Versatile fabrication of vascularizable scaffolds for large tissue engineering in bioreactor

Despite significant progresses were achieved in tissue engineering over the last 20 years, a number of unsolved problems still remain. One of the most relevant issues is the lack of a proper vascularization that is limiting the size of the engineered tissues to smaller than clinically relevant dimensions. Sacrificial molding holds great promise to engineered construct with perfusable vascular architectures, but there is still the need to develop more versatile approaches able to be independent of the nature and dimensions of the construct. In this work we developed a versatile sacrificial molding technique for fabricating bulk, cell-laden and porous scaffolds with embedded vascular fluidic networks. These branched fluidic architectures are created by highly resistant thermoplastic sacrificial templates, made of poly(vinyl alcohol), representing a remarkable progress in manufacturability and scalability. The obtained architecture, when perfused in bioreactor, has shown to prevent the formation of a necrotic core in thick cell-laden constructs and enabled the rapid fabrication of hierarchically branched endothelium. In conclusion we demonstrate a novel strategy towards the engineering of vascularized thick tissues through the integration of the PVA-based microfabrication sacrificial approach and perfusion bioreactors. This approach may be able to scale current engineered tissues to clinically relevant dimensions, opening the way to their widespread clinical applications.     

Stem cell-based tissue engineering with silk biomaterials

Silks are naturally occurring polymers that have been used clinically as sutures for centuries. When naturally extruded from insects or worms, silk is composed of a filament core protein, termed fibroin, and a glue-like coating consisting of sericin proteins. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility, slow degradability and remarkable mechanical properties of the material. In addition, the ability to now control molecular structure and morphology through versatile processability and surface modification options have expanded the utility for this protein in a range of biomaterial and tissue-engineering applications. Silk fibroin in various formats (films, fibers, nets, meshes, membranes, yarns, and sponges) has been shown to support stem cell adhesion, proliferation, and differentiation in vitro and promote tissue repair in vivo. In particular, stem cell-based tissue engineering using 3D silk fibroin scaffolds has expanded the use of silk-based biomaterials as promising scaffolds for engineering a range of skeletal tissues like bone, ligament, and cartilage, as well as connective tissues like skin. To date fibroin from Bombyx mori silkworm has been the dominant source for silk-based biomaterials studied. However, silk fibroins from spiders and those formed via genetic engineering or the modification of native silk fibroin sequence chemistries are beginning to provide new options to further expand the utility of silk fibroin-based materials for medical applications.     

注射型组织再生支架的研究进展

在组织修复与再生中,可注射型支架具有微创的优点,并能够为细胞的增殖与分化提供更接近于天然细胞外基质的化学与物理环境。因此,相比于传统的硬支架具有更大的优势,是细胞支架的一个重要组成部分与发展方向。目前,可注射型支架材料是以水凝胶为基础,包括温敏型水凝胶、交联型水凝胶和以水凝胶为载体的混合物。这类支架已被广泛地用于骨和软骨的修复。这些支架在体内实验中表现出良好的生物和细胞相容性,能够促进组织的形成。本文综述了可注射型组织再生支架近年来的发展动向,分析了各类可注射支架的优缺点。     

大鼠BMSCs成骨诱导及复合支架材料构建组织工程骨组织的研究*

目的利用诱导成骨分化的骨髓间充质干细胞(bone marrowmesenchymal stem cells,BMSCs)复合生物支架材料构建组织工程骨组织。方法采用密度梯度离心法获取大鼠骨髓间充质干细胞,原代培养扩增后,条件培养基诱导成骨分化作为实验组,并设非条件培养基培养为对照组。诱导培养后,通过碱性磷酸酶、钙结节染色;I型胶原、骨钙素检测鉴定成骨性。将诱导的BMSCs利用滴加法种入自制组织工程生物支架复合培养,采取扫描电镜、HE切片染色观察培养8天时细胞在支架内部的生长情况。结果密度梯度离心法获取培养的原代骨髓间充质干细胞呈梭形或三角形贴壁生长,以梭形为主;经成骨诱导剂诱导后细胞呈多角形贴壁生长,碱性磷酸酶染色呈阳性、茜素红染色出现阳性的钙化结节;Western blotting检测Ⅰ型胶原蛋白表达较对照组明显增加(<0.05);ELISA法检测骨钙素结果较对照组明显升高(<0.01)。HE切片染色可见支架内部有细胞长入,细胞呈圆形或椭圆形。扫描电镜可见支架内部有大量细胞长入,细胞粘附、生长良好,呈现完全伸展状态,细胞-支架-细胞之间有基质连接。结论本实验获取的原代细胞为骨髓间充质干细胞,诱导剂诱导后成功分化为成骨细胞。采用经诱导成骨后的细胞作为组织工程骨构建的种子细胞,与三维支架材料复合后共培养,使构建的组织复合物更接近骨组织,为临床大段骨缺损的修复增加可能性。     

