仿生型人工小血管支架的构建与体外力学研究

目的 制备具有三层管壁结构的可降解人工小血管支架,对其生物力学性能进行测试,并与正常生理血管对比以检测其是否符合体内移植试验的要求.方法 以可降解的聚对二氧环己酮(PDS)缝合线编织成网管状织物作为血管内支架,模仿天然血管的三层结构,内层共混硫酸软骨素-胶原,外层包被小肠黏膜下层,缝线加固,构建内径<4 mm的小血管支架,检测血管支架的生物力学性能(爆破压力、抗拉伸能力、顺应性等),并与正常生理血管进行比较.结果 所制备的人工小血管支架平均内径3.83 mm,爆破压为(43.50±8.30)kPa,断裂强度为(19.10±1.56)N,应变率为(42.88±3.16)%,径向顺应性为(5.96±0.87)%/100 mm Hg(1 mm Hg=0.133 kPa).结论 所制备人工血管的力学性能优良,可以满足动物体内移植试验的要求.     

聚对二氧环己酮网制备及重建犬胸壁缺损的研究

目的 通过生物材料的选择和制备,研制新型可降解人工胸壁修复材料,并通过动物实验探讨其用于胸壁重建的可行性.方法 采用聚对二氧环己酮(PDO)纤维编织成网状结构人工胸壁,应用于犬胸壁缺损重建动物模型,8、16、24周处死实验犬,观察人工材料降解变化、材料与组织结合界面、胸壁再生情况.结果 PDO网可以重建胸壁稳定性,并在24周内逐步降解吸收,由机体再生组织完全取代.结论 PDO网具备适宜的可降解特性,作为胸壁重建材料,可获得有效胸壁稳定,具有良好临床应用前景.     

仿生型人工小血管支架的构建与体内降解研究

目的 以复合成型工艺构建可降解仿生型人工小血管支架,观察其体内降解过程及组织反应.方法 模仿天然血管的三层结构,使用可降解的聚埘二氧环己酮(PDS)编织支架作为中间弹力层、以小肠黏膜F层(SIS)为外层、内层用共混硫酸软骨素胶原海绵涂层构建复合型人工小血管支架,将支架材料植入比格犬背部脊柱两侧肌肉内,于2、4、12、24周取材,行组织学和透视电镜观察.结果 所有实验动物术后活动正常,切口I期愈合.2～4周时,SIS与胶原结构基本保持,日J见较多的炎细胞浸润,胶原海绵网孔结构开始破坏融合,形成板层结构.12周时,自身纤维结缔组织完全取代胶原海绵材料,纤维组织广泛长人网材孔间隙内,PDS发生了部分的降解,炎细胞浸润明显消退.24周时,支架材料完全吸收,被新生纤维组织填充,炎细胞和和异物巨噬细胞少见.降解过程中未见材料周围组织变性、坏死或肉芽肿异常增生现象.结论 本实验制备的仿生型人工小血管支架组织反应轻,降解时间合适,满足体内组织再生要求,可用于下一步实验.     

自体组织构建小口径血管的实验研究

目的在大鼠自体肌肉内预置硅胶棒后形成结缔组织管,观察将其作为血管替代物移植于腹主动脉后的组织结构变化。方法取Wistar大鼠48只随机分为对照组和2、4、6个月移植组。先在腹壁肌肉内埋植直径0 .2cm、长1.5cm硅胶棒,6周后将硅胶棒周围形成的结缔组织管状物作为血管替代物移植于大鼠腹主动脉,在移植术后2、4、6个月分别观察血管通畅情况,光镜、透射及扫描电镜观察替代物的组织结构。结果1.血管替代物移植于腹主动脉后2、4、6个月血管全部通畅。2 .组织学观察平滑肌细胞及内皮细胞在2个月组替代物仅中段少部分未覆盖,4个月组替代物内壁覆盖连续,6个月组与对照组相近,且在近吻合口端可见弹力膜形成。结论1.自体组织所形成的替代物移植于腹主动脉后6个月后管腔通畅良好。2 .移植后6个月的替代物管壁厚度及组织结构已接近正常血管。     

