Design and fabrication of porous biodegradable scaffolds: a strategy for tissue engineering

Current strategies of tissue engineering are focused on the reconstruction and regeneration of damaged or deformed tissues by grafting of cells with scaffolds and biomolecules. Recently, much interest is given to scaffolds which are based on mimic the extracellular matrix that have induced the formation of new tissues. To return functionality of the organ, the presence of a scaffold is essential as a matrix for cell colonization, migration, growth, differentiation and extracellular matrix deposition, until the tissues are totally restored or regenerated. A wide variety of approaches has been developed either in scaffold materials and production procedures or cell sources and cultivation techniques to regenerate the tissues/organs in tissue engineering applications. This study has been conducted to present an overview of the different scaffold fabrication techniques such as solvent casting and particulate leaching, electrospinning, emulsion freeze-drying, thermally induced phase separation, melt molding and rapid prototyping with their properties, limitations, theoretical principles and their prospective in tailoring appropriate micro-nanostructures for tissue regeneration applications. This review also includes discussion on recent works done in the field of tissue engineering.     

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。     

细胞外基质来源支架在软骨组织工程中的应用

BACKGROUND: At present, a variety of extracellular matrix-derived scaffolds have been successfully applied for cartilage tissue engineering in experiment and clinical practice. OBJECTIVE: To summarize the application and research status of extracellular matrix-derived scaffolds in cartilage tissue engineering. METHODS: A computer-based online search in PubMed, CNKI, CqVip and WanFang databases was performed using the keywords of “tissue engineering, cartilage, extracellular matrix, scaffolds” in English and Chinese, respectively. A total of 1 140 literatures were retrieved, and finally 65 eligible literatures were included. RESULTS AND CONCLUSION: In terms of the components, extracellular matrix-derived scaffolds are divided into monomeric natural polymers, mixed natural polymers, natural polymers compositing with synthetic polymers as well as acellular extracellular matrix-derived materials. Extracellular matrix-derived scaffolds hold good biocompatibility and degradability, and can promote proliferation and differentiation of choncrodytes; therefore, they as good bionic scaffolds have been applied for cartilage tissue engineering in clinical practice, However, poor mechanical properties and difficulty to molding should never be ignored. Further research should focus on improving the preparation technology by combining synthetic materials with extracellular matrix-derived scaffolds for cartilage tissue engineering.      

Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels

Fibrin is a biopolymer that has been used in a variety of biomaterial, cell delivery and tissue engineering applications. The enzyme thrombin catalyzes the formation of fibrin microfibrils, which form a three-dimensional mesh in which cells can be directly embedded at the time of gel formation. In this study, fibrin hydrogels containing vascular smooth muscle cells were created using varying concentrations of thrombin. Over 7 days in culture, all gels decreased in volume as the fibrin matrix compacted, and the degree of gel compaction increased as thrombin concentration decreased. The material modulus and ultimate tensile stress of the gels also increased with decreasing thrombin concentration. Addition of thrombin to similar constructs made using collagen Type I did not show an effect on gel compaction or mechanical properties, suggesting that these effects were a result of thrombin's action on fibrin polymerization, and not cellular functions. Cell proliferation in fibrin hydrogels was not significantly affected by thrombin addition. Matrix examination using scanning electron microscopy showed increasing fibrin fiber diameters as thrombin concentration decreased. Confocal microscopic imaging of the actin cytoskeleton showed that cell morphology on two-dimensional substrates of fibrin showed marked changes, with higher thrombin concentrations producing cells with longer cellular projections. However, these morphological changes were not as apparent in cells embedded in three-dimensional (3-D) matrices, in which cells exhibited a similar morphology independent of thrombin concentration. These results relate features of the matrix and cellular components of 3-D fibrin constructs to mechanical properties, and contribute to the understanding of structure-function relationships in cell-seeded, 3-D protein hydrogels.     

