Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering

Electrospinning using natural proteins or synthetic polymers is a promising technique for the fabrication of fibrous scaffolds for various tissue engineering applications. However, one limitation of scaffolds electrospun from natural proteins is the need to cross-link with glutaraldehyde for stability, which has been postulated to lead to many complications in vivo including graft failure. In this study, we determined the characteristics of hybrid scaffolds composed of natural proteins including collagen and elastin, as well as gelatin, and the synthetic polymer poly(epsilon-caprolactone) (PCL), so to avoid chemical cross-linking. Fiber size increased proportionally with increasing protein and polymer concentrations, whereas pore size decreased. Electrospun gelatin/PCL scaffolds showed a higher tensile strength when compared to collagen/elastin/PCL constructs. To determine the effects of pore size on cell attachment and migration, both hybrid scaffolds were seeded with adipose-derived stem cells. Scanning electron microscopy and nuclei staining of cell-seeded scaffolds demonstrated the complete cell attachment to the surfaces of both hybrid scaffolds, although cell migration into the scaffold was predominantly seen in the gelatin/PCL hybrid. The combination of natural proteins and synthetic polymers to create electrospun fibrous structures resulted in scaffolds with favorable mechanical and biological properties.     

丝素蛋白材料在组织工程中的新进展

背景：丝素蛋白支架已被建议运用在组织工程骨和软骨重建、肌腱重建、血管重建,神经重建以及膀胱重建等各方面。 目的：总结丝素蛋白作为支架在生物材料和组织工程领域的应用与发展。 方法：由第一作者应用计算机检索PubMed数据库及中国期刊数据库2000年1月至2011年11月有关丝素蛋白支架制备工艺,丝素蛋白支架修饰方法及丝素蛋白在组织工程中的应用等方面的文献。 结果与结论：丝素蛋白具有机械强度高、生物降解性慢、生物相容性良好、制备工艺多样等特点,支持多种细胞黏附、分化和生长,可应用于人工韧带、血管、骨、神经组织等方面。近期以丝素蛋白支架作为载体,通过多种方式添加各种生物制剂,比如各种生长因子和细胞因子,进一步扩大丝素蛋白在组织工程中的应用范围。     

A strategy for the development of tissue engineering scaffolds that regulate cell behavior

The cellular scaffold represents an extracellular matrix (ECM) in vivo and a culture substratum in vitro, and provides microenvironmental signaling cues based upon the architecture and component for cells. This review article discusses the development of ideal cellular scaffolds for maintaining the activity of functional cells, for regulating cell behavior, and for reconstructing three-dimensional multicellular masses (3-DMMs). Four culture technologies devising the materials of cellular scaffolds developed by author's group are also presented; the preparation of a multicellular spheroid utilizing a thermo-responsive polymer, the preparation of a three-dimensionally reconstructed multicellular mass (3-DRMM) with a medium circulating system utilizing cotton-gauze, a concept for organ engineering that can remodel an organ into a reconstructed organ by a continuous three-step perfusion to change the cellular scaffold, and a concept for cellomics study to culture cells on a substratum made of tissue/organ sections for histopathology (TOSHI-substratum) that conserve the microarchitecture and component of the original tissue in vivo. The former two tissue engineering technologies still lacked the method to design 3-DMMs with hierarchical tissue architectures based on cell lineages. To overcome this task, the latter two technologies that can convert tissue architecture in vivo into the three-dimensional culture mode in vitro were innovated.     

几丁聚糖在组织工程中的应用

支架材料作为组织工程的生物学植入替代物,对细胞移植与引导新组织生长有重要的作用.几丁聚糖可制成无毒性、无刺激性、生物相容性和生物可降解性良好的生物医用材料,在人工皮肤、骨修复材料、手术缝线等方面已广泛应用.本文分析了纯几丁聚糖支架结构和它与其他天然或合成材料复合后的支架结构的物理、化学性质及其独特的生物学功能,同时还进一步介绍了其应用的范例并探讨了发展前景.     