大孔隙非限制性血管外支架抑制移植静脉内膜增生的实验研究

目的探讨大孔隙非限制性聚对二氧环己酮血管外支架抑制移植静脉内膜增生的实验效果及其作用机制。方法建立兔自体颈外静脉移植至颈总动脉动物模型,48只新西兰大白兔随机分为对照组(单纯静脉移植,n=24)和实验组(静脉移植 血管外支架,n=24)。于术后4周、12周分别取出移植静脉,行病理学检查测量内膜、中膜的厚度和面积,对增殖细胞核抗原行免疫组织化学检查计算细胞增殖率。结果48只兔无手术和术后死亡,移植血管全部通畅。实验组血管外支架与移植静脉壁之间形成富含毛细血管的"新生外膜"。术后4周和12周实验组管腔面积大于对照组,而中膜面积、内膜面积、中膜厚度和内膜厚度均小于对照组(P<0.05)。术后4周实验组细胞增殖率(22%±11%)低于对照组(37%±15%,P<0.05);术后12周实验组和对照组细胞增殖率均较术后4周降低,而实验组细胞增殖率(8%±5%)低于对照组(16%±9%,P<0.05)。结论大孔隙非限制性聚对二氧环己酮血管外支架通过刺激外膜滋养血管生成、改善静脉壁缺血和缺氧、减小移植静脉壁的轴向应力,而达到减轻内膜和中膜增生的作用。     

小口径人造血管支架的研究进展

近年来,随着血管疾病增加,对功能动脉置换的需要也日益增加,小口径动脉如膝下动脉、冠状动脉,使用合成材料移植不像大口径动脉移植物那样有较高的成功率。目前,自身静脉仍是主要移植物。然而,由于血管疾病、截肢术或血管以前已被采集,许多病人无适合的血管可使用,组织工程血管的出现为解决这一问题带来了希望。     

四肢小口径组织工程血管的实验研究

目的研究以小肠黏膜下层组织（small—calibu tissue，SIS）为血管支架，在体内血流条件下培养小口径组织工程血管的可行性。方法自犬隐动脉分离出血管种子细胞，与Ⅰ型胶原蛋白凝胶均匀混合，种植于SIS膜表面，包绕3mm聚乙烯棒制成3层管状支架，为实验组；制成单层无细胞管状支架，为对照组。两种支架作为血管移植物，分别植入15只犬两侧股动脉缺损处进行桥接吻合。术后进行彩超、组织学评价血管的形成过程。结果术后12周，14个3层血管支架保持通畅，通畅率为93．3％，有明显的血管生物结构形成。单层血管支架5个出现部分狭窄，只有3个完全通畅，通畅率为52．6％。结论SIS膜制成的3层血管支架能直接修复血管缺损，并能在体内环境中培形成生物化的小口径组织工程血管。     

新型聚β-羟基丁酯作为血管组织工程支架材料的实验研究

目的：探讨经改性处理后的可降解生物材料聚β-羟基丁酯(PHB)作为血管组织工程支架材料与血管平滑肌细胞的细胞相容性。方法：采用体外培养的免血管平滑肌细胞接种在经胶原包埋处理后的管型PHB支架材料上，用相差显微镜和扫描电镜观察细胞的粘附和生长情况，并行HE染色观察。结果：免血管平滑肌细胞在改性后的管型PHB支架材料上粘附生长良好，扫描电镜下可见细胞生长融合成片状，HE染色示细胞在PHB材料上生长良好。结论：改性后的聚β-羟基丁酯材料与兔血管平滑肌细胞有较好的生物相容性。     

中胚层疗法联合聚对二氧环己酮面部埋线改善眶周老化的效果

目的观察注射透明质酸填充剂联合聚对二氧环己酮面部可吸收线改善眶周老化的效果。方法收集2017年5月至2019年5月大连市中心医院整形美容科眶周老化女性患者104例, 年龄20～55(36±5)岁。分为轻度组48例、中度组56例。通过注射填充剂和聚对二氧环己酮可吸收线联合治疗改善眶周老化, 治疗后随访6～12个月。结果轻度组在术后即刻满意度明显高于中度组, 且6个月医师评分及患者满意度均高于其他月份。术后6个月, 轻度组满意度评分(7.48±1.29)分, 中度组满意度评分(6.32±1.03)分。随着时间延长两组治疗效果满意度均有不同程度下降。结论依据透明质酸钠凝胶流变学特性及泪沟形成的解剖学特点, 用多点注射填充技术联合小线治疗轻、中度眶周老化, 可获得较好的临床效果。     