小转速髓芯减压结合自体红骨髓组织工程复合物治疗早中期股骨头缺血性坏死

目的探讨小转速髓芯减压结合自体红骨髓组织工程复合物治疗早中期股骨头缺血性坏死的临床疗效。方法选取2010年6月至2015年6月在我院住院的股骨头缺血坏死患者47例(52髋),采用随机数字表法分为2组:A组采用小转速髓芯减压+自体红骨髓组织工程复合物的方法治疗25例(28髋),B组采用髓芯减压+人工骨治疗22例(24髋),所有患者术前行髋关节X线片、CT及MRI检查,术后6、12、18个月行髋关节X线片、CT、MRI检查观察股骨头修复及坏死进展情况。结果所有病例平均随访34个月(18~60个月)。经过治疗,两组患者的临床坏死体积对比,影像学稳定率,股骨头生存率,有统计学差异(P0.05),两组患者的性别、年龄、病因基础资料差异无统计学意义(P0.05)。结论小转速髓芯减压+自体红骨髓组织工程复合物治疗早中期股骨头缺血性坏死操作简单,临床疗效满意,早期患者术后优良率更高。     

Incorporation of exudates of human platelet-rich fibrin gel in biodegradable fibrin scaffolds for tissue engineering of cartilage

The goal of this study was to assess the incorporation of exudates of human platelet-rich fibrin (hPRF) that is abundant in platelet cytokines and growth factors into biodegradable fibrin (FB) scaffolds as a regeneration matrix for promoting chondrocyte proliferation and re-differentiation. hPRF was obtained from human blood by centrifugation without an anticoagulant, and the exudate of hPRF was collected and mixed with bovine fibrinogen, and then thrombin was added to form the FB scaffold. Proliferation and differentiation of human primary chondrocytes and a human chondrosarcoma cell line, the SW-1353, embedded in the three-dimensional (3D) scaffolds and on the two-dimensional (2D) surface of the FB scaffolds so produced were evaluated in comparison with an agarose (AG) scaffold serving as the control. Results demonstrated that the amounts of these cytokines and growth factors in hPRF exudates were higher than those in the blood-derived products except for TGF-β1. Chondrocytes and SW1353 cells on the 2D and 3D FB scaffolds with the addition of the exudates of PRF exhibited more-available proliferation and differentiation than cells on 2D and 3D FB and AG scaffolds. It was concluded that FB scaffolds can provide an appropriate environment for chondrocyte proliferation and re-differentiation, and it could be improved by adding exudates of hPRF. These 3D scaffolds have great promise for cartilage tissue engineering.     

基于石墨烯构筑的三维组织工程支架材料

背景：基于石墨烯纳米片的化学成分和物理结构,可形成与细胞外生物基质十分相似的微观环境。目的：综述近年来基于石墨烯构筑三维支架的制备、细胞相容性及与细胞的相互作用。方法：应用计算机检索中国知网CNKI数据库、ACS-ACS Publications、Elsevier Science, Nature及PNAS美国国家科学院院刊等,有关生物医用石墨烯材料的种类和制备方法及其在组织工程中的应用研究。结果与结论：将石墨烯及其衍生物作为模块,利用石墨烯二维平面结构特点或氧化石墨烯含有丰富的官能团特性,不仅能够将石墨烯纳米片以不同的方式组装构筑三维宏观结构,而且易于复合其他功能性材料。与单个石墨烯片或传统的碳纳米材料相比,三维宏观石墨烯及复合材料将有可能形成更为新奇独特的结构及性能,具有更加实际的应用价值,包括拥有组织工程支架的功能。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     

Regulation of endothelial cell phenotype by biomimetic matrix coated on biomaterials for cardiovascular tissue engineering