Natural origin scaffolds with in situ pore forming capability for bone tissue engineering applications

This work describes the development of a biodegradable matrix, based on chitosan and starch, with the ability to form a porous structure in situ due to the attack by specific enzymes present in the human body (alpha-amylase and lysozyme). Scaffolds with three different compositions were developed: chitosan (C100) and chitosan/starch (CS80-20, CS60-40). Compressive test results showed that these materials exhibit very promising mechanical properties, namely a high modulus in both the dry and wet states. The compressive modulus in the dry state for C100 was 580+/-33MPa, CS80-20 (402+/-62MPa) and CS60-40 (337+/-78MPa). Degradation studies were performed using alpha-amylase and/or lysozyme at concentrations similar to those found in human serum, at 37 degrees C for up to 90 days. Scanning electron micrographs showed that enzymatic degradation caused a porous structure to be formed, indicating the potential of this methodology to obtain in situ forming scaffolds. In order to evaluate the biocompatibility of the scaffolds, extracts and direct contact tests were performed. Results with the MTT test showed that the extracts of the materials were clearly non-toxic to L929 fibroblast cells. Analysis of cell adhesion and morphology of seeded osteoblastic-like cells in direct contact tests showed that at day 7 the number of cells on CS80-20 and CS60-40 was noticeably higher than that on C100, which suggests that starch containing materials may promote cell adhesion and proliferation. This combination of properties seems to be a very promising approach to obtain scaffolds with gradual in vivo pore forming capability for bone tissue engineering applications.     

Biodegradable porous scaffolds for tissue engineering

 Three-dimensional scaffolds play an important role in tissue engineering as an adhesive substrate for implanted cells and a physical support to guide the formation of new organs. The scaffolds should facilitate cell adhesion, promote cell growth, allow the retention of differentiated cell functions, and be biocompatible, biodegradable, highly porous with a large surface-to-volume ratio, mechanically strong, and malleable. A number of biodegradable three-dimensional scaffolds have been developed for tissue engineering. This paper reviews some of the recent events in the development of these scaffolds. Received: March 6, 2002     

Bilayered scaffolds for osteochondral tissue engineering

Osteoarthritis (OA) is a prevalent degenerative joint disease that places a significant burden on the socioeconomic efficacy of communities around the world. Tissue engineering repair of articular cartilage in synovial joints represents a potential OA treatment strategy superior to current surgical techniques. In particular, osteochondral tissue engineering, which promotes the simultaneous regeneration of articular cartilage and underlining subchondral bone, may be a clinically relevant approach toward impeding OA progression. The unique and complex functional demands of the two contrasting tissues that comprise osteochondral tissue require the use of bilayered scaffolds to promote individual growth of both on a single integrated implant. This paper reviews the three current bilayered scaffold strategies applied to solve this challenging problem, with a focus on the need for an innovative approach to design and fabrication of new optimized scaffold combinations to reinforce materials science as an important element of osteochondral tissue engineering.     

牙髓牙本质组织工程生物支架材料的研究进展

背景：组织工程方法中选择合适的支架是关键性的步骤。 目的：回顾分析牙髓牙本质组织工程中支架材料的应用研究。 方法：由第一作者检索1993至2012年 PubMed数据及万方数据库有关牙髓牙本质组织工程中支架材料应用研究等方面的文献。 结果与结论：在牙髓牙本质组织工程中有包括天然生物、人工合成材料和复合材料在内的大量生物材料可供选择,每一种材料都有各自的生物学特点。其中胶原、聚酯、羟基磷灰石等是研究较多的支架材料。自组装多肽水凝胶是由氨基酸制成的新型支架材料,满足理想牙髓牙本质组织工程支架材料的大部分要求,是一种前景广阔的牙髓牙本质组织工程支架材料。     

Injectable poly(lactic-co-glycolic) acid scaffolds with in situ pore formation for tissue engineering

Appropriate porosity is an important biomaterial design criterion for scaffolds used in tissue engineering applications as it can permit increased cell adhesion, migration, proliferation and extracellular matrix production within the scaffold at a tissue defect site. Tissue engineering scaffolds can either be injected in a minimally invasive manner or implanted through surgical procedures. Many injectable scaffolds are hydrogel-based; these materials often possess nanoscale porosity, which is suboptimal for cell migration and proliferation. Solid scaffolds with engineered micron-scale porosity are widely used, but these scaffolds are usually pre-formed and then must be implanted. Here we report on the development of a solid, injectable, biomaterial scaffold that solidifies in situ via phase inversion with microporous, interconnected architecture on the surface and within the bulk. This injectable system utilizes the biodegradable polymer poly(lactic-co-glycolic acid), a nontoxic FDA-approved solvent, and biocompatible porogens. Various scaffold formulations are examined in terms of morphology, porosity, degradation, elastic modulus, and ability to support cellular adhesion and growth. Furthermore, the ability to form a microporous architecture upon injection in vivo is verified. This technology is a promising noninvasive approach for in vivo formation of porous biodegradable scaffolds.     