应用脂肪干细胞构建组织工程化小口径血管平滑肌层的实验研究

目的探索利用脂肪干细胞在生物反应器内构建组织工程血管平滑肌层的可行性。方法用抽吸的脂肪获取脂肪干细胞,在生长因子TGF-β1和BMP4作用下诱导成平滑肌细胞,然后将诱导的平滑肌细胞接种于PGA上,将细胞-材料复合物置于生物反应器内进行培养,在模拟胚胎发育血流动力学的刺激下(搏动频率:75次/分;扩展量<5%),构建小口径的血管平滑肌组织。培养8周后,取材行组织学和生物力学检测并与正常血管对比。结果脂肪干细胞在TGF-β1和BMP4的诱导下,细胞呈现平滑肌细胞特有的"波峰-波谷"样生长特点,并表达平滑肌细胞的特异性标记物α-SMA、SM22α、calponin和SM-MHC;反应器内培养8周后,构建的管状组织胶原分泌旺盛,具有一定的力学强度和弹性。结论利用脂肪干细胞可在体外生物反应器内构建组织工程化小口径血管平滑肌层,为临床上小血管病变的修复提供了一种新的可能的途径。     

利用同种异体脱细胞血管基质预构小口径血管的研究

目的通过观察狗腹腔内预植同种异体脱细胞血管基质（ACVM）所形成的结缔组织管作为替代血管的力学性能，以及移植于自体股动脉后的组织结构变化，探讨利用同种异体ACVM预构小口径血管的可行性。方法在狗腹腔内埋植长8～12cm用硅胶棒支撑的已制备好的同种异体ACVM，3周后将硅胶棒周围形成的管状物作为血管替代物桥接移植于自体股动脉后，进行力学、组织学检查及与颈动脉、股静脉自体移植的对比分析。于移植术后6个月观测作为替代血管的通畅情况，组织学观察其组织结构变化。结果血管替代物的力学性能弱于正常动脉而强于正常静脉。组织学观察：形成的管状物内腔有少量的间皮细胞黏附；弹性纤维、胶原纤维结构较完整；纤维网中充满成纤维细胞。6个月后内皮细胞在替代物内壁连续覆盖，平滑肌细胞与对照组相近。移植6个月后异体ACVM组血管全部通畅。结论用同种异体ACVM预构的血管替代物，力学性能符合血管移植、血运重建的需求。所形成的结缔组织管作为血管替代物移植6个月后，管壁厚度及组织结构已接近正常血管。     

利用脱细胞基质预构小口径组织工程血管的实验研究

目的 在犬腹腔内预置脱细胞基质后形成结缔组织管,观察将其作为血管替代物的力学特征及移植于自体股动脉后的组织结构变化.方法 在犬腹腔内埋置长8～12 cm用硅胶棒支撑的制备好的脱细胞基质,3周后将硅胶棒周围形成的管状物作为血管替代物移植于自体股动脉,同时对此管状物作力学、组织学检测及与颈动脉、股静脉的对比分析.在移植术后6个月观察血管通畅情况.光镜、透射及扫描电镜观察替代物的组织结构.结果 ①血管替代物的力学性能弱于正常动脉而强于正常静脉.②组织学观察:形成的管状物内腔有少量的间皮细胞黏附;弹性纤维、胶原纤维结构较完整;纤维网中充满成纤维母细胞.6个月后内皮细胞在替代物内壁覆盖连续,平滑肌细胞与对照组相近.③移植6个月后脱细胞基质组血管全部通畅.结论 ①用脱细胞基质预构的血管替代物力学性能符合血管移植、血运重建的需要.②所形成的血管替代物移植6个月后组织相容性良好,替代物管壁厚度及组织结构已接近正常血管.     

