Electrospun nanofibrous 3D scaffold for bone tissue engineering

Tissue engineering aims at developing functional substitutes for damaged tissues by mimicking natural tissues. In particular, tissue engineering for bone regeneration enables healing of some bone diseases. Thus, several methods have been developed in order to produce implantable biomaterial structures that imitate the constitution of bone. Electrospinning is one of these methods. This technique produces nonwoven scaffolds made of nanofibers which size and organization match those of the extracellular matrix. Until now, seldom electrospun scaffolds were produced with thickness exceeding one millimeter. This article introduces a new kind of electrospun membrane called 3D scaffold of thickness easily exceeding one centimeter. The manufacturing involves a solution of poly(ε-caprolactone) in DMF/DCM system. The aim is to establish parameters for electrospinning in order to characterize these 3D scaffolds and, establish whether such scaffolds are potentially interesting for bone regeneration.     

Orthopaedic tissue engineering and bone regeneration.

Orthopaedic tissue engineering combines the application of scaffold materials, cells and the release of growth factors. It has been described as the science of persuading the body to reconstitute or repair tissues that have failed to regenerate or heal spontaneously. In the case of bone regeneration 3-D scaffolds are used as a framework to guide tissue regeneration. Mesenchymal cells obtained from the patient via biopsy are grown on biomaterials in vitro and then implanted at a desired site in the patient's body. Medical implants that encourage natural tissue regeneration are generally considered more desirable than metallic implants that may need to be removed by subsequent intervention. Numerous polymeric materials, from natural and artificial sources, are under investigation as substitutes for skeletal elements such as cartilage and bone. For bone regeneration, cells (obtained mainly from bone marrow aspirate or as primary cell outgrowths from bone biopsies) can be combined with biodegradable polymeric materials and/or ceramics and absorbed growth factors so that osteoinduction is facilitated together with osteoconduction; through the creation of bioactive rather than bioinert scaffold constructs. Relatively rapid biodegradation enables advantageous filling with natural tissue while loss of polymer strength before mass is disadvantageous. Innovative solutions are required to address this and other issues such as the biocompatibility of material surfaces and the use of appropriate scaffold topography and porosity to influence bone cell gene expression.     

Fabrication and characterization of chitosan/OGP coated porous poly(ε-caprolactone) scaffold for bone tissue engineering

As one of the stimulators on bone formation, osteogenic growth peptide (OGP) improves both proliferation and differentiation of the bone cells in vitro and in vivo. The aim of this work was the preparation of three dimensional porous poly(ε-caprolactone) (PCL) scaffold with high porosity, well interpore connectivity, and then its surface was modified by using chitosan (CS)/OGP coating for application in bone regeneration. In present study, the properties of porous PCL and CS/OGP coated PCL scaffold, including the microstructure, water absorption, porosity, hydrophilicity, mechanical properties, and biocompatibility in vitro were investigated. Results showed that the PCL and CS/OGP-PCL scaffold with an interconnected network structure have a porosity of more than 91.5, 80.8%, respectively. The CS/OGP-PCL scaffold exhibited better hydrophilicity and mechanical properties than that of uncoated PCL scaffold. Moreover, the results of cell culture test showed that CS/OGP coating could stimulate the proliferation and growth of osteoblast cells on CS/OGP-PCL scaffold. These finding suggested that the surface modification could be a effective method on enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering application and the developed porous CS/OGP-PCL scaffold should be considered as alternative biomaterials for bone regeneration.     

Biomaterials for bone regeneration: an orthopedic and dentistry overview

Because bone-associated diseases are increasing, a variety of tissue engineering approaches with bone regeneration purposes have been proposed over the last years. Bone tissue provides a number of important physiological and structural functions in the human body, being essential for hematopoietic maintenance and for providing support and protection of vital organs. Therefore, efforts to develop the ideal scaffold which is able to guide the bone regeneration processes is a relevant target for tissue engineering researchers. Several techniques have been used for scaffolding approaches, such as diverse types of biomaterials. On the other hand, metallic biomaterials are widely used as support devices in dentistry and orthopedics, constituting an important complement for the scaffolds. Hence, the aim of this review is to provide an overview of the degradable biomaterials and metal biomaterials proposed for bone regeneration in the orthopedic and dentistry fields in the last years.     

