Presence of pores and hydrogel composition influence tensile properties of scaffolds fabricated from well-defined sphere templates

Sphere templating is an attractive method to produce porous polymeric scaffolds with well-defined and uniform pore structures for applications in tissue engineering. While high porosity is desired to facilitate cell seeding and enhance nutrient transport, the incorporation of pores will impact gross mechanical properties of tissue scaffolds and will likely be dependent on pore size. The goals of this study were to evaluate the effect of pores, pore diameter, and polymer composition on gross mechanical properties of hydrogels prepared from crosslinked poly(ethylene glycol) (PEG) and poly(2-hydroxyethyl methacrylate) (pHEMA). Sphere templates were fabricated from uncrosslinked poly(methyl methacrylate) spheres sieved between 53-63 and 150-180 μm. Incorporating pores into hydrogels significantly decreased the quasi-static modulus and ultimate tensile stress, but increased the ultimate tensile strain. For pHEMA, decreases in gel crosslinking density and increases in pore diameters followed similar trends. Interestingly, the mechanical properties of porous PEG hydrogels were less sensitive to changes in pore diameter for a given polymer composition. Additionally, pore diameter was shown to affect skeletal myoblast adhesion whereby many cells cultured in porous hydrogels with smaller pores were seen spanning across multiple pores, but lined the inside of larger pores. In summary, incorporation of pores and changes in pore diameter significantly affect the gross mechanical properties, but in a manner that is dependent on gel chemistry, structure, and composition. Together, these findings will help to design better hydrogel scaffolds for applications where gross mechanical properties and porosity are critical.     

Rapid prototyping of tissue-engineering constructs, using photopolymerizable hydrogels and stereolithography

One of the most important aspects of tissue engineering is the design of the scaffold providing the mechanical strength and access to nutrients for the new tissue. For customized tissue engineering, it is essential to be able to fabricate three-dimensional scaffolds of various geometric shapes, in order to repair defects caused by accidents, surgery, or birth. Rapid prototyping or solid free-form fabrication (SFF) techniques hold great promise for designing three-dimensional customized scaffolds, yet traditional cell-seeding techniques may not provide enough cell mass for larger constructs. This article presents a novel attempt to fabricate three-dimensional scaffolds, using hydrogels combined with cell encapsulation to fabricate high-density tissue constructs. A commercially available stereolithography technique was applied to fabricate scaffolds using poly(ethylene oxide) and poly(ethylene glycol)dimethacrylate photopolymerizable hydrogels. Mechanical characterization shows the constructs to be comparable with soft tissues in terms of elasticity. High cell viability was achieved and high-density constructs fabricated.     

Three-dimensional plotted scaffolds with controlled pore size gradients: Effect of scaffold geometry on mechanical performance and cell seeding efficiency

Scaffolds produced by rapid prototyping (RP) techniques have proved their value for tissue engineering applications, due to their ability to produce predetermined forms and structures featuring fully interconnected pore architectures. Nevertheless, low cell seeding efficiency and non-uniform distribution of cells remain major limitations when using such types of scaffold. This can be mainly attributed to the inadequate pore architecture of scaffolds produced by RP and the limited efficiency of cell seeding techniques normally adopted. In this study we aimed at producing scaffolds with pore size gradients to enhance cell seeding efficiency and control the spatial organization of cells within the scaffold. Scaffolds based on blends of starch with poly(ε-caprolactone) featuring both homogeneously spaced pores (based on pore sizes of 0.75 and 0.1 mm) and pore size gradients (based on pore sizes of 0.1-0.75-0.1 and 0.75-0.1-0.75 mm) were designed and produced by three-dimensional plotting. The mechanical performance of the scaffolds was characterized using dynamic mechanical analysis (DMA) and conventional compression testing under wet conditions and subsequently characterized using scanning electron microscopy and micro-computed tomography. Osteoblast-like cells were seeded onto such scaffolds to investigate cell seeding efficiency and the ability to control the zonal distribution of cells upon seeding. Scaffolds featuring continuous pore size gradients were originally produced. These scaffolds were shown to have intermediate mechanical and morphological properties compared with homogenous pore size scaffolds. The pore size gradient scaffolds improved seeding efficiency from ～35% in homogeneous scaffolds to ～70% under static culture conditions. Fluorescence images of cross-sections of the scaffolds revealed that scaffolds with pore size gradients induce a more homogeneous distribution of cells within the scaffold.     