Heparin-functionalized polymeric biomaterials in tissue engineering and drug delivery applications

Heparin plays an important role in many biological processes via its interaction with various proteins, and hydrogels and nanoparticles comprising heparin exhibit attractive properties, such as anticoagulant activity, growth factor binding, and antiangiogenic and apoptotic effects, making them great candidates for emerging applications. Accordingly, this review summarizes recent efforts in the preparation of heparin-based hydrogels and formation of nanoparticles, as well as the characterization of their properties and applications. The challenges and future perspectives for heparin-based materials are also discussed. Prospects are promising for heparin-containing polymeric biomaterials in diverse applications ranging from cell carriers for promoting cell differentiation to nanoparticle therapeutics for cancer treatment.     

Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review

Once damaged, articular cartilage has very little capacity for spontaneous healing because of the avascular nature of the tissue. Although many repair techniques have been proposed over the past four decades, none has sucessfully regenerated long-lasting hyaline cartilage tissue to replace damaged cartilage. Tissue engineering approaches, such as transplantation of isolated chondrocytes, have recently demonstrated tremendous clinical potential for regeneration of hyaline-like cartilage tissue and treatment of chondral lesions. As such a new approach emerges, new important questions arise. One of such questions is: what kinds of biomaterials can be used with chondrocytes to tissue-engineer articular cartilage? The success of chondrocyte transplantation and/or the quality of neocartilage formation strongly depend on the specific cell-carrier material. The present article reviews some of those biomaterials, which have been suggested to promote chondrogenesis and to have potentials for tissue engineering of articular cartilage. A new biomaterial, a chitosan-based polysaccharide hydrogel, is also introduced and discussed in terms of the biocompatibility with chondrocytes.     

New biotextiles for tissue engineering: Development,characterization and in vitro cellular viability

This work proposes biodegradable textile-based structures for tissue engineering applications. We describe the use of two polymers, polybutylene succinate (PBS) proposed as a viable multifilamentand silk fibroin (SF), to produce fibre-based finely tuned porous architectures by weft knitting. PBS is here proposed as a viable extruded multifilament fibre to be processed by a textile-based technology. A comparative study was undertaken using a SF fibre with a similar linear density. The knitted constructs obtained are described in terms of their morphology, mechanical properties, swelling capability, degradation behaviour and cytotoxicity. The weft knitting technology used offers superior control over the scaffold design (e.g. size, shape, porosity and fibre alignment), manufacturing and reproducibility. The presented fibres allow the processing of a very reproducible intra-architectural scaffold geometry which is fully interconnected, thus providing a high surface area for cell attachment and tissue in-growth. The two types of polymer fibre allow the generation of constructs with distinct characteristics in terms of the surface physico-chemistry, mechanical performance and degradation capability, which has an impact on the resulting cell behaviour at the surface of the respective biotextiles. Preliminary cytotoxicity screening showed that both materials can support cell adhesion and proliferation. These results constitute a first validation of the two biotextiles as viable matrices for tissue engineering prior to the development of more complex systems. Given the processing efficacy and versatility of the knitting technology and the interesting structural and surface properties of the proposed polymer fibres it is foreseen that the developed systems could be attractive for the functional engineering of tissues such as skin, ligament, bone or cartilage.     