One major weakness that all cardiovascular replacements have in common is the lack of endothelial cell (EC) growth and post-implant remodeling of the device. The emerging field of tissue engineering focuses on the in vitro generation of functional organ replacements using living endothelial cells and other vascular cells for which nondegradable or biodegradable scaffold base materials are used. In this paper, it is demonstrated that some of the cardiovascular device materials in clinical use lack the ability to promote endothelial cell growth in vitro. We previously established a biomimetic matrix composition which supports the growth of human umbilical vein endothelial cells (HUVECs) while maintaining normal physiology in vitro. Here the effectiveness of the same coating to preserve the normal antithrombotic phenotype of endothelial cells grown on biomaterials was evaluated. The up/down-regulation of two prothrombotic and two antithrombotic molecules by HUVECs grown on bare material surfaces were compared with that on composite-coated materials. The suitability of this approach for blood-contacting applications was investigated by in vitro blood compatibility studies as recommended in ISO10993 part 4, by putting an EC-seeded surface in contact with human whole blood. It is demonstrated that EC-seeded bare material surfaces are prothrombotic, whereas surfaces pre-coated with biomimetic molecules facilitated maintenance of the normal EC phenotype and reduced the risk of platelet adhesion and activation of blood coagulation. The results presented here suggest that matrix composed of biomimetic adhesive proteins and growth factors is suitable for cardiovascular tissue engineering to improve biological function, irrespective of the material chosen to meet the mechanical properties of the device.     

骨组织工程应用可降解聚合物的可能性探讨

成骨细胞细胞外基质材料的选择是骨组织工程研究中一项重要而紧迫的任务。本文阐述了理想骨组织工程基质材料的研制要求 ,介绍了几种可降解聚合物在骨组织工程中的应用现状 ,并重点介绍了 PL A、PGA及其共聚物的优缺点和相应的改进方法。作者认为 ,以合成可降解聚合物为主要组成成分的新型基质材料必将在未来骨组织工程研究中展示出可喜的应用前景     

纤维蛋白在软骨组织工程中的应用

纤维蛋白是纤维蛋白原经激活后形成的聚合物凝胶，作为软骨组织工程研究的载体材料，在相关的生物学和物理特性上具有其一定的优势。对纤维蛋白在软骨组织工程中应用的现状和发展方向进行综述。     

Bone tissue engineering in osteoporosis

Osteoporosis is a polygenetic, environmentally modifiable disease, which precipitates into fragility fractures of vertebrae, hip and radius and also confers a high risk of fractures in accidents and trauma. Aging and the genetic molecular background of osteoporosis cause delayed healing and impair regeneration. The worldwide burden of disease is huge and steadily increasing while the average life expectancy is also on the rise. The clinical need for bone regeneration applications, systemic or in situ guided bone regeneration and bone tissue engineering, will increase and become a challenge for health care systems. Apart from in situ guided tissue regeneration classical ex vivo tissue engineering of bone has not yet reached the level of routine clinical application although a wealth of scaffolds and growth factors has been developed. Engineering of complex bone constructs in vitro requires scaffolds, growth and differentiation factors, precursor cells for angiogenesis and osteogenesis and suitable bioreactors in various combinations. The development of applications for ex vivo tissue engineering of bone faces technical challenges concerning rapid vascularization for the survival of constructs in vivo. Recent new ideas and developments in the fields of bone biology, materials science and bioreactor technology will enable us to develop standard operating procedures for ex vivo tissue engineering of bone in the near future. Once prototyped such applications will rapidly be tailored for compromised conditions like vitamin D and sex hormone deficiencies, cellular deficits and high production of regeneration inhibitors, as they are prevalent in osteoporosis and in higher age.     

