脱细胞猪主动脉瓣叶的生物力学特性和免疫反应性

目的：探讨脱细胞猪主动脉瓣叶构建组织工程心脏瓣膜支架的可行性。方法：经胰酶-EDTA、表面活性剂、核酸酶处理,去除瓣叶的细胞成分,测定脱细胞瓣叶的生物力学特性,同时行大鼠皮下包埋实验,观察其免疫反应性。结果：瓣叶中的细胞成分能完全去除,获得无细胞的纤维网状支架。脱细胞瓣叶与新鲜瓣叶有基本相同的应力-应变曲线及应力-松弛曲线,而弹性模量、面积比、松弛强度、断裂强度和断裂伸长率两者无显著差异。脱细胞瓣叶的免疫反应性明显降低。结论：猪主动脉瓣叶经脱细胞处理后可以作为组织工程心脏瓣膜的支架材料。     

The susceptibility of bioprosthetic heart valve leaflets to oxidation

The clinical use of bioprosthetic heart valves (BHV) is limited due to device failure caused by structural degeneration of BHV leaflets. In this study we investigated the hypothesis that oxidative stress contributes to this process. Fifteen clinical BHV that had been removed for device failure were analyzed for oxidized amino acids using mass spectrometry. Significantly increased levels of ortho-tyrosine, meta-tyrosine and dityrosine were present in clinical BHV explants as compared to the non-implanted BHV material glutaraldehyde treated bovine pericardium (BP). BP was exposed in vitro to oxidizing conditions (FeSO₄/H₂O₂) to assess the effects of oxidation on structural degeneration. Exposure to oxidizing conditions resulted in significant collagen deterioration, loss of glutaraldehyde cross-links, and increased susceptibility to collagenase degradation. BP modified through covalent attachment of the oxidant scavenger 3-(4-hydroxy-3,5-di-tert-butylphenyl) propyl amine (DBP) was resistant to all of the monitored parameters of structural damage induced by oxidation. These results indicate that oxidative stress, particularly via hydroxyl radical and tyrosyl radical mediated pathways, may be involved in the structural degeneration of BHV, and that this mechanism may be attenuated through local delivery of antioxidants such as DBP.     

组织工程心脏瓣膜及干细胞应用前景

组织工程心脏瓣膜是一种具有活力、能够自我修复和增生的人工瓣膜。理想的组织工程心脏瓣膜由于具有优良的血流动力学特征,低或甚至无免疫反应,不需要长期抗凝治疗以及耐用性好等特性,能够很好地克服目前临床上使用的机械瓣和生物瓣的缺点。综述了近年来国外组织工程心脏瓣膜在生物材料、培养环境和种子细胞等方面的新进展,并展望了干细胞作为种子细胞的应用前景。     

Electrospun scaffolds for tissue engineering of vascular grafts

There is a growing demand for off-the-shelf tissue engineered vascular grafts (TEVGs) for the replacement or bypass of damaged arteries in various cardiovascular diseases. Scaffolds from the decellularized tissue skeletons to biopolymers and biodegradable synthetic polymers have been used for fabricating TEVGs. However, several issues have not yet been resolved, which include the inability to mimic the mechanical properties of native tissues, and the ability for long-term patency and growth required for in vivo function. Electrospinning is a popular technique for the production of scaffolds that has the potential to address these issues. However, its application to human TEVGs has not yet been achieved. This review provides an overview of tubular scaffolds that have been prepared by electrospinning with potential for TEVG applications.     