Diffusion in Musculoskeletal Tissue Engineering Scaffolds: Design Issues Related to Porosity,Permeability, Architecture,and Nutrient Mixing

The field of tissue engineering continues to advance with the discovery of new biomaterials, growth factors and scaffold fabrication techniques. However, for the ultimate success of a tissue engineered construct the issue of nutrient transport to the scaffold interior needs to be addressed. Often, the requirements for adequate nutrient supply are at odds with other scaffold design parameters such as mechanical properties as well as scaffold fabrication techniques, leading to incongruities in finding optimal solutions. The goal of this review article is to provide an overview of the various engineering design factors that promote movement of nutrients, waste and other biomolecules in scaffolds for musculoskeletal tissue engineering applications. The importance of diffusion in scaffolds and how it is influenced by porosity, permeability, architecture, and nutrient mixing has been emphasized. Methods for measuring porosity and permeability have also been outlined. The different types of biomaterials used, scaffold fabrication techniques implemented and the pore sizes/porosities obtained over the past 5 years have also been addressed.     

不同生物材料复合支架对关节软骨缺损的修复评价

背景：不同生物材料制备的复合软骨支架其修复软骨缺损也各具特点。 目的：探讨不同生物材料制备复合支架的组织工程学特性及其修复关节软骨缺损的性能评价。 方法：以“软骨组织工程,生物材料,工程软骨,复合支架”为中文关键词,以“tissue enginneering,articular cartilage,scaffold material”为英文关键词,采用计算机检索中国期刊全文数据库、PubMed数据库(1993-01/2010-11)相关文章。纳入复合支架材料-细胞复合物修复关节软骨损伤等相关的文章,排除重复研究或Meta分析类文章。 结果与结论：复合支架是当前软骨组织工程中应用较多的支架,它是将具有互补特征的生物相容性可降解支架,按一定比例和方式组合,设计出结构与性能优化的复合支架。较单一支架材料具有更好的生物相容性和一定强度的韧性,较好的孔隙和机械强度。复合支架的制备不仅包括同一类生物材料的复合,还包括不同类别生物材料之间的交叉复合。可分为纯天然支架材料、纯人工支架材料以及天然与人工支架材料的复合等3类。复合支架使生物材料具有互补特性,一定程度上满足了理想生物材料支架应具的综合特点,但目前很多研究仍处于实验阶段,还有一些问题有待于解决,如不同材料的复合比例、复合工艺等。     

Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering

Biodegradable polymers and bioactive ceramics are being combined in a variety of composite materials for tissue engineering scaffolds. Materials and fabrication routes for three-dimensional (3D) scaffolds with interconnected high porosities suitable for bone tissue engineering are reviewed. Different polymer and ceramic compositions applied and their impact on biodegradability and bioactivity of the scaffolds are discussed, including in vitro and in vivo assessments. The mechanical properties of today's available porous scaffolds are analyzed in detail, revealing insufficient elastic stiffness and compressive strength compared to human bone. Further challenges in scaffold fabrication for tissue engineering such as biomolecules incorporation, surface functionalization and 3D scaffold characterization are discussed, giving possible solution strategies. Stem cell incorporation into scaffolds as a future trend is addressed shortly, highlighting the immense potential for creating next-generation synthetic/living composite biomaterials that feature high adaptiveness to the biological environment.     

Self-assembling short oligopeptides and the promotion of angiogenesis

Because an adequate blood supply to and within tissues is an essential factor for successful tissue regeneration, promoting a functional microvasculature is a crucial factor for biomaterials. In this study, we demonstrate that short self-assembling peptides form scaffolds that provide an angiogenic environment promoting long-term cell survival and capillary-like network formation in three-dimensional cultures of human microvascular endothelial cells. Our data show that, in contrast to collagen type I, the peptide scaffold inhibits endothelial cell apoptosis in the absence of added angiogenic factors, accompanied by enhanced gene expression of the angiogenic factor VEGF. In addition, our results suggest that the process of capillary-like network formation and the size and spatial organization of cell networks may be controlled through manipulation of the scaffold properties, with a more rigid scaffold promoting extended structures with a larger inter-structure distance, as compared with more dense structures of smaller size observed in a more compliant scaffold. These findings indicate that self-assembling peptide scaffolds have potential for engineering vascularized tissues with control over angiogenic processes. Since these peptides can be modified in many ways, they may be uniquely valuable in regeneration of vascularized tissues.     