Chemical sintering generates uniform porous hyaluronic acid hydrogels

The implantation of scaffolds for tissue repair has achieved only limited success due primarily to the inability to achieve vascularization within the construct. Many strategies have therefore moved to incorporate pores into the scaffolds to encourage rapid cellular infiltration and subsequent vascular ingrowth. We utilized an efficient chemical sintering technique to create a uniform network of polymethyl methacrylate (PMMA) microspheres for porous hyaluronic acid hydrogel formation. The porous hydrogels generated from chemical sintering possessed pore uniformity and interconnectivity comparable to the commonly used non- and heat sintering techniques. Moreover, a similar cell response to the porous hydrogels generated from each sintering approach was observed in cell viability, spreading and proliferation in vitro, as well as cellular invasion in vivo. We propose chemical sintering of PMMA microspheres using a dilute acetone solution as an alternative method to generate porous hyaluronic acid hydrogels since it requires equal or 10-fold less processing time as the currently used non-sintering or heat sintering technique, respectively.     

DNA delivery from matrix metalloproteinase degradable poly(ethylene glycol) hydrogels to mouse cloned mesenchymal stem cells

The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation and to study molecular pathways in three dimensions than protein delivery. In this report, we explored gene transfer to infiltrating MSCs into matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP-degradable peptides via Michael addition chemistry. A large fraction of encapsulated polyplexes remained active after encapsulation (65%) and the mechanical properties of the hydrogels were unchanged by the encapsulation of the polyplexes. Cells were seeded inside the hydrogel scaffolds using two different approaches: clustered and homogeneous. The viability of MSCs was similar in hydrogels with and without polyplexes. Transgene expression was characterized with time using a secreted reporter gene and showed different profiles for clustered and homogeneously seeded cells. Clustered cells resulted in cumulative transgene expression that increased through the 21-day incubation, while homogeneously seeded cells resulted in cumulative transgene expression that plateaued after 7 days of culture. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long lasting signals in vivo, which are essential for the regeneration of functional tissues.     

制备软骨组织工程支架的材料和方法

背景：软骨组织工程的研究为修复软骨缺损提供了新的思路和方法,其中如何获得理想的组织工程支架是这一研究的核心和难点。 目的：回顾性分析软骨组织工程支架的材料选择和制备方法。 方法：由第一作者检索2000至2012年 PubMed数据库、ELSEVIER SCIENCEDIRECT、万方数据库、中国知网库有关制备软骨组织工程支架的材料选择和方法等方面的文献。 结果与结论：软骨支架材料分为天然生物材料、人工合成高分子材料和复合材料。可采用相分离法、溶剂浇铸/粒子沥滤技术、气体发泡技术、快速成型技术及静电纺丝法制备支架材料。由于胶原、琼脂糖和藻酸盐等水凝胶类天然材料可提供足够的生物相容性、增殖和黏附能力及亲水性,电纺的人工合成高分子材料复合支架又可以保证支架的力学强度、塑形要求、孔隙率、可降解性等,将天然材料利用包埋技术和表面修饰技术复合于电纺的高分子复合材料支架上将更有利于支架性能的发挥。     

Strong fiber-reinforced hydrogel

In biological hydrogels, the gel matrix is usually reinforced with micro- or nanofibers, and the resulting composite is tough and strong. In contrast, synthetic hydrogels are weak and brittle, although they are highly elastic. The are many potential applications for strong synthetic hydrogels in medical devices, including as scaffolds for tissue growth. This work describes a new class of hydrogel composites reinforced with elastic fibers, giving them a cartilage-like structure. A three-dimensional rapid prototyping technique was used to form crossed “log-piles” of elastic fibers that are then impregnated with an epoxy-based hydrogel in order to form the fiber-reinforced gel. The fibrous construct improves the strength, modulus and toughness of the hydrogel, and also constrains the swelling. By altering the construct geometry and studying the effect on mechanical properties, we will develop the understanding needed to design strong hydrogels for biomedical devices and soft machines.     

Cell immobilization in gelatin–hydroxyphenylpropionic acid hydrogel fibers

Gelatin–hydroxyphenylpropionic acid (Gtn–HPA) hydrogels are highly porous and biodegradable materials. Herein we report a fiber spinning method that can produce cell-seeded solid and hollow hydrogel fibers by enzymatically cross-linking Gtn–HPA in solutions flowing within a capillary tube. The cell-immobilized hydrogel fibers, with feature sizes down to 20 μm, are formed as a result of continuous cross-linking of cell-mixed hydrogel precursors in a multiphase laminar flow. This fiber formation process is mild enough to retain the cell viability. The continuous fiber formation, simultaneous cell encapsulation, as well as versatile combination of fiber structures provided by this approach make it a promising and effective technique for the preparation of cell-seeded hydrogel scaffolds and carriers for tissue engineering.     

