应用猪主动脉脱细胞基质制备新型组织工程血管支架:生物相容性及力学性能评价(英文)

背景:组织工程血管构建的关键依赖于理想的支架。猪血管作为组织工程血管构建材料已有广泛应用,但其较高的免疫原性及较差的力学强度限制了该材料作为组织工程支架的应用。目的:应用猪主动脉脱细胞基质制备一种新的具有良好机械性能及生物相容性的组织工程血管支架。方法:对猪主动脉进行脱细胞处理和热交联改性制备脱细胞血管基质支架,采用苏木精-伊红染色及生物力学分析评估其脱细胞效果及血管基质的力学性能。将人脐静脉血管内皮细胞接种于脱细胞血管基质支架中进行体外培养,评估其生物相容性。结果与结论:用1%的TritonX-100溶液处理猪主动脉84h可完全脱除血管细胞,同时不破坏血管基质结构;经真空下120℃热交联处理12h,脱细胞基质的拉伸断裂强度得到明显提高,达到1.70MPa。在该改性血管基质支架上接种人脐带静脉内皮细胞体外培养7d,扫描电镜显示内皮细胞呈现典型的血管内皮层状结构。表明猪主动脉经过脱细胞处理能够维持血管基质完整,冷冻干燥和真空热交联处理可有效提高其拉伸强度,且对血管内皮细胞具有良好的相容性。     

组织工程血管基质的制备与改性

目的：完全脱除猪胸主动脉细胞，对脱细胞血管基质进行改性，增强基质的力学强度，制备组织工程血管支架材料。方法：取家猪的新鲜去除外膜胸主动脉20根，随机分成4组，分别采用胰蛋白酶、TritonX-100及十二烷基硫酸钠（SDS）作为脱细胞试剂对猪胸主动脉进行脱细胞处理，并对脱细胞后的血管基质进行改性，采用HE染色、弹力纤维染色、力学性能测试，以评价脱细胞效果以及材料的力学性能。结果：采用1％TritonX-100单独作用84h既能完全脱除细胞，同时又可完整保留血管基质的三维结构，对胶原纤维、弹力纤维的损伤小，是一种较理想的脱细胞方法。对脱细胞后的基质进行冷冻干燥及真空热交联处理，能有效提高材料的机械强度，冷冻干燥24h后真空120℃下热交联处理12h所获得的材料的机械强度最好，断裂强度可达到1．70MPa。结论：以脱细胞血管基质经冷冻干燥和真空热交联处理后，可以作为血管组织工程的支架材料。     

应用脱细胞血管基质构建组织工程血管的初步研究

目的： 采用脱细胞处理的猪胸主动脉血管基质作为支架材料，接种人脐带血管内皮细胞，构建组织工程血管。方法: 以新鲜猪胸主动脉为原材料， 1% Triton X-100为脱细胞试剂，制备脱细胞血管基质；采用冷冻干燥和热交联法对脱细胞血管基质进行改性，并进行组织学观察及力学性能测定。用人脐带内皮细胞经培养和扩增后，再与所制备的脱细胞血管基质进行复合培养，光学显微镜及扫描电镜观察内皮细胞生长状况。结果: 1% Triton X-100处理84h的猪胸主动脉，既能完全脱除血管中细胞，同时又完整保留血管基质的三维结构；对脱细胞血管基质冷冻干燥24 h，120 ℃下热交联12 h，能有效提高材料的机械强度，断裂强度可达到1.70 MPa。扫描电镜下可见，脱细胞血管基质与内皮细胞复合培养7 d，已形成典型血管内膜样结构。结论: 经改性后的脱细胞血管基质与内皮细胞具有良好的相容性，用其作为支架材料与内皮细胞复合培养，有望应用于构建组织工程化血管。     

EDC交联改性的脱细胞异种(猪)肌腱生物学特性研究

目的探讨EDC交联的脱细胞异种(猪)肌腱生物学特性,为其作为肌腱移植材料的应用研究提供依据。方法猪肌腱脱细胞后进行EDC改性处理,进行组织形态学、生物力学、体外降解性能、免疫性能和细胞相容性检测,并与新鲜肌腱、深低温冷冻肌腱和单纯脱细胞肌腱进行比较。结果 EDC改性的脱细胞肌腱免疫原性最低,且力学性能、体外降解特性显著优于单纯脱细胞肌腱,而与新鲜肌腱、冻存肌腱无显著性差异,各组之间的细胞相容性无显著性差异。结论 EDC改性的脱细胞肌腱具有较低的免疫原性和较好的生物学力学特性,是一种具有潜在应用价值的肌腱支架材料。     