Janus magnetic cellular spheroids for vascular tissue engineering

Cell aggregates, or spheroids, have been used as building blocks to fabricate scaffold-free tissues that can closely mimic the native three-dimensional in vivo environment for broad applications including regenerative medicine and high throughput testing of drugs. The incorporation of magnetic nanoparticles (MNPs) into spheroids permits the manipulation of spheroids into desired shapes, patterns, and tissues using magnetic forces. Current strategies incorporating MNPs often involve cellular uptake, and should therefore be avoided because it induces adverse effects on cell activity, viability, and phenotype. Here, we report a Janus structure of magnetic cellular spheroids (JMCS) with spatial control of MNPs to form two distinct domains: cells and extracellular MNPs. This separation of cells and MNPs within magnetic cellular spheroids was successfully incorporated into cellular spheroids with various cellular and extracellular compositions and contents. The amount of cells that internalized MNPs was quantified and showed that JMCSs resulted in significantly lower internalization (35%) compared to uptake spheroids (83%, p < 0.05). Furthermore, the addition of MNPs to cellular spheroids using the Janus method has no adverse effects on cellular viability up to seven weeks, with spheroids maintaining at least 82% viability over 7 weeks when compared to control spheroids without MNPs. By safely incorporating MNPs into cellular spheroids, results demonstrated that JMCSs were capable of magnetic manipulation, and that magnetic forces used during magnetic force assembly mediate fusion into controlled patterns and complex tissues. Finally, JMCSs were assembled and fused into a vascular tissue construct 5 mm in diameter using magnetic force assembly.     

浓集自体骨髓基质干细胞组织工程复合物治疗早期股骨头坏死的实验疗效

目的研究浓集自体骨髓基质干细胞组织工程复合物治疗兔早期股骨头坏死的实验疗效，为临床应用提供依据。方法24只新西兰大白兔随机分为两组，建立右侧股骨头骨缺损模型，并用液氮从缺损内将股骨头冷冻坏死，A组为对照组，仅植入空白明胶海绵，B组为实验组，植入复合物。术后每组分别于2、4、6、8周各处死3只动物，做影像学及组织学检查。进行有关指标检测。结果①影像学结果：随着时间延长无论是X线、CT表现还是MRI表现，实验组钻孔区由低密度逐渐增高，8周时有骨小梁结构；对照组钻孔区无新骨形成表现。②组织学结果：A组2周标本缺损区内，充满坏死的组织碎片；4周标本，缺损区内为疏松的纤维肉芽组织；6周标本缺损区内为纤维组织；8周标本缺损区内仍为纤维组织，无新生骨组织。B组2周标本缺损区内，有大量的成骨细胞；4周标本，缺损区内有大量骨小梁及类骨质填充；6周标本缺损区内出现比较成熟的骨小梁；8周标本缺损区内骨小梁成熟，骨髓组织形成。结论浓集自体骨髓基质干细胞组织工程复合物对兔股骨头坏死有极好的修复作用。     

血管组织工程的发展现状和趋势

心脑血管疾病是全球发病率和死亡率最高的疾病,其主要病因是动脉粥样硬化。临床上主要采用血管移植物重建病损血管,人造合成血管在大口径血管修复中取得了满意的效果,但在小口径血管修复中效果并不理想。近30年来,血管组织工程发展极其迅速,从再生的角度为血管修复提供了新的途径。本文综述血管组织工程的最新进展(体外、体内、原位血管组织工程),并对未来发展趋势进行了前瞻性展望。     

纤维蛋白在软骨组织工程中的应用

纤维蛋白是纤维蛋白原经激活后形成的聚合物凝胶,作为软骨组织工程研究的载体材料,在相关的生物学和物理特性上具有其一定的优势。对纤维蛋白在软骨组织工程中应用的现状和发展方向进行综述。     

Design of a composite biomaterial system for tissue engineering applications

Biomaterials that regulate vascularized tissue formation have the potential to contribute to new methods of tissue replacement and reconstruction. The goal of this study was to develop a porous, degradable tissue engineering scaffold that could deliver multiple growth factors and regulate vessel assembly within the porous structure of the material. Porous hydrogels of poly(ethylene glycol)-co-(l-lactic acid) (PEG–PLLA) were prepared via salt leaching. The degradation time of the hydrogels could be controlled between 1 and 7 weeks, based on hydrogel composition. Fibrin was incorporated into the interconnected pores of the hydrogels to promote neovascularization and as a reservoir for rapid (<5 days) growth factor delivery. Poly(lactic-co-glycolic acid) (PLGA) microspheres were incorporated into the degradable polymeric hydrogel scaffold to allow sustained (>30 days) growth factor delivery. Fibroblast growth factor-1 (FGF-1) and platelet-derived growth factor-BB (PDGF-BB) were delivered from the system owing to their roles in the promotion of angiogenesis and vascular stabilization, respectively. Hydrogels tested in vivo with a subcutaneous implantation model were selected based on the results from in vitro degradation and growth factor release kinetics. Dual growth factor delivery promoted significantly more tissue ingrowth in the scaffold compared with blank or single growth factor delivery. The sequential delivery of FGF-1 following PDGF-BB promoted more persistent and mature blood vessels. In conclusion, a biomaterials system was developed to provide structural support for tissue regeneration, as well as delivery of growth factors that stimulate neovascularization within the structure prior to complete degradation.     