Design of tissue engineering scaffolds based on hyperbolic surfaces: Structural numerical evaluation

Tissue engineering represents a new field aiming at developing biological substitutes to restore, maintain, or improve tissue functions. In this approach, scaffolds provide a temporary mechanical and vascular support for tissue regeneration while tissue in-growth is being formed. These scaffolds must be biocompatible, biodegradable, with appropriate porosity, pore structure and distribution, and optimal vascularization with both surface and structural compatibility. The challenge is to establish a proper balance between porosity and mechanical performance of scaffolds.This work investigates the use of two different types of triple periodic minimal surfaces, Schwarz and Schoen, in order to design better biomimetic scaffolds with high surface-to-volume ratio, high porosity and good mechanical properties. The mechanical behaviour of these structures is assessed through the finite element method software Abaqus. The effect of two parametric parameters (thickness and surface radius) is also evaluated regarding its porosity and mechanical behaviour.     

Collagen-based biomaterials as 3D scaffold for cell cultures: applications for tissue engineering and gene therapy

Many substances are used in the production of biomaterials: metals (titanium), ceramics (alumina), synthetic polymers (polyurethanes, silicones, polyglycolic acid (PGA), polylactic acid (PLA), copolymers of lactic and glycolic acids (PLGA), polyanhydrides, polyorthoesters) and natural polymers (chitosan, glycosaminoglycans, collagen). With the rapid development in tissue engineering, these different biomaterials have been used as three-dimensional scaffolds and cell transplant devices. The principal biochemical and biological characteristics of the collagen-based biomaterials are presented, including their interactions with cells (fibroblasts), distinct from those of synthetic polymers, and their potential use in gene therapy through the formation of neo-organs or organoids.     

Simulation of tissue differentiation in a scaffold as a function of porosity, Young's modulus and dissolution rate: application of mechanobiological models in tissue engineering

Numerous experimental studies have attempted to determine the optimal properties for a scaffold for use in bone tissue engineering but, as yet, no computational or theoretical approach has been developed that suggests how best to combine the various design parameters, e.g. scaffold porosity, Young's modulus, and dissolution rate. Previous research has shown that bone regeneration during fracture healing and osteochondral defect repair can be simulated using mechanoregulation algorithms based on computing strain and/or fluid flow in the regenerating tissue. In this paper a fully three-dimensional approach is used for computer simulation of tissue differentiation and bone regeneration in a regular scaffold as a function of porosity, Young's modulus, and dissolution rate—and this is done under both low and high loading conditions. The mechanoregulation algorithm employed determines tissue differentiation both in terms of the prevailing biophysical stimulus and number of precursor cells, where cell number is computed based on a three-dimensional random-walk approach. The simulations predict that all three design variables have a critical effect on the amount of bone regenerated, but not in an intuitive way: in a low load environment, a higher porosity and higher stiffness but a medium dissolution rate gives the greatest amount of bone whereas in a high load environment the dissolution rate should be lower otherwise the scaffold will collapse—at lower initial porosities however, higher dissolution rates can be sustained. Besides showing that scaffolds may be optimised to suit the site-specific loading requirements, the results open up a new approach for computational simulations in tissue engineering.     

Combinatorial scaffold morphologies for zonal articular cartilage engineering

Articular cartilage lesions are a particular challenge for regenerative medicine strategies as cartilage function stems from a complex depth-dependent organization. Tissue engineering scaffolds that vary in morphology and function offer a template for zone-specific cartilage extracellular matrix (ECM) production and mechanical properties. We fabricated multi-zone cartilage scaffolds by the electrostatic deposition of polymer microfibres onto particulate-templated scaffolds produced with 0.03 or 1.0 mm³ porogens. The scaffolds allowed ample space for chondrocyte ECM production within the bulk while also mimicking the structural organization and functional interface of cartilage’s superficial zone. Addition of aligned fibre membranes enhanced the mechanical and surface properties of particulate-templated scaffolds. Zonal analysis of scaffolds demonstrated region-specific variations in chondrocyte number, sulfated GAG-rich ECM, and chondrocytic gene expression. Specifically, smaller porogens (0.03 mm³) yielded significantly higher sGAG accumulation and aggrecan gene expression. Our results demonstrate that bilayered scaffolds mimic some key structural characteristics of native cartilage, support in vitro cartilage formation, and have superior features to homogeneous particulate-templated scaffolds. We propose that these scaffolds offer promise for regenerative medicine strategies to repair articular cartilage lesions.     