动物血管脱细胞方法及细胞外基质材料评价研究

采用独立设计的反复冻融方法,去除兔颈总动脉中的血管细胞,对所获得的细胞外基质进行组织、生化、力学等分析。在分离兔颈总动脉后,首先用低渗缓冲液处理,然后经低温反复冻融,最后用非离子型去污剂处理。所得的支架行组织染色和扫描电镜分析,显示基质的微观结构;荧光染色和基因组DNAPCR分析,检测细胞DNA残留;分别行羟脯氨酸定量分析和轴向拉伸强度分析,检测胶原蛋白和血管生物力学性能的变化;支架没有明显的细胞毒性,溶血率低于医用材料的标准;支架随时间缓慢降解。种植兔的骨髓干细胞,研究细胞在支架上的粘附生长能力。应用本方法能彻底去除血管细胞,没有细胞碎片和DNA的残留,并保留了较完整的细胞外基质和力学性能,具有开放的大孔径结构,细胞生物相容性好,是一种理想的组织工程血管支架材料。     

冻干韧带脱细胞支架材料的生物相容性及优势

背景：韧带脱细胞基质是通过各种脱细胞方法将韧带组织内的细胞成分清除,降低免疫原性,同时对纤维支架结构破坏轻微,保留了细胞外基质的机械性能。 目的：评价冻干兔髌韧带脱细胞支架的生物相容性及优势。 方法：取兔髌韧带,利用1%脱氧胆酸钠行脱细胞处理,制备冻干和未冻干脱细胞韧带支架。 结果与结论：冻干韧带脱细胞支架保持了冻干前的胶原结构与力学特征,无细胞残留,其浸提液对细胞生长无抑制作用,无全身急性毒性反应,无热原存在,且原发刺激指数为0分,皮内刺激实验阴性。体内埋植实验显示冻干韧带脱细胞支架表现为免疫原性小,炎症反应轻的特点。说明冻干韧带脱细胞支架具有较好的生物相容性,且制作简单,便于消毒、包装、保存。     

全生物化组织工程血管构建的初步研究

为探索在体外初步构建全生物化组织工程血管,采用以酶消化为主的方法制备猪颈总动脉脱细胞支架,再种植犬胸主动脉的平滑肌细胞,培养四周。经组织学染色和电镜观察显示:在猪颈总动脉脱细胞支架上,犬血管平滑肌细胞大量生长,组织学和电镜结构与正常血管壁结构类似。结果表明,我们初步成功地构建了全生物化组织工程血管,为临床血管替代物的基础研究提供了有意义的实验资料。     

比较两种方法制备脱细胞小血管支架

目的:比较0.5%Triton X-100 0.05%NH4OH和酶法制备脱细胞小血管支架的效果.方法:分别用含0.5%Triton X-100 0.05%NH4OH处理3 d或1.0H Triton X-100 0.125%胰蛋白酶 Dnase Rnase孵育48 h,脱去犬股静脉中的细胞成分,50Co辐照消毒,血清浸泡24 h,将平滑肌细胞和内皮细胞接种到脱细胞支架中;进行H-E染色、胶原纤维和弹力纤维染色,扫描电镜观察及力学检测.结果:0.5%Triton X-100 0.05%NH4OH法完全地脱去了血管细胞;细胞外基质较完整地保留下来,其形态结构与脱细胞前无明显改变;见种植细胞在支架内生长良好,连成片,支架具有良好的生物相容性;力学结果显示其弹性回复率和最大断裂强度好于酶组.结论:两种方法比较,0.5%Triton X-100 0.05%NH4OH法简便易行,成本低,脱细胞效果好,组织相容性佳,对力学性状影响小,是比较理想的制备脱细胞小血管支架的方法.     

