Three-dimensional scaffold containing EGF incorporated biodegradable polymeric nanoparticles for stem cell based tissue engineering applications

Stem cell-based tissue engineering holds much hope for the development of multifunctional tissues to replace diseased organs. The attachment and survival of stem cells on a three-dimensional (3D) scaffold must be enhanced for faster progression of stem cell based tissue engineering. This study evaluate the stability of mesenchymal stem cells (MSCs) in 3D porous scaffolds composed of a collagen and chitosan blend impregnated with epidermal growth factor incorporated chitosan nanoparticles (EGF-CNP). The EGF-CNP scaffolds were characterized by transmission electron microscopy, which revealed that the nanoparticles were round in shape and 20 ∼ 50 nm in size. The scaffolds were prepared by freeze drying. A Fourier-transform infrared spectrum study revealed that the linkage between collagen and chitosan was through an ionic interaction. Thermal analysis and degradation studies showed that the scaffold could be used in tissue engineering application. MSCs proliferated well in the EGF-CNP impregnated scaffold. A scanning electron microscope study showed anchored and elongated MSCs on the EGF-CNP impregnated scaffold. A 3D biodegradable collagen chitosan scaffold impregnated with EGF-CNP is a promising transportable candidate for MSC-based tissue engineering, and this scaffold could be used as an in vitro model for subsequent clinical applications.     

基于天然水凝胶的生物材料在骨组织工程中的应用*

天然水凝胶是指原材料来自于天然生物材料的水凝胶。由于这种天然的聚合物含有构成生物体的天然成分,与天然组织具有生物学和化学相似性,而受到特别关注。天然水凝胶由于其与细胞外基质高度的相似性被认为是骨组织工程中优良的仿生基质材料。而针对天然水凝胶机械性能差、成骨诱导性能弱等缺陷,通常需要对天然水凝胶进行改性、引入其他材料或生物活性因子,以此来获得更适用于骨组织工程支架材料。对近年来基于天然水凝胶的生物材料在骨组织工程的应用,与其不同的应用形式(可注射水凝胶、多孔水凝胶支架、3D生物打印水凝胶支架等)进行了概述,以期对这类基于天然水凝胶的生物材料在未来骨组织工程中的应用提供参考。     

Electrospun poly-l-lactic acid/polyvinyl alcohol nanofibers improved insulin-producing cell differentiation potential of human adipose-derived mesenchymal stem cells

Combination of adipose-derived mesenchymal stem cells (ADSCs) and synthetic materials in terms of pancreatic tissue engineering can be considered as a treatment of diabetes. This study aimed to evaluate the differentiation of human ADSCs to pancreatic cells on poly-l -lactic acid/polyvinyl alcohol (PLLA/PVA) nanofibers as a three-dimensional (3D) scaffold. Mesenchymal stem cells (MSCs) were characterized for mesenchymal surface markers by flow cytometry. Then ADSCs were seeded on 3D scaffolds and treated with pancreatic differentiation medium. Immunostaining assay showed that ADSCs were very efficiently differentiated into a relatively homogeneous population of insulin-producing cells. Moreover, real-time RT-PCR results revealed that pancreas-specific markers were highly expressed in 3D scaffolds compared with their expression in tissue culture plates and this difference in expression level was significant. In addition, insulin and C-peptide secreted in response to varying concentrations of glucose in the 3D scaffold group was significantly higher than that in 2D culture. The results of the present study confirmed that PLLA/PVA scaffold seeded with ADSCs could be a suitable option in pancreatic tissue engineering.     

Evaluation of biological protein-based collagen scaffolds in cartilage and musculoskeletal tissue engineering--a systematic review of the literature

The term tissue engineering is the technology that combines cells, engineering and biological/synthetic material in order to repair, replace or regenerate biological tissues such as bone, muscle, tendons and cartilage. The major human applications of tissue engineering are: skin, bone, cartilage, corneas, blood vessels, left mainstem bronchus and urinary structures. In this systematic review several criteria were identified as the most desirable characteristics of an ideal scaffold. These state that an ideal scaffolds needs to be biodegradable, possess mechanical strength, be highly porous, biocompatible, non-cytotoxic, non antigentic, stuitable for cell attachment, proliferation and differentiation, flexible and elastic, three dimensional, osteoconductive and support the transport of nutrients and metabolic waste. Subsequently, studies reporting on the various advantages and disadvantages of using collagen based scaffolds in musculoskeletal and cartilage tissue engineering were identified. The purpose of this review is to 1) provide a list of ideal characteristics of a scaffold as identified in the literature 2) identify different types of biological protein-based collagen scaffolds used in musculoskeletal and cartilage tissue engineering 3) assess how many of the criteria each scaffold type meets 4) weigh different scaffolds against each other according to their relative properties and shortcomings. The rationale behind this approach is that the ideal scaffold material has not yet been identified. Hence, this review will define how many of the identified ideal characteristics are fulfilled by natural collagen-based scaffolds and address the shortcomings of its use as found in the literature.     

Cell colonization in degradable 3D porous matrices

Cell colonization is an important in a wide variety of biological processes and applications including vascularization, wound healing, tissue engineering, stem cell differentiation, and biosensors. During colonization porous 3D structures are used to support and guide the ingrowth of cells into the matrix. In this review, we summarize our understanding of various factors affecting cell colonization in 3-dimensional environment. The structural, biological, and degradation properties of the matrix all play key roles during colonization. Further, specific scaffold properties such as porosity, pore size, fiber thickness, topography, and scaffold stiffness as well as important cell material interactions such as cell adhesion and mechanotransduction also influence colonization.     