Chitosan-alginate hybrid scaffolds for bone tissue engineering

A biodegradable scaffold in tissue engineering serves as a temporary skeleton to accommodate and stimulate new tissue growth. Here we report on the development of a biodegradable porous scaffold made from naturally derived chitosan and alginate polymers with significantly improved mechanical and biological properties as compared to its chitosan counterpart. Enhanced mechanical properties were attributable to the formation of a complex structure of chitosan and alginate. Bone-forming osteoblasts readily attached to the chitosan-alginate scaffold, proliferated well, and deposited calcified matrix. The in vivo study showed that the hybrid scaffold had a high degree of tissue compatibility. Calcium deposition occurred as early as the fourth week after implantation. The chitosan-alginate scaffold can be prepared from solutions of physiological pH, which may provide a favorable environment for incorporating proteins with less risk of denaturation. Coacervation of chitosan and alginate combined with liquid-solid separation provides a scaffold with high porosity, and mechanical and biological properties suitable for rapid advancement into clinical trials.     

Biodegradable polymeric scaffolds for musculoskeletal tissue engineering

Biodegradable scaffolds have played an important role in a number of tissue engineering attempts over the past decade. The goal of this review article is to provide a brief overview of some of the important issues related to scaffolds fabricated from synthetic biodegradable polymers. Various types of such materials are available; some are commercialized and others are still in the laboratories. The properties of the most common of these polymers are discussed here. A variety of fabrication techniques were developed to fashion polymeric materials into porous scaffolds, and a selection of these is presented. The very important issue of scaffold architecture, including the topic of porosity and permeability, is discussed. Other areas such as cell growth on scaffolds, surface modification, scaffold mechanics, and the release of growths factors are also reviewed. A summary outlining the common themes in scaffold-related science that are found in the literature is presented.     

Jellyfish collagen scaffolds for cartilage tissue engineering

Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans.     

Multilayered silk scaffolds for meniscus tissue engineering

Removal of injured/damaged meniscus, a vital fibrocartilaginous load-bearing tissue, impairs normal knee function and predisposes patients to osteoarthritis. Meniscus tissue engineering solution is one option to improve outcomes and relieve pain. In an attempt to fabricate knee meniscus grafts three layered wedge shaped silk meniscal scaffold system was engineered to mimic native meniscus architecture. The scaffolds were seeded with human fibroblasts (outside) and chondrocytes (inside) in a spatial separated mode similar to native tissue, in order to generate meniscus-like tissue in vitro. In chondrogenic culture in the presence of TGF-b3, cell-seeded constructs increased in cellularity and extracellular matrix (ECM) content. Histology and Immunohistochemistry confirmed maintenance of chondrocytic phenotype with higher levels of sulfated glycosaminoglycans (sGAG) and collagen types I and II. Improved scaffold mechanical properties along with ECM alignment with time in culture suggest this multiporous silk construct as a useful micro-patterned template for directed tissue growth with respect to form and function of meniscus-like tissue.     

Biomimetic nanofibrous scaffolds for bone tissue engineering

Bone tissue engineering is a highly interdisciplinary field that seeks to tackle the most challenging bone-related clinical issues. The major components of bone tissue engineering are the scaffold, cells, and growth factors. This review will focus on the scaffold and recent advancements in developing scaffolds that can mimic the natural extracellular matrix of bone. Specifically, these novel scaffolds mirror the nanofibrous collagen network that comprises the majority of the non-mineral portion of bone matrix. Using two main fabrication techniques, electrospinning and thermally-induced phase separation, and incorporating bone-like minerals, such as hydroxyapatite, composite nanofibrous scaffolds can improve cell adhesion, stem cell differentiation, and tissue formation. This review will cover the two main processing techniques and how they are being applied to fabricate scaffolds for bone tissue engineering. It will then cover how these scaffolds can enhance the osteogenic capabilities of a variety of cell types and survey the ability of the constructs to support the growth of clinically relevant bone tissue.     