改进构建后的全生物化组织工程血管的组织结构与生物力学特性

目的改进体外构建的生物化组织工程血管(TEBV),研究其生物力学特性,获得能承载生理血流力学作用的血管替代物。方法用酶消化法制备猪颈总动脉脱细胞支架,加压灌注结合散点注射种植犬胸主动脉平滑肌细胞(VSMC),用自制的血管自动旋转流体培养系统培养3周,随后再用加压灌注法种植犬胸主动脉内皮细胞(EC),继续培养至4周,取材做HE染色。光镜和电镜观察VSMC及EC在支架内的生长情况。用力学测试仪器检测TEBV的应力-应变关系、拉伸弹性回复率以及最大断裂强度和长度。结果在血管自动旋转流体培养系统,VSMC种植3周在中膜层已经大量均匀分布,EC种植7天后已形成连续完整的EC单层;而应用单独的加压灌注法VSMC种植3周时仍不能均匀分布,EC种植1周后仍分布不均匀,未能形成连续的EC单层。TEBV培养至第4周,中膜层有大量VSMC生长,内膜层则形成连续的EC单层;透射电镜下可见VSMC、EC亚微结构与生理状态的细胞结构相似,可见缝隙连接等细胞连接方式,VSMC可产生新的胶原蛋白。扫描电镜可见EC在支架上生长良好,细胞轮廓清晰,形成连续的单层。力学特性检测结果显示:TEBV的粘弹性、拉伸弹性回复率以及最大断裂强度均接近生理血管。结论血管自动旋转流体培养系统在脱细胞血管支架上联合种植VSMC和EC,有利于种子细胞在支架腔面和管壁内均匀生长,改进了TEBV的组织结构。改进构建的TEBV生物力学特性接近生理血管,将有利于TEBV更好地承载血流动力学的作用。     

犬平滑肌样细胞和胶原包埋PGA支架组织的相容性

目的 研究骨髓间充质干细胞所诱导的犬平滑肌样细胞和胶原包埋聚羟基乙酸（PGA）支架的组织相容性。方法 胶原包埋PGA构建复合支架,犬骨髓间充质干细胞诱导血管平滑肌样细胞,评价组织相容性。结果 HE染色见胶原包埋PGA组有平滑肌样细胞生长;甲苯胺蓝染色见平滑肌样细胞被染成浅蓝色,胶原包埋PGA组较单纯PGA组明显增多;电镜观察,胶原包埋PGA组可见到细胞在支架上贴附和生长良好。结论 细胞和胶原包埋PGA的支架组织相容性良好。     

A prototype tissue engineered blood vessel using amniotic membrane as scaffold

In this study, we used amniotic membrane (AM), a natural extracellular matrix, as a scaffold for the fabrication of tissue engineered blood vessels (TEBVs). The inner surface of the denuded glutaraldehyde cross-linked AM tube was endothelialized with porcine vascular endothelial cells (ECs) and subjected to a physiological (12 dynecm(-2)) shear stress (SS) for 2 and 4 days. The results showed that after applying SS, an intact EC monolayer was maintained in the lumen surface of the TEBV. The ECs were aligned with their long axis parallel to the blood flow. The immunofluorescent microscopy showed that the intercellular junctional proteins, PECAM-1 and VE-cadherin, were surrounding the EC periphery and were better developed and more abundant in SS-treated TEBVs than the static controls. The Western blot indicated that the expressions of PECAM-1 and VE-cadherin were increased by 72 ± 9% and 67 ± 7%, respectively, after shear stress treatment. The distribution pattern of integrin β1 was mainly at the interface of ECs and AM in static TEBVs but it was extended to the cell-cell junctions after SS treatment. The SS promoted the expression of integrin α(v)β(3) without altering its distribution in TEBV. The results suggest that glutaraldehyde cross-linked AM tube can potentially be used as a scaffold biomaterial for TEBV fabrication. Most importantly, the use of an AM tube shortened the TEBV fabrication.     

Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern

It was reported that acellular biological tissues can provide a natural microenvironment for host cell migration and may be used as a scaffold for tissue regeneration. To reduce antigenicity, biological tissues have to be fixed with a crosslinking agent before implantation. As a tissue-engineering scaffold, it is speculated that the crosslinking degree of an acellular tissue may affect its tissue regeneration pattern. In the study, a cell extraction process was employed to remove the cellular components from bovine pericardia. The acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completely degraded before the infiltrated cells began to secrete their own extracellular matrix. In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern.     