Preparation and characterization of gelatin-bioactive glass ceramic scaffolds for bone tissue engineering

Macroporous composite scaffolds comprising of gelatin and glass ceramic has been fabricated and characterized for bone tissue engineering applications. Gelatin scaffold with varying glass-ceramic content was fabricated using lyophilization technique. The microstructure, compressive strength, bioactivity, biodegradation and biocompatibility of the fabricated scaffolds were evaluated. The scaffolds presented macroporous pore size with porosity varying from 79 to 84%. The compressive strength was enhanced by glass ceramic addition and the scaffolds exhibited strength in the range of 1.9 to 5.7?MPa. The obtained strength and porosity was in the range of cancellous bone. The dissolution of gelatin scaffolds was optimized by an additional in situ glutaraldehyde crosslinking step and further by glass-ceramic addition. The composite scaffolds showed good apatite-forming ability in vitro. Biocompatibility and osteogenic ability of the scaffolds were analyzed in vitro by cell adhesion study, alkaline phosphatase activity and Alizarin S staining. The obtained results revealed the composite scaffolds possessed enhanced osteogenic ability and good cell adhesion properties. The developed scaffold is a prospective candidate as a biomaterial for bone tissue engineering.     

Mechanical and structural response of a hybrid hydrogel based on chitosan and poly(vinyl alcohol) cross-linked with epichlorohydrin for potential use in tissue engineering

The development and characterization of a hybrid hydrogel based on chitosan (CS) and poly(vinyl alcohol) (PVA) chemically cross-linked with epichlorohydrin (ECH) is presented. The mechanical response of these hydrogels was evaluated by uniaxial tensile tests; in addition, their structural properties such as average molecular weight between cross-link points (M_crl), mesh size (D_N), and volume fraction (v_s) were determined. This was done using the equivalent polymer network theory in combination with the obtained results from tensile and swelling tests. The films showed Young’s modulus values of 11?±?2?MPa and 9?±?1?MPa for none irradiated and ultraviolet (UV) irradiated hydrogels, respectively. The cell viability was assessed using Calcein AM and Ethidium homodimer-1 assay and environmental scanning electron microscopy. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan thiazolyl blue formazan (MTT Formazan assay) results did not show cytotoxic effects; this was in good agreement with nuclear magnetic resonance and fourier transform infrared spectroscopies; their results did not show traces of ECH. This indicated that after the crosslinking process, there was no free ECH; furthermore, any possibility of ECH release in the construct during cell culture was discarded. The CS-PVA-ECH hybrid hydrogel allowed cell growth and extracellular matrix formation and showed adequate mechanical, structural, and biological properties for potential use in tissue engineering applications.     

Mechanical enhancement and in vitro biocompatibility of nanofibrous collagen-chitosan scaffolds for tissue engineering

The collagen–chitosan complex with a three-dimensional nanofiber structure was fabricated to mimic native ECM for tissue repair and biomedical applications. Though the three-dimensional hierarchical fibrous structures of collagen–chitosan composites could provide more adequate stimulus to facilitate cell adhesion, migrate and proliferation, and thus have the potential as tissue engineering scaffolding, there are still limitations in their applications due to the insufficient mechanical properties of natural materials. Because poly (vinyl alcohol) (PVA) and thermoplastic polyurethane (TPU) as biocompatible synthetic polymers can offer excellent mechanical properties, they were introduced into the collagen–chitosan composites to fabricate the mixed collagen/chitosan/PVA fibers and a sandwich structure (collagen/chitosan-TPU-collagen/chitosan) of nanofiber in order to enhance the mechanical properties of the nanofibrous collagen–chitosan scaffold. The results showed that the tensile behavior of materials was enhanced to different degrees with the difference of collagen content in the fibers. Besides the Young’s modulus had no obvious changes, both the break strength and the break elongation of materials were heightened after reinforced by PVA. For the collagen–chitosan nanofiber reinforced by TPU, both the break strength and the Young’s modulus of materials were heightened in different degrees with the variety of collagen content in the fibers despite the decrease of the break elongation of materials to some extent. In vitro cell test demonstrated that the materials could provide adequate environment for cell adhesion and proliferation. All these indicated that the reinforced collagen–chitosan nanofiber could be as potential scaffold for tissue engineering according to the different mechanical requirements in clinic.     