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.     

组织工程神经支架的研究进展

周围神经损伤在临床上非常多见,周围神经损伤给患者带来了高致残率,并给社会及患者家庭带来了巨大的经济负担.这些都使得周围神经损伤成为全球所面临的严峻的健康问题之一.目前,随着神经组织工程的发展,为临床上神经缺损的修复带来了新的希望.神经支架在修复神经缺损方面具有重要作用,可为神经细胞提供暂时的支持、黏附、生长环境,促进神...     

Delivery of dimethyloxallyl glycine in mesoporous bioactive glass scaffolds to improve angiogenesis and osteogenesis of human bone marrow stromal cells

Development of hypoxia-mimicking bone tissue engineering scaffolds is of great importance in stimulating angiogenesis for bone regeneration. Dimethyloxallyl glycine (DMOG) is a cell-permeable, competitive inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH), which can stabilize hypoxia-inducible factor 1α (HIF-1α) expression. The aim of this study was to develop hypoxia-mimicking scaffolds by delivering DMOG in mesoporous bioactive glass (MBG) scaffolds and to investigate whether the delivery of DMOG could induce a hypoxic microenvironment for human bone marrow stromal cells (hBMSC). MBG scaffolds with varied mesoporous structures (e.g. surface area and mesopore volume) were prepared by controlling the contents of mesopore-template agent. The composition, large-pore microstructure and mesoporous properties of MBG scaffolds were characterized. The effect of mesoporous properties on the loading and release of DMOG in MBG scaffolds was investigated. The effects of DMOG delivery on the cell morphology, cell viability, HIF-1α stabilization, vascular endothelial growth factor (VEGF) secretion and bone-related gene expression (alkaline phosphatase, ALP; osteocalcin, OCN; and osteopontin, OPN) of hBMSC in MBG scaffolds were systematically investigated. The results showed that the loading and release of DMOG in MBG scaffolds can be efficiently controlled by regulating their mesoporous properties via the addition of different contents of mesopore-template agent. DMOG delivery in MBG scaffolds had no cytotoxic effect on the viability of hBMSC. DMOG delivery significantly induced HIF-1α stabilization, VEGF secretion and bone-related gene expression of hBMSC in MBG scaffolds in which DMOG counteracted the effect of HIF-PH and stabilized HIF-1α expression under normoxic condition. Furthermore, it was found that MBG scaffolds with slow DMOG release significantly enhanced the expression of bone-related genes more than those with instant DMOG release. The results suggest that the controllable delivery of DMOG in MBG scaffolds can mimic a hypoxic microenvironment, which not only improves the angiogenic capacity of hBMSC, but also enhances their osteogenic differentiation.     

自组装多肽构建神经组织工程支架材料的研究进展

神经组织工程是利用组织工程支架材料、种子细胞等工程学技术重建神经以治疗神经系统损伤与疾病,恢复病变或损伤神经的解剖结构与功能.神经组织工程支架能诱导轴突再生.减少或隔离局部胶质瘢痕形成,帮助重建轴突与靶细胞之间的连接.自组装多肽是构建神经组织工程支架的优秀材料,也是目前研究的热点.对自组装多肽构建神经组织工程支架的研究现状进行综述,并提出面临问题及今后的研究方向.     

Current trends in the design of scaffolds for computer-aided tissue engineering

Advances introduced by additive manufacturing have significantly improved the ability to tailor scaffold architecture, enhancing the control over microstructural features. This has led to a growing interest in the development of innovative scaffold designs, as testified by the increasing amount of research activities devoted to the understanding of the correlation between topological features of scaffolds and their resulting properties, in order to find architectures capable of optimal trade-off between often conflicting requirements (such as biological and mechanical ones). The main aim of this paper is to provide a review and propose a classification of existing methodologies for scaffold design and optimization in order to address key issues and help in deciphering the complex link between design criteria and resulting scaffold properties.     

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.     

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。