脉动式张应力环境下培养组织工程血管的初步实验

目的探讨脉动式张应力环境下,在生物反应器内进行组织工程血管（TEBV）培养的可行性。方法从人脐动脉获取血管平滑肌细胞（VSMC）,经体外培养扩增后接种到聚乙醇酸（PGA）支架并置于生物反应器内进行三维培养。实验组利用气动式左心循环辅助泵对VSMC施加模拟人体内血管环境（压力120mmHg,1mmHg=0．133kPa,频率60次／rain）的脉动式张应力作用。对照组无张力环境下进行三维培养。2周后收获TEBV分别行扫描电镜（SEM）检测、组织切片HE染色和Masson染色。结果实验组TEBV外观色泽鲜亮,具有一定厚度和弹性,能自我维持管腔形状。SEM显示实验组TEBV管壁表面光滑平整,横切面有丰富的细胞外基质（ECM）,ECM分布均匀,把PGA完全包裹。HE染色管壁均匀致密,ECM及细胞的排列具有一定方向性,其中含有未降解的PGA碎片。Masson染色管壁胶原纤维丰富。对照组TEBV外观色泽暗淡,管壁薄,弹性差,从支架取下后管腔塌陷。SEM显示管壁表面欠光滑,横切面上ECM含量少,大部分PGA没有被包裹。组织切片染色管壁结构疏松,ECM和胶原纤维含量较少,层次感差。结论在生物反应器内的脉动式张应力环境下,可培养出具有良好形态结构的组织工程血管样组织。     

The long head of the biceps tendon is a suitable cell source for tendon tissue regeneration

Introduction

Tendon tissue engineering (TTE) tries to produce tendinous tissue of high quality to replace dysfunctional tissue. One possible application of TTE might be the replacement of ruptured tissue of the rotator cuff. Autologous tenocytes seem to be most suitable as no differentiation in vitro is necessary. Today it is still uncertain if there is a difference between tendon-derived cells (TDC) of different native tissues. Moreover, the search for suitable scaffolds is another important issue in TTE.

Material and methods

This study compared TDC of the long head of the biceps tendon (LHB), the anterior cruciate ligament (ACL) and the tendon of the musculus semitendinosus (TMS). The TDC were isolated using the cell migration method. Cell morphology was assessed using light microscopy and gene expression was performed using polymerase chain reaction (PCR). Afterwards, cell seeding efficiency and proliferation were tested on a collagen I scaffold using the WST-1 assay. Results were confirmed using H + E staining.

Results

The TDC of the LHB showed higher expression levels of collagen type I and decorin (p < 0.01) compared to TDC of other origin. Results showed efficient cell seeding and proliferation within the scaffold. Proliferation within the scaffold was not as high as when cells were cultivated without a scaffold.

Conclusions

The TDC of the LHB seems to be the most suitable cell source. Further research is necessary to find out if the results can be transferred to an in vivo model. The new collagen I scaffold seems to offer an opportunity to combine good biocompatibility and mechanical strength.     

生物组织工程血管的体外构建及其生物力学特性的研究

目的:探索生物组织工程血管(TEBV)的体外构建,观察血管壁形成过程中血管平滑肌细胞的变化,检测TEBV的生物力学特性,为构建理想的临床血管替代物提供资料。方法采用酶消化法制备猪颈总动脉脱细胞支架,再种植犬胸主动脉的平滑肌细胞,体外培养4周,分别于2周和4周取材作形态结构观察和平滑肌肌动蛋白-α(SMA)、金属蛋白酶-2(MMP-2)及血小板衍生生长因子-AA(PDGF-AA)的免疫组化染色并作图象分析。将幼年犬和成年犬的胸主动脉作对照。用INSTRON1122型万能电子强力仪检测TEBV的断裂强度、应力应变关系和松弛试验。结果体外培养2周和4周,血管平滑肌细胞大量增殖并迁移至支架全层,细胞间出现桥粒、缝隙连接。TEBV培养2周和4周,SMA的表达高于幼年犬,但明显低于成年犬;PDGF-AA的表达低于幼年犬,但明显高于成年犬;MMP-2高于成年犬,培养4周时MMP-2的表达接近幼年犬。TEBV的应力应变曲线呈典型的粘弹性特征,松弛应力和断裂强度均与生理血管相似。结论生物组织工程血管体外构建中平滑肌细胞分泌PDGF-AA、SMA的表达逐渐减少,平滑肌细胞由收缩表型逐渐转变为合成表型,大量增殖和分泌胶原,有细胞连接形成;同时MMP-2分泌增加,细胞发生迁移,重建TEBV的管壁结构,且血管壁的生物力学特性逐渐接近于生理血管。     