组织工程支架的力学分析及相关模式探讨

目的探讨Tensegrity模式描述天然骨衍生材料的结构并进行力学性能分析。方法制备天然骨衍生材料,测定力学性能,用MurakamiNishimura的Tensegrity模式作分析。结果骨中压力杆羟基磷灰石受到5.91×108dyn/cm2压应力时若尺寸保持不变,由预应力为4.4×108dyn/cm2十二根水平胶原纤维和预应力为2.7×108dyn/cm2六根垂直胶原纤维组成的一阶Tensegrity结构为基本单位形成的n阶右螺旋圆筒结构的极限强度和极限应变等同于骨单元的力学性质。结论n阶多重右圆筒Tensegrity结构适合作为组织工程支架材料设计的模型。     

应用生物反应器体外构建组织工程化血管平滑肌层

目的探讨生物反应器内构建具有一定强度的组织工程化血管的可行性。方法将体外培养扩增的猪血管平滑肌细胞接种于聚羟基乙酸(PGA)上,形成细胞材料复合物,将其置于生物反应器内培养。实验组(n=5)为动态力学刺激培养(搏动频率:75次/分;扩张量:<5%);对照组(n=5)为静态培养。3周后行大体观察及组织学检测。结果血管样组织直径5mm,长10mm,厚1mm。实验组具有良好的弹性,管腔圆;平滑肌纤维与弹力纤维较多,排列有层次。对照组弹性欠佳,管腔塌陷;平滑肌纤维与弹力纤维较少且排列紊乱。结论应用生物反应器可构建具有良好弹性的血管平滑肌层组织。     

Laparoscopy in ureteral engineering: a feasibility study

Objective

We recently bioengineered a ureter substitute from a seeded scaffold implanted by open surgery in the omentum. In view of the development of laparoscopy in the treatment of benign conditions of the ureter, obtaining a ureter substitute by minimally invasive techniques would be a desirable objective. However, conflicting results about the biological impact of carbon dioxide insufflation on the microcirculation of intra-abdominal organs prompted us to investigate first whether the results obtained by open surgery, in terms of vascular supply and maturation, could be reproduced laparoscopically.

Materials and methods

Bladder full-thickness tissue was harvested laparoscopically from three pigs for urothelial and smooth muscle cell primary cultures subsequently used to seed a small intestinal submucosa (SIS) matrix. After 2 wk, the in vitro seeded constructs were shaped around silicone drains and transferred laparoscopically into the abdomen for omental maturation. Three weeks later, the constructs were harvested for histological, immunohistochemical, and electron microscopic analysis.

Results

The laparoscopic procedures were performed successfully in all animals. After omental maturation, the constructs were vascularized and comprised of a well-differentiated multilayered urothelium with umbrella cells, over connective tissue and smooth muscle cells, with no evidence of fibrosis or inflammation. Electron microscopic analysis showed characteristics of a terminally differentiated urothelium.

Conclusion

As shown by conventional microscopy, immunochemistry, and electron microscopy, carbon dioxide insufflation does not impact cell growth and differentiation. These findings validate the laparoscopic approach for omental maturation of ureter substitutes.     

Biomechanical researches on tissue engineering bone constructed by deproteinated bone

Objective： To study biomechanical changes of newly formed bones 24 weeks after repairing large defects of long bones of goats using heterogeneous deproteinated bone （DPB） prepared by modified methods as an engineering scaffold. Methods： According to a fully randomized design, 18 goats were evenly divided into three groups： normal bone control group （Group A）, autologous bone group （Group B） and experimental group （Group C）. Each goat in Groups B and C were subjected to the periosteum and bone defect at middle-lower part of the right tibia （20% of the whole tibia in length）, followed by autologous bone or DPB plus autolognus MSCs ＋ rhBMP2 implantation, respectively and semi- ring slot fixation; while goats in Group A did not perform osteotomy. At 24 weeks after surgery, biomechanical tests were carried out on the tibias. Results： At 24 weeks after surgery, the results of anticompression test on tibias in three groups were recorded by a functional recorder presented as linear pressure-deformation curve. The shapes of the curves and their change tendency were similar among three groups. The ultimate pressure values were 10.74 MPa±1.23 MPa, 10. 11 MPa±1.35 MPa and 10.22 MPa±1.32 MPa and fracture compression rates were 26.82%±0.87%, 27.17%±0.75% and 28.22%±1.12% in Groups A, B and C, respectively. Comparisons of anti-compression ultimate pressures and fracture compression rates among three groups demonstrated no significant difference （PAB=0.415, PBC=0.494）. Three-point antibend test on tibias was recorded as load-deformation curves, and the shapes of the curves and their change tendency were similar among three groups. The ultimate pressure values of the anti-bend test were 481.52 N±12.45 N, 478.34 N±14.68 N and 475.62 N±13.41 N and the fracture bend rates were 2.62 mm±0.12 mm, 2.61 mm±0.15 mm and 2.81 mm±0.13 mm in Groups A, B and C, respectively. There was no significant difference between groups （PAB=0.7, PBc=0.448）. The ultimate anti-torsion torque values were 6.55 N.mi-0.25 N.m, 6.34 N＇m~0.18 N＇m and 6.42 N＇m~0.21 N＇m and fracture torsion rates were 29.51°±1.64°, 28.88±1.46° and 28.81°±1.33° in Groups A, B and C, respectively. There was no significant difference between groups （PAB=0.123, PBc=0.346）. Conclusions： The biomechanical characteristics of newly formed bones from heterogeneous DPB for repairing large segmental long bone defect are comparable to those of normal bones and autologous bones. DPB has the potential for clinical usage as bone graft material.     