Strong and tough mineralized PLGA nanofibers for tendon-to-bone scaffolds

Engineering complex tissues such as the tendon-to-bone insertion sites require a strong and tough biomimetic material system that incorporates both mineralized and unmineralized tissues with different strengths and stiffnesses. However, increasing strength without degrading toughness is a fundamental challenge in materials science. Here, we demonstrate a promising nanofibrous polymer–hydroxyapatite system, in which, a continuous fibrous network must function as a scaffold for both mineralized and unmineralized tissues. It is shown that the high toughness of this material system could be maintained without compromising on the strength with the addition of hydroxyapatite mineral. Individual electrospun poly (lactide-co-glycolide) (PLGA) nanofibers demonstrated outstanding strain-hardening behavior and ductility when stretched uniaxially, even in the presence of surface mineralization. This highly desirable hardening behavior which results in simultaneous nanofiber strengthening and toughening was shown to depend on the initial cross-sectional morphology of the PLGA nanofibers. For pristine PLGA nanofibers, it was shown that ellipsoidal cross-sections provide the largest increase in fiber strength by almost 200% compared to bulk PLGA. This exceptional strength accompanied by 100% elongation was shown to be retained for thin and strongly bonded conformal mineral coatings, which were preserved on the nanofiber surface even for such very large extensions.     

Tissue engineering for myocardial regeneration

 Recent progress in stem cell biology has shown the possibility of implantable human myocardial cell sources. It has encouraged myocardial tissue engineering for rescuing damaged hearts. The present strategy is to repair not all of the myocardial tissue, but part of it. There are two approaches. The first is direct injection of dissociated cell suspensions via the pericardium, coronary arteries, or endocardium. Studies using animal models have found improved heart function after transplantation of various types of cells. Myoblasts or bone marrow cells have already been transplanted into patients suffering from severe ischemic heart disease. In direct transplantation of dissociated cells, it is difficult to control the shape, size, and location of the grafts. To solve these problems, further therapies to transplant tissue-engineered three-dimensional (3-D) heart grafts have been investigated. The most popular technique in tissue engineering is to use 3-D biodegradable scaffolds as alternatives to the extracellular matrix. On the basis of this concept, poly(glycolic acid)(PGA), gelatin, alginate, and collagen have been used as scaffolds to fabricate 3-D heart tissues. A new method consisting of layering cell sheets to construct 3-D tissues without any artificial scaffolds has also been applied to myocardial tissue engineering. Electrically communicative pulsatile heart tissues have been achieved both in vitro and in vivo by layering cardiomyocyte sheets. Although myocardial tissue engineering has rapidly progressed, there are several problems to be solved with regard to the source of myocardial cells, tissue reconstruction, neovascularization, and transplantation technology. Further interdisciplinary research will solve these problems, and transplantation of cells or engineered heart tissues will become one of the major treatments for severe heart failure in the near future. Received: October 1, 2002 Correspondence to:T. Okano     

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     

组织工程心脏瓣膜细胞生物学研究进展

由于现有的机械瓣和生物瓣仍存在种种不足，如不具备生长性、需抗凝、易感染、不能生长和自我修复等。组织工程心脏瓣膜是一新兴的研究领域，涉及多门学科。构建组织工程心脏瓣膜应包括支架的制作、细胞的种植、瓣膜的体外培养和最终移植人人体。其中种植的细胞是组织工程心脏瓣膜的基本要素。就组织工程心脏瓣膜的细胞生物学研究进展做一综述。     

低渗冻融联合生物酶法制备组织工程化静脉瓣脱细胞支架

目的:采用低渗冻融联合生物酶法制备组织工程化静脉瓣脱细胞支架,并与先前方法制备的静脉瓣脱细胞支架比较,以期获得一种性能较好的带瓣静脉脱细胞支架材料.方法:手术获得Beagle犬带瓣静脉分为4组,对照组、脱氧胆酸钠组、Triton组和冻融+生物酶组.处理后对各组标本行H-E染色,透射电镜观察,DAPI荧光染色,细胞相容性测试等观察.结果:3种脱细胞方法均能彻底去除细胞,DAPI荧光检测显示各组支架材料均无DNA成分残留;H-E染色及透射电镜显示,冻融+生物酶组胶原纤维排列整齐,未见明显的胶原纤维结构改变,其他2组可见胶原纤维断裂及结构紊乱现象;与其他2组脱细胞支架材料相比,冻融+生物酶组的组织细胞相容性好,可见内皮祖细胞在支架材料上生长良好.结论:结合渗透压改变的反复冻融加生物酶法,既可以较彻底除去带瓣膜静脉细胞成分,又保留了较完整的细胞外基质结构,具有良好的组织和细胞相容性,是较理想的带瓣膜静脉脱细胞支架制备方法.     