Engineering functionally graded tissue engineering scaffolds

Tissue Engineering (TE) aims to create biological substitutes to repair or replace failing organs or tissues due to trauma or ageing. One of the more promising approaches in TE is to grow cells on biodegradable scaffolds, which act as temporary supports for the cells to attach, proliferate and differentiate; after which the scaffold will degrade, leaving behind a healthy regenerated tissue. Tissues in nature, including human tissues, exhibit gradients across a spatial volume, in which each identifiable layer has specific functions to perform so that the whole tissue/organ can behave normally. Such a gradient is termed a functional gradient. A good TE scaffold should mimic such a gradient, which fulfils the biological and mechanical requirements of the target tissue. Thus, the design and fabrication process of such scaffolds become more complex and the introduction of computer-aided tools will lend themselves well to ease these challenges. This paper reviews the needs and characterization of these functional gradients and the computer-aided systems used to ease the complexity of the scaffold design stage. These include the fabrication techniques capable of building functionally graded scaffolds (FGS) using both conventional and rapid prototyping (RP) techniques. They are able to fabricate both continuous and discrete types of FGS. The challenge in fabricating continuous FGS using RP techniques lies in the development of suitable computer aided systems to facilitate continuous FGS design. What have been missing are the appropriate models that relate the scaffold gradient, e.g. pore size, porosity or material gradient, to the biological and mechanical requirements for the regeneration of the target tissue. The establishment of these relationships will provide the foundation to develop better computer-aided systems to help design a suitable customized FGS.     

Maintenance of phenotype and function of cryopreserved bone-derived cells

The emerging fields of tissue engineering and regenerative medicine require large numbers of cells for therapy. Although the properties of cells obtained from a variety of fresh tissues have been delineated, the knowledge regarding cryopreserved grafts-derived cells remains elusive. Previous studies have shown that living cells could be isolated from cryopreserved bone grafts. However, whether cryopreserved bone-derived cells can be applied in regenerative medicine is largely unknown. The present study was to evaluate the potential application of cryopreserved grafts-derived cells for tissue regeneration. We showed that cells derived from cryopreserved bone grafts could maintain good proliferation activity and osteogenic phenotype. The biological phenotype of these cells could be well preserved. The transplantation of cryopreserved bone-derived cells on scaffold could promote new bone formation in nude mice and enhance the osteointegration for dental implants in canine, which confirmed their osteogenic capacity, and showed that cells derived from cryopreserved bone were comparable to that of fresh bone in terms of the ability to promote osteogenesis in vivo. This work demonstrates that cryopreserved bone grafts may represent a novel, accessible source of cells for tissue regeneration therapy, and the results of our study may also stimulate the development of other cryopreservation techniques in basic and clinical studies.     

Designed hybrid scaffolds consisting of polycaprolactone microstrands and electrospun collagen-nanofibers for bone tissue regeneration

Biomedical scaffolds used in bone tissue engineering should have various properties including appropriate bioactivity, mechanical strength, and morphologically optimized pore structures. Collagen has been well known as a good biomaterial for various types of tissue regeneration, but its usage has been limited due to its low mechanical property and rapid degradation. In this work, a new hybrid scaffold consisting of polycaprolactone (PCL) and collagen is proposed for bone tissue regeneration. The PCL enhances the mechanical properties of the hybrid scaffold and controls the pore structure. Layered collagen nanofibers were used to enhance the initial cell attachment and proliferation. The results showed that the hybrid scaffold yielded better mechanical properties of pure PCL scaffold as well as enhanced biological activity than the pure PCL scaffold did. The effect of pore size on bone regeneration was investigated using two hybrid scaffolds with pore sizes of 200 ± 20 and 300 ± 27 μm. After post-seeding for 7 days, the cell proliferation with pore size, 200 ± 20 μm, was greater than that with pore size, 300 ± 27 μm, due to the high surface area of the scaffold.     

修复运动性肌腱损伤组织工程化肌腱及种子细胞和支架材料

背景：获得大规模、具有再生活力的种子细胞以及具有与正常人体肌腱组织相接近的力学性能的理想支架材料是当前组织工程化肌腱研究面临的最为关键的限制性因素。 目的：总结和分析组织工程肌腱研究中的种子细胞和支架材料的研究进展。 方法：查阅近年来肌腱组织工程研究的相关文献,综合国内外最新研究成果,就肌腱组织工程中合适的种子细胞来源、研究更为理想的支架材料及组织相容性等方面的进展进行概述。 结果与结论：肌腱组织工程中常用的种子细胞有间充质干细胞、肌腱干细胞及胚胎干细胞等,可以向骨、软骨和脂肪分化,修复肌腱损伤的理想细胞。肌腱组织工程支架材料有天然材料及人工合成材料等,肌腱组织工程支架材料应有良好的生物相容性和适度的机械性能,复合材料将是肌腱组织工程支架材料研究的重点。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程