Current status of tissue engineering for cardiovascular structures

Various vascular and valvlular grafts are commonly used in the treatment of cardiovascular disease. Current prosthetic or bioprosthetic materials lack growth potential, and therefore, subsequent replacement further defeats the concept of primary repair early in pediatric cardiac patients. Tissue engineering is a new discipline that offers the potential to create replacement structures from autologous cells and biodegradable polymer scaffolds. Because tissue-engineering constructs contain living cells, they may have the potential for growth, self-repair, and self-remodeling. Cardiac valve leaflets and large conduits in the pulmonary ciruulation have been made with this tissue-engineering approach in lambs. Venous conduits were also created in dogs. Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted peripheral arteries or vein. A synthetic biodegradable scaffold con-sisting of polyglactin and polyglycolic acid fibers was seeded in vitro with mixed cultured cells. After one week, these autologous cell/polymer constructs were reimplanted in animals. Each animal was then followed periodically by echocardiography and angiography. The animals were sacrificed, and the implanted tissues were examined histologically, biochemically, and biomechanically. A 4-hydroxyproline assay was performed to evaluate the collagen content. The implanted conduit diameters increased as the animals grew during the study period. Histologically, the biodegradable polymer scaffold was completely degraded. Collagen analysis of the constructs showed the development of an extracellular matrix. Immunohistochemical staining demonstrated elastin fiber in the matrix and factor VIII on the inner surface of the conduits. In conclusion, a tissue-engineering approach to constructing cardiovascular structures is feasible using cells of either arterial or venous origin. In these tissue-engineered autografts, transplanted autologous cells generated the proper matrix over the polymer scaffold under physiologic conditions.     

Development of photosynthetic biomaterials for in vitro tissue engineering

Tissue engineering has opened a new therapeutic avenue that promises a revolution in regenerative medicine. To date, however, the translation of engineered tissues into clinical settings has been highly limited and the clinical results are often disappointing. Despite decades of research, the appropriate delivery of oxygen into three-dimensional cultures still remains one of the biggest unresolved problems for in vitro tissue engineering. In this work, we propose an alternative source of oxygen delivery by introducing photosynthetic scaffolds. Here we demonstrate that the unicellular and photosynthetic microalga Chlamydomonas reinhardtii can be cultured in scaffolds for tissue repair; this microalga shows high biocompatibility and photosynthetic activity. Moreover, Chlamydomonas can be co-cultured with fibroblasts, decreasing the hypoxic response under low oxygen culture conditions. Finally, results showed that photosynthetic scaffolds are capable of producing enough oxygen to be independent of external supply in vitro. The results of this study represent the first step towards engineering photosynthetic autotrophic tissues.     

Scaffolds for tissue engineering of cardiac valves

Tissue engineered heart valves offer a promising alternative for the replacement of diseased heart valves avoiding the limitations faced with currently available bioprosthetic and mechanical heart valves. In the paradigm of tissue engineering, a three-dimensional platform – the so-called scaffold – is essential for cell proliferation, growth and differentiation, as well as the ultimate generation of a functional tissue. A foundation for success in heart valve tissue engineering is a recapitulation of the complex design and diverse mechanical properties of a native valve. This article reviews technological details of the scaffolds that have been applied to date in heart valve tissue engineering research.