Application of microstereolithography in the development of three-dimensional cartilage regeneration scaffolds

Conventional methods for fabricating three-dimensional (3-D) tissue engineering scaffolds have substantial limitations. In this paper, we present a method for applying microstereolithography in the construction of 3-D cartilage scaffolds. The system provides the ability to fabricate scaffolds having a pre-designed internal structure, such as pore size and porosity, by stacking photopolymerized materials. To control scaffold structure, CAD/CAM technology was used to generate a scaffold pattern algorithm. Since tissue scaffolds must be constructed using a biocompatible, biodegradable material, scaffolds were synthesized using liquid photocurable TMC/TMP, followed by acrylation at the terminal ends, and photocured under UV light irradiation. The solidification properties of the TMC/TMP polymer were also assessed. To assess scaffold functionality, chondrocytes were seeded on two types of 3-D scaffold and characterized for cell adhesion. Results indicate that scaffold geometry plays a critical role in chondrocyte adhesion, ultimately affecting the tissue regeneration utility of the scaffolds. These 3-D scaffolds could eventually lead to optimally designed constructs for the regeneration of various tissues, such as cartilage and bone.     

制备软骨组织工程支架的方法

背景：软骨组织工程支架作为软骨细胞外基质的替代物,其外形和孔结构对实现其作用和功能具有非常重要的意义。 目的：回顾目前若干种常用软骨组织工程中三维多孔支架的制备方法。 方法：由第一作者检索2000至2013年PubMed数据库,ELSEVIER SCIENCEDIRECT、万方数据库、中国知网数据库。英文检索词为“Cartilage tissue engineering;scaffolds;fabrication”,中文检索词为“软骨组织工程;制备方法;支架材料;多孔支架”。 结果与结论：制备软骨组织工程支架的方法有相分离/冷冻干燥法、水凝胶技术、快速成型技术、静电纺丝法、溶剂浇铸/粒子沥滤法及气体发泡法等。目前研究发现,支架中孔径的大小对组织的重建有着直接的影响,孔径为100-250 μm的孔有益于骨及软骨组织的再生。通过溶液浇铸/粒子沥滤法、气体发泡法所制备的支架孔径大小在这一范围内,因此比较适合用于骨、软骨组织工程支架的构建。研究人员通常将多种方法结合起来,以期能制备出生物和力学性能方面更加仿生的组织工程多孔支架。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Non-destructive quantitative 3D analysis for the optimisation of tissue scaffolds

In tissue engineering, porous scaffolds are often used as three-dimensional (3D) supports for tissue growth. In scaffold design, it is imperative to be able to quantify the pore sizes and more importantly the interconnects between the pores. X-ray micro-computed tomography (microCT) has become a popular tool for obtaining 3D images of scaffold biomaterials, however images are only qualitative. In this work, methods were developed for obtaining pore size distributions for both the macropores and their interconnects. Scaffolds have been developed, by foaming sol-gel derived bioactive glasses, which have the potential to fulfil the criteria for an ideal scaffold for bone tissue engineering. MicroCT images were obtained from scaffolds with different pore structures. The images were thresholded and three algorithms were applied in 3D to identify pores and interconnects and to obtain pore size distributions. The results were validated against mercury intrusion porosimetry and manual 3D image analysis. The microCT data were then meshed such that predictions of permeability as a function of changes in the pore network could be made. Such predictions will be useful for optimising bioreactor conditions for tissue engineering applications. These techniques would be suitable for many other types of scaffolds.     

Chitosan/silk fibroin-based,Schwann cell-derived extracellular matrix-modified scaffolds for bridging rat sciatic nerve gaps

Extracellular matrix (ECM) plays a prominent role in establishing and maintaining an ideal microenvironment for tissue regeneration, and ECM scaffolds are used as a feasible alternative to cellular and molecular therapy in the fields of tissue engineering. Because of their advantages over tissue-derived ECM scaffolds, cultured cell-derived ECM scaffolds are beginning to attract attention, but they have been scarcely studied for peripheral nerve repair. Here we aimed to develop a tissue engineered nerve scaffold by reconstituting nerve cell-derived ECM with natural biomaterials. A protocol was adopted to prepare and characterize the cultured Schwann cell (SC)-derived ECM. A chitosan conduit and silk fibroin (SF) fibers were prepared, cultured with SCs for ECM deposition, and subjected to decellularization, followed by assembly into a chitosan/SF-based, SC-derived ECM-modified scaffold, which was used to bridge a 10 mm rat sciatic nerve gap. The results from morphological analysis as well as electrophysiological examination indicated that regenerative outcomes achieved by our developed scaffold were similar to those by an acellular nerve graft (namely a nerve tissue-derived ECM scaffold), but superior to those by a plain chitosan/SF scaffold. Moreover, blood and histopathological parameters confirmed the safety of scaffold modification by SC-derived ECM. Therefore, a hybrid scaffold based on joint use of acellular and classical biomaterials represents a promising approach to nerve tissue engineering.     