Fabrication and Mechanical Evaluation of Anatomically-Inspired Quasilaminate Hydrogel Structures with Layer-Specific Formulations

A major tissue engineering challenge is the creation of multilaminate scaffolds with layer-specific mechanical properties representative of native tissues, such as heart valve leaflets, blood vessels, and cartilage. For this purpose, poly(ethylene glycol) diacrylate (PEGDA) hydrogels are attractive materials due to their tunable mechanical and biological properties. This study explored the fabrication of trilayer hydrogel quasilaminates. A novel sandwich method was devised to create quasilaminates with layers of varying stiffnesses. The trilayer structure was comprised of two “stiff” outer layers and one “soft” inner layer. Tensile testing of bilayer quasilaminates demonstrated that these scaffolds do not fail at the interface. Flexural testing showed that the bending modulus of acellular quasilaminates fell between the bending moduli of the “stiff” and “soft” hydrogel layers. The bending modulus and swelling of trilayer scaffolds with the same formulations were not significantly different than single layer gels of the same formulation. The encapsulation of cells and the addition of phenol red within the hydrogel layers decreased bending modulus of the trilayer scaffolds. The data presented demonstrates that this fabrication method can make quasilaminates with robust interfaces, integrating layers of different mechanical properties and biofunctionalization, and thus forming the foundation for a multilaminate scaffold that more accurately represents native tissue.     

Cell encapsulation in biodegradable hydrogels for tissue engineering applications

Encapsulating cells in biodegradable hydrogels offers numerous attractive features for tissue engineering, including ease of handling, a highly hydrated tissue-like environment for cell and tissue growth, and the ability to form in vivo. Many properties important to the design of a hydrogel scaffold, such as swelling, mechanical properties, degradation, and diffusion, are closely linked to the crosslinked structure of the hydrogel, which is controlled through a variety of different processing conditions. Degradation may be tuned by incorporating hydrolytically or enzymatically labile segments into the hydrogel or by using natural biopolymers that are susceptible to enzymatic degradation. Because cells are present during the gelation process, the number of suitable chemistries and formulations are limited. In this review, we describe important considerations for designing biodegradable hydrogels for cell encapsulation and highlight recent advances in material design and their applications in tissue engineering.     

快速成型方法制备组织工程支架的研究与应用

背景：快速成型是基于材料堆积法,结合计算机、数控、激光和材料技术于一体的高新制造技术。 目的：综述快速成型技术在组织工程支架制备中的应用。 方法：由第一作者检索万方数据库、中国知网数据库和Elsevier Science Direct Online有关支架材料的生物力学性能、支架材料发展前景及快速成型技术在支架材料制备领域中应用研究等方面的文献。 结果与结论：快速成型技术应用于组织工程支架的制备已经越来越成熟,快速成型技术不但克服了传统制造方法中存在的支架复杂外形制造困难和内部微结构无法控制的缺陷,而且还可以通过有限元分析预先对支架的结构进行优化,以实现改善支架机械强度等某些特殊的要求。但是,由于组织器官的特殊性和排外性及细胞的黏附条件,不但要从结构上改善支架,而且需要快速成型技术与具有组织相容性及可降解的材料相结合,使支架植入生物体后,细胞能更好地增殖和分化,促进组织再生,修复缺损组织。     

组织工程支架材料在脊髓损伤中的应用

背景：近年来,随着基础研究的发展,许多新方法、新策略已经开始用于脊髓损伤修复,其中组织工程学的发展开辟了一条新的途径,利用组织工程学的方法治疗脊髓损伤已逐渐成为当前新的研究热点。 目的：探讨组织工程支架材料在脊髓损伤中的应用的研究进展。 方法：检索SCI数据库2002/2011有关组织工程支架材料在脊髓损伤中的应用的文献,检索词为“组织工程(tissue engineering);脊髓损伤(spinal cord injury);支架材料(scaffold material);胶原(collagen);壳聚糖(chitosan);藻酸盐凝胶(alginate hydrogel);纤维蛋白凝胶(fibrin glue);聚羟基丁酸酯(poly-b-hydroxybutyrate);琼脂糖凝胶(agarose);聚乳酸(poly lactic acid);合成水凝胶(synthetic hydrogels);聚乙二醇(polyethylene glycol)”,对组织工程支架材料修复脊髓损伤的临床及基础文献进行深入分析。 结果与结论：组织工程支架是组织工程修复脊髓损伤研究的重点内容。组织工程支架的材料包括天然材料和人工合成材料,天然材料具有良好的细胞和组织相容性,人工合成的聚合物支架在结构形状、机械强度及规模化生产方面均具有很大的优势。近年来,组织工程支架材料在脊髓损伤中的应用有了很大发展,相继出现了新型支架材料。     

Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering

In the year 2000 a new rapid prototyping (RP) technology was developed at the Freiburg Materials Research Center to meet the demands for desktop fabrication of scaffolds useful in tissue engineering. A key feature of this RP technology is the three-dimensional (3D) dispensing of liquids and pastes in liquid media. In contrast to conventional RP systems, mainly focused on melt processing, the 3D dispensing RP process (3D plotting) can apply a much larger variety of synthetic as well as natural materials, including aqueous solutions and pastes, to fabricate scaffolds for application in tissue engineering. For the first time, hydrogel scaffolds with a designed external shape and a well-defined internal pore structure were prepared by this RP process. Surface coating and pore formation were achieved to facilitate cell adhesion and cell growth. The versatile application potential of new hydrogel scaffolds was demonstrated in cell culture.     

Synthesis,properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels

Gelatin methacryloyl (GelMA) hydrogels have been widely used for various biomedical applications due to their suitable biological properties and tunable physical characteristics. GelMA hydrogels closely resemble some essential properties of native extracellular matrix (ECM) due to the presence of cell-attaching and matrix metalloproteinase responsive peptide motifs, which allow cells to proliferate and spread in GelMA-based scaffolds. GelMA is also versatile from a processing perspective. It crosslinks when exposed to light irradiation to form hydrogels with tunable mechanical properties. It can also be microfabricated using different methodologies including micromolding, photomasking, bioprinting, self-assembly, and microfluidic techniques to generate constructs with controlled architectures. Hybrid hydrogel systems can also be formed by mixing GelMA with nanoparticles such as carbon nanotubes and graphene oxide, and other polymers to form networks with desired combined properties and characteristics for specific biological applications. Recent research has demonstrated the proficiency of GelMA-based hydrogels in a wide range of tissue engineering applications including engineering of bone, cartilage, cardiac, and vascular tissues, among others. Other applications of GelMA hydrogels, besides tissue engineering, include fundamental cell research, cell signaling, drug and gene delivery, and bio-sensing.     

Versatile click alginate hydrogels crosslinked via tetrazine–norbornene chemistry

Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.     

The development of silk fibroin scaffolds using an indirect rapid prototyping approach: Morphological analysis and cell growth monitoring by spectral-domain optical coherence tomography

To date, naturally derived biomaterials are rarely used in advanced tissue engineering (TE) methods despite their superior biocompatibility. This is because these native materials, which consist mainly of proteins and polysaccharides, do not possess the ability to withstand harsh processing conditions. Unlike synthetic polymers, natural materials degrade and decompose rapidly in the presence of chemical solvents and high temperature, respectively. Thus, the fabrication of tissue scaffolds using natural biomaterials is often carried out using conventional techniques, where the efficiency in mass transport of nutrients and removal of waste products within the construct is compromised. The present study identified silk fibroin (SF) protein as a suitable material for the application of rapid prototyping (RP) or additive manufacturing (AM) technology. Using the indirect RP method, via the use of a mould, SF tissue scaffolds with both macro- and micro-morphological features can be produced and qualitatively examined by spectral-domain optical coherence tomography (SD-OCT). The advanced imaging technique showed the ability to differentiate the cells and SF material by producing high contrasting images, therefore suggesting the method as a feasible alternative to the histological analysis of cell growth within tissue scaffolds.     

组织工程多孔支架材料制备技术的研究进展

组织工程多孔支架材料作为组织工程学的三大要素之一,除本身的性质外,支架材料的形状、孔径大小和孔隙率都直接影响着种子细胞的黏附、增殖和分化,因此如何制备具有高孔隙率、孔径大小合适且内部联通的多孔支架材料.为种子细胞的生长提供良好的微环境是非常重要的.回顾了近年来发展的组织工程多孔支架材料制备技术:纤维粘接法、乳液冷冻干燥法、溶液浇注,沥滤法、气体发泡法、热致相分离法及静电纺丝法.并重点介绍了目前国内外研究较多的快速成形技术;总结分析认为各种基本制备技术的联合应用和具备结构高度可控性、个体化制备特点的快速成形技术将是今后组织工程多孔支架材料制备技术的发展方向.     

Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration

Basic fibroblast growth factor (bFGF) was immobilized to hydrogel scaffolds with retention of mitogenic and chemotactic activity. The bFGF was functionalized in order to incorporate it covalently within polyethylene glycol (PEG) hydrogel scaffolds by reaction with acryloyl-PEG-NHS. Hydrogels were formed by exposing aqueous solutions of PEG diacrylate, acryloyl-PEG-RGDS, and acryloyl-PEG-bFGF to long-wavelength ultraviolet light in the presence of a photoinitiator. These bFGF-modified hydrogels with RGD adhesion sites were evaluated for their effect on vascular smooth muscle cell (SMC) behavior, increasing SMC proliferation by approximately 41% and migration by approximately 15%. A covalently immobilized bFGF gradient was formed using a gradient maker to pour the hydrogel precursor solutions and then photopolymerizing to lock in the concentration gradient. Silver staining was used to detect the bFGF gradient, which increased linearly along the hydrogel's length. Cells were observed to align on hydrogels modified with a bFGF gradient in the direction of increasing tethered bFGF concentration as early as 24 h after seeding. SMCs also migrated differentially, up the concentration gradient, on bFGF-gradient hydrogels compared to control hydrogels with and without a constant bFGF concentration. These hydrogel scaffolds may be useful for studying protein gradient effects on cell behavior and for directing cell migration in tissue-engineering applications.     

Temporal and spatially controllable cell encapsulation using a water-soluble phospholipid polymer with phenylboronic acid moiety

Temporal and spatially controllable cell encapsulation based on a water-soluble phospholipid polymer is reported in this study. Phospholipid polymers, i.e., poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV), were synthesized. A series of hydrogels was prepared between the water-soluble PMBV and other water-soluble polymers having multi-valent alcoholic groups, such as poly(vinyl alcohol) (PVA). The PMBV/PVA hydrogels were formed not only in water, but also in a cell culture medium, and dissociated by the excess addition of low molecular weight di-valent hydroxyl compounds, such as d-glucose. The PMBV/PVA hydrogel was applied as a cell-container which has three-dimensional matrices for the reversible encapsulation of living cells without any response in it. Uniform cell seeding can be achieved using the hydrogels due to the homogenous gel formation of PMBV and PVA in the cell culture medium. Fibroblast cells were encapsulated in the PMBV/PVA hydrogel and maintained for 1 week. After dissociation of the PMBV/PVA hydrogel, the cells were seeded on conventional tissue culture polystyrene. The cells adhered and proliferated as usual on the plate. That is, the PMBV/PVA hydrogel will be useful as a cell-container, which can maintain the cells without any significant adverse effect on the entrapped cells.     

Hydrogel based on interpenetrating polymer networks of dextran and gelatin for vascular tissue engineering

Hydrogel networks are highly desirable as three-dimensional (3-D) tissue engineering scaffolds for cell encapsulation due to the high water content and ability to mimick the native extracellular matrix. However, their application is limited by their nanometer-scale mesh size, which restricts the spreading and proliferation of encapsulated cells, and their poor mechanical properties. This study seeks to address both limitations through application of a novel cell-encapsulating hydrogel family based on the interpenetrating polymer network (IPN) of gelatin and dextran bifunctionalized with methacrylate (MA) and aldehyde (AD) (Dex-MA-AD). The chemical structure of the synthesized Dex-MA-AD was verified by (1)H-NMR and the degrees of substitution of MA and AD were found to be 14 and 13.9+/-1.3 respectively. The water contents in all these hydrogels were approximately 80%. Addition of 40 mg/ml to 60 mg/ml gelatin to neat Dex-MA-AD increased the compressive modulus from 15.4+/-3.0 kPa to around 51.9+/-0.1 kPa (about 3.4-fold). Further, our IPN hydrogels have higher dynamic storage moduli (i.e. on the order of 10(4)Pa) than polyethylene glycol-based hydrogels (around 10(2)-10(3)Pa) commonly used for smooth muscle cells (SMCs) encapsulation. Our dextran-based IPN hydrogels not only supported endothelial cells (ECs) adhesion and spreading on the surface, but also allowed encapsulated SMCs to proliferate and spread in the bulk interior of the hydrogel. These IPN hydrogels appear promising as 3-D scaffolds for vascular tissue engineering.