组织工程学方法体外构建血管的研究

目的 探讨用组织工程学方法构建血管的可行性。方法 实验组：将体外培养、扩增的人脐带动脉平滑肌细胞与用可降解的生物材料聚乙醇酸(PGA)制成的管状结构结合，体外培养1周后植入裸鼠背部皮下。分别于2、4、5、6、11周取材进行大体及组织学检测；对照组：将没有细胞的单纯管状PGA植入裸鼠背部皮下，于相同时间点进行检测。结果 实验组在观测点均形成一内壁光滑的管状结构，组织学检测表明，该结构随观测时间的延长组织学成分也有明显的变化，从开始以PGA为主向平滑肌细胞及其分泌的胶原为主要成分过渡。而对照组则随着PGA的降解管状结构逐渐丧失。结论 组织工程学方法可形成具有一定张力及组织成分的管状结构。该方法用于构建血管是可行的。     

Tissue engineering human small-caliber autologous vessels using a xenogenous decellularized connective tissue matrix approach: preclinical comparative biomechanical studies

Suggesting that bioartificial vascular scaffolds cannot but tissue-engineered vessels can withstand biomechanical stress, we developed in vitro methods for preclinical biological material testings. The aim of the study was to evaluate the influence of revitalization of xenogenous scaffolds on biomechanical stability of tissue-engineered vessels. For measurement of radial distensibility, a salt-solution inflation method was used. The longitudinal tensile strength test (DIN 50145) was applied on bone-shaped specimen: tensile/tear strength (SigmaB/R), elongation at maximum yield stress/rupture (DeltaB/R), and modulus of elasticity were determined of native (NAs; n = 6), decellularized (DAs; n = 6), and decellularized carotid arteries reseeded with human vascular smooth muscle cells and human vascular endothelial cells (RAs; n = 7). Radial distensibility of DAs was significantly lower (113%) than for NAs (135%) (P < 0.001) or RAs (127%) (P = 0.018). At levels of 120 mm Hg and more, decellularized matrices burst (120, 160 [n = 2] and 200 mm Hg). Although RAs withstood levels up to 300 mm Hg, ANOVA revealed a significant difference from NA (P = 0.018). Compared with native vessels (NAs), SigmaB/R values were lower in DAs (44%; 57%) (P = 0.014 and P = 0.002, respectively) and were significantly higher in RAs (71%; 83%) (both P < 0.001). Similarly, DeltaB/R values were much higher in DAs compared with NAs (94%; 88%) (P < 0.001) and RAs (87%; 103%) (P < 0.001), but equivalent in NAs and RAs. Modulus of elasticity (2.6/1.1/3.7 to 16.6 N/mm(2)) of NAs, DAs, RAs was comparable (P = 0.088). Using newly developed in vitro methods for small-caliber vascular graft testing, this study proved that revitalization of decellularized connective tissue scaffolds led to vascular graft stability able to withstand biomechanical stress mimicking the human circulation. This tissue engineering approach provides a sufficiently stable autologized graft.