffolds with anti-CD34 antibody were superior to bare scaffolds in early postoperative anticoagulation and patency rate.It is not impossible for anti-CD34 antibody crosslinked scaffolds to recruit endothelial progenitor cells from the circulation to participate recellularization.This method simpilify the procedure of tissue engineering blood vessel fabricating in vitro and take the research one step forward.
Objective To study the method and process of obtaining decellularized vascular scaffolds and evaluate its mechanical characterization and biocompatibility.To evaluate the effects of the decellularized vascular scaffolds coated with anti-CD34 antibodies capturing endothelial progenitor cells from the circulation to participate recellularization.Methods Fresh caprine carotids and brachial arteries were treated with repeated frozen/thawing,cold isostatic pressing(5000 MPa,4℃)and 0.125% sodium dodecyl sulfate(SDS) separately for preparation of decellularized vascular scaffolds.Biological characteristics were compared with the raw caprine carotids using hematoxylin and eosin staining,Masson staining,scanning electron microscopy and mechanical test.Using fluorescence staining and DNA remains test to assess the cell extracting results.Biocompatibility were detected using cell adhesion test,MTT assay,and subcutaneously embedding test.After reacted with photochemical crosslinker SANPAH,anti-rabbit CD34 antibody was coated onto the decellularized vascular scaffolds using ultraviolet ray.20 New Zealand white rabbits were performed right lower-abdominal pedicled skin flap which supplyed by branches of femoral artery.Whole defect 1cm in femoral artery were made and end-to-end anastomosis repaired by 10 crosslinked and 10 non-crosslinked scaffolds in experimental group and control group separately.The patency of the pedicle was observed through color and appearance of the flap postoperatively.After 4 weeks of transplantation,patency rate and cell seeding were detected by Color Doppler,DSA and pathological test. Results Hematoxylin and eosin staining and Masson staining revealed that no nucleus was detected in the decellularized vascular scaffolds.Scanning electron microscopy demonstrated that a lot of collagen fibers were preserved which was beneficial for cell adhesion.Fluorescence staining and DNA remains test showed that the cells were removed completely.Mechanical test revealed that the decellularized vascular scaffolds reserved mechanical properties of the raw caprine arteries.Cell adhesion test and MTT assay confirmed that cytotoxicity was grade 0-1,and the decellularized vascular scaffolds had a good compatibility to endothelial cells.After subcutaneously embedding for 8 weeks,negligible moderately lymphocyte infiltration was observed. Flaps of experimental group had good blood supply after transplantation while swelling and necrosis could be found in the control group.Doppler and DSA showed that the 7 of 10 crosslinked scaffolds remained patent for 4 weeks and also no stenosis had been developed,but bare scaffolds were obstructed in the control group 1 week. Hematoxylin and eosin staining revealed that the inner layers of crosslinked scaffolds were partly covered with endothelial cells in one month.In contrast,no cell coverage was observed in the bare scaffolds but thrombosis.Conclusion The cells were removed completely after multistep processes and no debris and DNA residue.The decellularized vascular scaffolds show negligible cell toxicity but favorable bio compatibility.The mechanical characterization of the decellularized vascular scaffolds is well-preserved by the combination of repeated frozen/thawing,ultrahigh pressure treatment and chemical detergent.Anti-CD34 antibody was firmly coated onto the decellularized vascular scaffolds using ultraviolet ray by crosslinker SANPAH.Crosslinked scaffolds with anti-CD34 antibody were superior to bare scaffolds in early postoperative anticoagulation and patency rate.It is not impossible for anti-CD34 antibody crosslinked scaffolds to recruit endothelial progenitor cells from the circulation to participate recellularization.This method simpilify the procedure of tissue engineering blood vessel fabricating in vitro and take the research one step forward.
引文
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[3]Pasic M,Muller-Glauser W,von Segesser L,Odermatt B,Lachat M and Turina M.Endothelial cell seeding improves patency of synthetic vascular grafts:manual versus automatized method.Eur J Cardiothorac Surg.1996;10:372-9.
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[6]Hirashima M,Kataoka H,Nishikawa S and Matsuyoshi N.Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.Blood.1999;93:1253-63.
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[9]Gimble JM.Adipose tissue-derived therapeutics.Expert Opin Biol Ther.2003;3:705-13.
[10]Mizuno H,Zuk PA,Zhu M,Lorenz HP,Benhaim P and Hedrick MR Myogenic differentiation by human processed lipoaspirate cells.Plast Reconstr Surg.2002;109:199-209;discussion 10-1.
[11]Jack GS,Almeida FG,Zhang R,Alfonso ZC,Zuk PAand Rodriguez LV Processed lipoaspirate cells for tissue engineering of the lower urinary tract:implications for the treatment of stress urinary incontinence and bladder reconstruction.J Urol.2005;174:2041-5.
[12]Martinez-Estrada OM,Munoz-Santos Y,Julve J,Reina M and Vilaro S.Human adipose tissue as a source of Flk-1+ cells:new method of differentiation and expansion.Cardiovasc Res. 2005; 65:328-33.
[13] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275:964-7.
[14] Gehling UM, Ergun S, Schumacher U, et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood. 2000; 95:3106-12.
[15] Kaushal S, Amiel G E, Guleserian KJ, et al. Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med. 2001; 7:1035-40.
[16] Griese DP, Ehsan A, Melo LG, et al. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy. Circulation. 2003; 108:2710-5.
[17] Hristov M, Erl W and Weber PC. Endothelial progenitor cells: isolation and characterization. Trends Cardiovasc Med. 2003; 13:201-6.
[18] Ceradini DJ and Gurtner GC. Homing to hypoxia: HIF-1 as a mediator of progenitor cell recruitment to injured tissue. Trends Cardiovasc Med. 2005; 15:57-63.
[19] Strehlow K, Werner N, Berweiler J, et al. Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation. Circulation. 2003; 107:3059-65.
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