Effect of polycaprolactone scaffold permeability on bone regeneration in vivo

Successful bone tissue engineering depends on the scaffold's ability to allow nutrient diffusion to and waste removal from the regeneration site, as well as provide an appropriate mechanical environment. Since bone is highly vascularized, scaffolds that provide greater mass transport may support increased bone regeneration. Permeability encompasses the salient features of three-dimensional porous scaffold architecture effects on scaffold mass transport. We hypothesized that higher permeability scaffolds will enhance bone regeneration for a given cell seeding density. We manufactured poly-?-caprolactone scaffolds, designed to have the same internal pore design and either a low permeability (0.688×10(-7)m(4)/N-s) or a high permeability (3.991×10(-7)m(4)/N-s), respectively. Scaffolds were seeded with bone morphogenic protein-7-transduced human gingival fibroblasts and implanted subcutaneously in immune-compromised mice for 4 and 8 weeks. Micro-CT evaluation showed better bone penetration into high permeability scaffolds, with blood vessel infiltration visible at 4 weeks. Compression testing showed that scaffold design had more influence on elastic modulus than time point did and that bone tissue infiltration increased the mechanical properties of the high permeability scaffolds at 8 weeks. These results suggest that for polycaprolactone, a more permeable scaffold with regular architecture is best for in vivo bone regeneration. This finding is an important step toward the end goal of optimizing a scaffold for bone tissue engineering.     

Healing potential of nanohydroxyapatite,gelatin, and fibrin-platelet glue combination as tissue engineered scaffolds in radial bone defects of rats

Different biomaterials have been used in orthopedic surgery. Evaluation of biomaterials for bone healing promotion has been a wide area of research of the orthopedic field. Sixty critical size defects of 5 mm long were bilaterally created in the radial diaphysis of 30 rats. The animals were randomly divided into six equal groups as empty defect, autograft, nanohydroxyapatite (nHA), Gelatin (Gel)-nHA, fibrin-platelet glue (FPG)-nHA, and Gel-FPG-nHA groups (n = 10 in each group). Radiographs of each forelimb were taken postoperatively on the 1st day and then at the 28th and 56th days post injury. After 56 days, the rats were euthanized and their harvested healing bone samples were evaluated by histopathology, scanning electron microscopy, and biomechanical testing.

All the treated defects demonstrated significantly superior new bone formation, remodeling, and bone tissue volume. Moreover, the defects treated with FPG-nHA showed significantly higher ultimate load, yield load, and stiffness. The Gel-FPG-nHA moderately improved bone regeneration that was not close to the autograft in some parameters, whereas FPG-nHA significantly improved bone healing closely comparable with the autograft group in most parameters. In conclusion, although all the nHA-containing scaffolds had some beneficial effects on bone regeneration, the FPG-nHA scaffold was more effective in improving the structural and functional properties of the newly formed bone and was more osteoinductive than the Gel and was comparable to the autograft. Therefore, the FPG can be regarded as a promising option to be used in conjunction with mineral scaffolds for bone tissue engineering.     

Applications of knitted mesh fabrication techniques to scaffolds for tissue engineering and regenerative medicine

Knitting is an ancient and yet, a fresh technique. It has a history of no less than 1,000 years. The development of tissue engineering and regenerative medicine provides a new role for knitting. Several meshes knitted from synthetic or biological materials have been designed and applied, either alone, to strengthen materials for the patching of soft tissues, or in combination with other kinds of biomaterials, such as collagen and fibroin, to repair or replace damaged tissues/organs. In the latter case, studies have demonstrated that knitted mesh scaffolds (KMSs) possess excellent mechanical properties and can promote more effective tissue repair, ligament/tendon/cartilage regeneration, pipe-like-organ reconstruction, etc. In the process of tissue regeneration induced by scaffolds, an important synergic relationship emerges between the three-dimensional microstructure and the mechanical properties of scaffolds. This paper presents a comprehensive overview of the status and future prospects of knitted meshes and its KMSs for tissue engineering and regenerative medicine.     