组织工程心脏瓣膜细胞生物学研究进展

由于现有的机械瓣和生物瓣仍存在种种不足,如不具备生长性、需抗凝、易感染、不能生长和自我修复等。组织工程心脏瓣膜是一新兴的研究领域,涉及多门学科。构建组织工程心脏瓣膜应包括支架的制作、细胞的种植、瓣膜的体外培养和最终移植入人体。其中种植的细胞是组织工程心脏瓣膜的基本要素。就组织工程心脏瓣膜的细胞生物学研究进展做一综述。     

Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering

Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.     

Mechanical heart valve prostheses:: identification and evaluation (erratum)

Mechanical heart value prostheses have been in use since the 1950s. Many prostheses have been used for a while and then discontinued. Today, there are a large number and variety of prostheses in use and an even larger variety that are in place in patients. These may be explanted at any time for a number of reasons. It is essential for the practicing pathologist to be able to identify the prosthesis and be aware of some of its reported complications and modes of failure. This article, and a second one on bioprosthetic heart valves, is designed as a ready reference guide to heart valve prostheses, their important identifying features, their common complications, and modes of failure. It should help in the accurate identification of explanted prosthetic valves and more definitive reports. This accuracy of identification as well as tracking of abnormalities noted will, we hope, permit the identification of new failure modes and the recording of causes of failure of new (or even modified) prosthetic heart valves.     

The potential of anisotropic matrices as substrate for heart valve engineering

Cells environment is increasingly recognized as an important function regulator through cell–matrix interactions. Extracellular matrix (ECM) anisotropy being a key component of heart valves properties, we have devised a method to create highly porous anisotropic nanofibrillar scaffolds and studied their suitability as cell-support and interactions with human adipose derived stem cells (hADSCs) and human valve interstitial cells (hVICs). Anisotropic nanofibrillar scaffolds were produced by a modified jet-spraying method that allows the formation of aligned nanofibres (600 nm) through air-stream diffraction of a polymer solution (poly (ε-caprolactone, PCL) and collection onto a variably rotating drum. The resulting matrices of high porosity (99%) mimicked valve mechanical anisotropy. Dynamically seeded hADSC and hVIC cultured on scaffolds up to 20 days revealed that hADSC and hVIC penetration within the matrices was improved by anisotropic organization. Within 10 days, cells populated the entire scaffolds thickness and produced ECM (collagen I, III and elastin). As a result, mechanical properties of the constructs were improved over culture, while remaining anisotropic. In contrast to isotropic matrices, anisotropy induced elongated hADSCs and hVICs morphology that followed nanofibres orientation. Interestingly, these morphological changes did not induce hADSC differentiation towards the mesoderm lineages while hVIC recovered a physiological phenotype over culture in the biomimetic matrices. Overall, this study indicates that highly porous anisotropic jet-sprayed matrices are interesting candidates for valve tissue engineering, through anisotropic mechanical properties, efficient cell population, conservation of stem cells phenotype and recovery of hVIC physiological phenotype.     