Electrospun nanostructured scaffolds for bone tissue engineering

The current challenge in bone tissue engineering is to fabricate a bioartificial bone graft mimicking the extracellular matrix (ECM) with effective bone mineralization, resulting in the regeneration of fractured or diseased bones. Biocomposite polymeric nanofibers containing nanohydroxyapatite (HA) fabricated by electrospinning could be promising scaffolds for bone tissue engineering. Nanofibrous scaffolds of poly-l-lactide (PLLA, 860 ± 110 nm), PLLA/HA (845 ± 140 nm) and PLLA/collagen/HA (310 ± 125 nm) were fabricated, and the morphology, chemical and mechanical characterization of the nanofibers were evaluated using scanning electron microscopy, Fourier transform infrared spectroscopy and tensile testing, respectively. The in vitro biocompatibility of different nanofibrous scaffolds was also assessed by growing human fetal osteoblasts (hFOB), and investigating the proliferation, alkaline phosphatase activity (ALP) and mineralization of cells on different nanofibrous scaffolds. Osteoblasts were found to adhere and grow actively on PLLA/collagen/HA nanofibers with enhanced mineral deposition of 57% higher than the PLLA/HA nanofibers. The synergistic effect of the presence of an ECM protein, collagen and HA in PLLA/collagen/HA nanofibers provided cell recognition sites together with apatite for cell proliferation and osteoconduction necessary for mineralization and bone formation. The results of our study showed that the biocomposite PLLA/collagen/HA nanofibrous scaffold could be a potential substrate for the proliferation and mineralization of osteoblasts, enhancing bone regeneration.     

Biomaterials and scaffolds for ligament tissue engineering

Tissue engineering has achieved much progress in an attempt to improve and recover impaired functions of tissues and organs. Although many studies have been done, progress for tissue-engineered anterior cruciate ligaments (ACLs) has been slow due to their complex structures and mechanical properties. In this review, the ACL anatomical structure, progresses achieved, material selection, structure design, and future direction have been discussed, while the challenges and requirements from materials and scaffolds are highlighted. There is a considerably huge amount work that needs to be carried out; as such, future direction in ligament tissue engineering is proposed in hope that this review will give information on future ligament tissue engineering.     

聚乙烯醇-壳聚糖-胶原组织工程支架的研究

目的 以聚乙烯醇（PVA）、壳聚糖（Cs）和胶原（Col）为主要原料制备PVA-Cs—Col复合支架,并研究其作为组织工程支架材料的可行性。方法 把聚乙烯醇、壳聚糖和胶原按一定配比复合,测定复合材料的含水率、膨胀率和力学性能,扫描电镜观察材料横截面的组织形态。结果 不同分子量PVA与不同质量的cs和Col复合,得到的复合支架材料湿态抗张强度为5．70MPa,含水率在60．15％-72．50％,膨胀率在185．33％～317．57％。不同配比的复合支架具有不同的内部组织形态结构。结论 PVA—Cs—Col复合支架材料具有较高的含水率和适宜的膨胀率,内部孔洞丰富,Cs：PVA：Col的质量比为30：15：0．20时,复合支架综合性能较佳,适合用于组织工程支架材料。     

Porous chitosan scaffolds for tissue engineering.

The wide array of tissue engineering applications exacerbates the need for biodegradable materials with broad potential. Chitosan, the partially deacetylated derivative of chitin, may be one such material. In this study, we examined the use of chitosan for formation of porous scaffolds of controlled microstructure in several tissue-relevant geometries. Porous chitosan materials were prepared by controlled freezing and lyophilization of chitosan solutions and gels. The materials were characterized via light and scanning electron microscopy as well as tensile testing. The scaffolds formed included porous membranes, blocks, tubes and beads. Mean pore diameters could be controlled within the range 1-250 microm, by varying the freezing conditions. Freshly lyophilized chitosan scaffolds could be treated with glycosaminoglycans to form ionic complex materials which retained the original pore structure. Chitosan scaffolds could be rehydrated via an ethanol series to avoid the stiffening caused by rehydration in basic solutions. Hydrated porous chitosan membranes were at least twice as extensible as non-porous chitosan membranes, but their elastic moduli and tensile strengths were about tenfold lower than non-porous controls. The methods and structures described here provide a starting point for the design and fabrication of a family of polysaccharide based scaffold materials with potentially broad applicability.     

Autologous extracellular matrix scaffolds for tissue engineering

Development of autologous scaffolds has been highly desired for implantation without eliciting adverse inflammatory and immune responses. However, it has been difficult to obtain autologous scaffolds by tissue decellularization because of the restricted availability of autologous donor tissues from a patient. Here we report a method to prepare autologous extracellular matrix (aECM) scaffolds by combining culture of autologous cells in a three-dimensional template, decellularization, and template removal. The aECM scaffolds showed excellent biocompatibility when implanted. We anticipate that "Full Autologous Tissue Engineering" can be realized to minimize undesirable host tissue responses by culturing the patient's own cells in an aECM scaffold.     

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.