In vitro and in vivo evaluation of a novel nanosize hydroxyapatite particles/poly(ester-urethane) composite scaffold for bone tissue engineering

Scaffolds for bone tissue engineering should provide an osteoconductive surface to promote the ingrowth of new bone after implantation into bone defects. This may be achieved by hydroxyapatite loading of distinct scaffold biomaterials. Herein, we analyzed the in vitro and in vivo properties of a novel nanosize hydroxyapatite particles/poly(ester-urethane) (nHA/PU) composite scaffold which was prepared by a salt leaching–phase inverse process. Microtomography, scanning electron microscopy and X-ray spectroscopy analyses demonstrated the capability of the material processing to create a three-dimensional porous PU scaffold with nHA on the surface. Compared to nHA-free PU scaffolds (control), this modified scaffold type induced a significant increase in in vitro adsorption of model proteins. In vivo analysis of the inflammatory and angiogenic host tissue response to implanted nHA/PU scaffolds in the dorsal skinfold chamber model indicated that the incorporation of nHA particles into the scaffold material did not affect biocompatibility and vascularization when compared to control scaffolds. Thus, nHA/PU composite scaffolds represent a promising new type of scaffold for bone tissue engineering, combining the flexible material properties of PU with the advantage of an osteoconductive surface.     

生物材料复合支架与运动性关节软骨缺损的修复

目的：探讨复合支架的组织工程学特性及其修复关节软骨缺损的性能评价。 方法：以“关节软骨、生物材料、工程软骨、复合材料、复合支架”为中文关键词,以“ tissue enginneering,articular cartilage,scaffold material”为英文关键词,采用计算机检索中国期刊全文数据库、PubMed数据库(1993-01/2010-11)相关文章。纳入复合支架材料-细胞复合物修复关节软骨损伤相关的文章,排除重复研究或Meta分析类文章。 结果：共入选18篇文章进入结果分析。复合支架是当前软骨组织工程中应用较多的支架,它是将具有互补特征的生物相容性可降解支架,按一定比例和方式组合,设计出结构与性能优化的复合支架。较单一支架材料具有显著优越性,具有更好的生物相容性和一定强度的韧性,较好的孔隙和机械强度。复合支架的制备不仅包括同一类生物材料的复合,还包括不同类别生物材料之间的交叉复合。可分为纯天然支架材料、纯人工支架材料以及天然与人工支架材料的复合等3类。 结论：复合支架使生物材料具有互补特性,一定程度上满足了理想生物支架材料应具有的综合特点,但目前很多研究仍处于实验阶段,还有一些问题有待于解决,如不同材料的复合比例、复合工艺等。     

Functional biomaterials for cartilage regeneration

The injury and degeneration of articular cartilage and associated arthritis are leading causes of disability worldwide. Cartilage tissue engineering as a treatment modality for cartilage defects has been investigated for over 20 years. Various scaffold materials have been developed for this purpose, but has yet to achieve feasibility and effectiveness for widespread clinical use. Currently, the regeneration of articular cartilage remains a formidable challenge, due to the complex physiology of cartilage tissue and its poor healing capacity. Although intensive research has been focused on the developmental biology and regeneration of cartilage tissue and a diverse plethora of biomaterials have been developed for this purpose, cartilage regeneration is still suboptimal, such as lacking a layered structure, mechanical mismatch with native cartilage and inadequate integration between native tissue and implanted scaffold. The ideal scaffold material should have versatile properties that actively contribute to cartilage regeneration. Functional scaffold materials may overcome the various challenges faced in cartilage tissue engineering by providing essential biological, mechanical, and physical/chemical signaling cues through innovative design. This review thus focuses on the complex structure of native articular cartilage, the critical properties of scaffolds required for cartilage regeneration, present strategies for scaffold design, and future directions for cartilage regeneration with functional scaffold materials.     

具有生物活性的多聚磷酸盐材料在骨修复中的应用：基础、进展与挑战

随着人们对骨修复和组织工程支架材料研究的深入,功能性骨科生物材料应运而生。无机多聚磷酸盐（polyphosphate, polyP）是一种具有高能磷酸键的聚合物,存在于成骨细胞和血小板中,其降解产物如磷酸根离子和产生的能量可以参与骨再生和代谢。受此启发,人们将polyP开发为新型骨修复材料,包括非晶态和结晶态两种结构,在体内外的实验研究都取得了丰富的成果。本文综合阐述了这种独特的能量自给型生物材料的生理功能、材料设计、制备方法、生物学效应及相关机制的进展,为其在骨修复中的应用提供依据。本文也同时讨论了polyP材料面临的挑战和争议,以期推动其临床转化。