Textile-templated electrospun anisotropic scaffolds for regenerative cardiac tissue engineering

For patients with end-stage heart disease, the access to heart transplantation is limited due to the shortage of donor organs and to the potential for rejection of the donated organ. Therefore, current studies focus on bioengineering approaches for creating biomimetic cardiac patches that will assist in restoring cardiac function, by repairing and/or regenerating the intrinsically anisotropic myocardium. In this paper we present a simplified, straightforward approach for creating bioactive anisotropic cardiac patches, based on a combination of bioengineering and textile-manufacturing techniques in concert with nano-biotechnology based tissue-engineering stratagems. Using knitted conventional textiles, made of cotton or polyester yarns as template targets, we successfully electrospun anisotropic three-dimensional scaffolds from poly(lactic-co-glycolic) acid (PLGA), and thermoplastic polycarbonate-urethane (PCU, Bionate^®). The surface topography and mechanical properties of textile-templated anisotropic scaffolds significantly differed from those of scaffolds electrospun from the same materials onto conventional 2-D flat-target electrospun scaffolds. Anisotropic textile-templated scaffolds electrospun from both PLGA and PCU, supported the adhesion and proliferation of H9C2 cardiac myoblasts cell line, and guided the cardiac tissue-like anisotropic organization of these cells in vitro. All cell-seeded PCU scaffolds exhibited mechanical properties comparable to those of a human heart, but only the cells on the polyester-templated scaffolds exhibited prolonged spontaneous synchronous contractility on the entire engineered construct for 10 days in vitro at a near physiologic frequency of ∼120 bpm. Taken together, the methods described here take advantage of straightforward established textile manufacturing strategies as an efficient and cost-effective approach to engineering 3D anisotropic, elastomeric PCU scaffolds that can serve as a cardiac patch.     

Heart Valve Tissue Engineering: Concepts, Approaches, Progress, and Challenges

Potential applications of tissue engineering in regenerative medicine range from structural tissues to organs with complex function. This review focuses on the engineering of heart valve tissue, a goal which involves a unique combination of biological, engineering, and technological hurdles. We emphasize basic concepts, approaches and methods, progress made, and remaining challenges. To provide a framework for understanding the enabling scientific principles, we first examine the elements and features of normal heart valve functional structure, biomechanics, development, maturation, remodeling, and response to injury. Following a discussion of the fundamental principles of tissue engineering applicable to heart valves, we examine three approaches to achieving the goal of an engineered tissue heart valve: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Lastly, we analyze challenges to the field and suggest future directions for both preclinical and translational (clinical) studies that will be needed to address key regulatory issues for safety and efficacy of the application of tissue engineering and regenerative approaches to heart valves. Although modest progress has been made toward the goal of a clinically useful tissue engineered heart valve, further success and ultimate human benefit will be dependent upon advances in biodegradable polymers and other scaffolds, cellular manipulation, strategies for rebuilding the extracellular matrix, and techniques to characterize and potentially non-invasively assess the speed and quality of tissue healing and remodeling.     

Biosynthetic activity in heart valve leaflets in response to in vitro flow environments

The development of bioreactors for tissue engineered heart valves would be aided by a thorough understanding of how mechanical forces impact cells within valve leaflets. The hypothesis of the present study is that flow may influence the biosynthetic activity of aortic valve leaflet cells. Porcine leaflets were exposed to one of several conditions for 48 h, including steady or pulsatile flow in a tubular flow system at 10 or 20 l/min, and steady shear stress in a parallel plate flow system at 1, 6, or 22 dyne/cm². Protein, glycosaminoglycan, and DNA synthesis increased during static incubation but remained at basal levels after exposure to flow. The modulation of synthetic activity was attributed to the presence of a shear stress on the leaflet surface, which may be transmitted to cells within the leaflet matrix through tensile forces. The -smooth muscle (-SM) actin distribution observed in fresh leaflets was proportionately decreased after exposure to antibiotics and not recovered by either static incubation or exposure to flow. These results indicate that exposure to flow maintains leaflet synthetic activity near normal levels, but that the inclusion of another force, such as bending or backpressure, may be necessary to preserve -SM actin immunoreactive cells. © 2001 Biomedical Engineering Society. PAC01: 8780Rb, 8719Hh, 8719Uv, 8715Rn, 8768+z, 8719Ff, 8714Gg, 8714Ee     

组织工程心脏瓣膜研究进展

目前组织工程心脏瓣膜研究已在支架的选材、种子细胞的选择、种子细胞的种植与瓣膜构建方法三个方面取得进展,并已构建出三种代表性组织工程心脏瓣膜。对它们各自的特点进行综述。