Carbon nanotubes leading the way forward in new generation 3D tissue engineering

Statistics from the NHS Blood and Transplant Annual Review show that total organ transplants have increased to 4213 in 2012, while the number of people waiting to receive an organ rose to 7613 that same year. Human donors as the origin of transplanted organs no longer meet the ever-increasing demand, and so interest has shifted to synthetic organ genesis as a form of supply. This focus has given rise to new generation tissue and organ engineering, in the hope of one day designing 3D organs in vitro. While research in this field has been conducted for several decades, leading to the first synthetic trachea transplant in 2011, scaffold design for optimising complex tissue growth is still underexplored and underdeveloped. This is mostly the result of the complexity required in scaffolds, as they need to mimic the cells’ native extracellular matrix. This is an intricate nanostructured environment that provides cells with physical and chemical stimuli for optimum cell attachment, proliferation and differentiation. Carbon nanotubes are a popular addition to synthetic scaffolds and have already begun to revolutionise regenerative medicine. Discovered in 1991, these are traditionally used in various areas of engineering and technology; however, due to their excellent mechanical, chemical and electrical properties their potential is now being explored in areas of drug delivery, in vivo biosensor application and tissue engineering. The incorporation of CNTs into polymer scaffolds displays a variety of structural and chemical enhancements, some of which include: increased scaffold strength and flexibility, improved biocompatibility, reduction in cancerous cell division, induction of angiogenesis, reduced thrombosis, and manipulation of gene expression in developing cells. Moreover CNTs’ tensile properties open doors for dynamic scaffold design, while their thermal and electrical properties provide opportunities for the development of neural, bone and cardiac tissue constructs.     

Polycaprolactone scaffolds fabricated with an advanced electrohydrodynamic direct-printing method for bone tissue regeneration

Electrohydrodynamic (EHD) direct writing has been used in diverse microelectromechanical systems and various supplemental methods for biotechnology and electronics. In this work, we expanded the use of EHD-induced direct writing to fabricate 3D biomedical scaffolds designed as porous structures for bone tissue engineering. To prepare the scaffolds, we modified a grounded target used in conventional EHD direct printing using a poly(ethylene oxide) solution bath, elastically cushioning the plotted struts to prevent crumbling. The fabricated scaffolds were assessed for not only physical properties including surface roughness and water uptake ability but also biological capabilities by culturing osteoblast-like cells (MG63) for the EHD-plotted polycaprolactone (PCL) scaffold. The EHD-scaffolds showed significantly roughened surface and enhanced water-absorption ability (400% increase) compared with the pure rapid-prototyped PCL. The results of cell viability, alkaline phosphatase activity, and mineralization analyses showed significantly enhanced biological properties of the scaffold (20 times the cell viability and 6 times the mineralization) compared with the scaffolds fabricated using RP technology. Because of the results, the modified EHD direct-writing process can be a promising method for fabricating 3D biomedical scaffolds in tissue engineering.     

Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems

One of the milestones in tissue engineering has been the development of 3D scaffolds that guide cells to form functional tissue. Recently, mouldless manufacturing techniques, known as solid free-form fabrication (SFF), or rapid prototyping, have been successfully used to fabricate complex scaffolds. Similarly, to achieve simultaneous addition of cells during the scaffold fabrication, novel robotic assembly and automated 3D cell encapsulation techniques are being developed. As a result of these technologies, tissue-engineered constructs can be prepared that contain a controlled spatial distribution of cells and growth factors, as well as engineered gradients of scaffold materials with a predicted microstructure. Here, we review the application, advancement and future directions of SFF techniques in the design and creation of scaffolds for use in clinically driven tissue engineering.     

Current and emerging vascularization strategies in skin tissue engineering

Vascularization is a key process in skin tissue engineering, determining the biological function of artificial skin implants. Hence, efficient vascularization strategies are a major prerequisite for the safe application of these implants in clinical practice. Current approaches include (i) modification of structural and physicochemical properties of dermal scaffolds, (ii) biological scaffold activation with growth factor-releasing systems or gene vectors, and (iii) generation of prevascularized skin substitutes by seeding scaffolds with vessel-forming cells. These conventional approaches may be further supplemented by emerging strategies, such as transplantation of adipose tissue-derived microvascular fragments, 3D bioprinting and microfluidics, miRNA modulation, cell sheet engineering, and fabrication of photosynthetic scaffolds. The successful translation of these vascularization strategies from bench to bedside may pave the way for a broad clinical implementation of skin tissue engineering.     

Biological matrices and bionanotechnology

A crucial step towards the goal of tissue engineering a heart valve will be the choice of scaffold onto which an appropriate cell phenotype can be seeded. Successful scaffold materials should be amenable to modification, have a controlled degradation, be compatible with the cells, lack cytotoxicity and not elicit an immune or inflammatory response. In addition, the scaffold should induce appropriate responses from the cells seeded onto it, such as cell attachment, proliferation and remodelling capacity, all of which should promote the formation of a tissue construct that can mimic the structure and function of the native valve. This paper discusses the various biological scaffolds that have been considered and are being studied for use in tissue engineering a heart valve. Also, strategies to enhance the biological communication between the scaffold and the cells seeded onto it as well as the use of bionanotechnology in the manufacture of scaffolds possessing the desired properties will be discussed.     

Fabricating tissues: Analysis of farming versus engineering strategies

Tissue Engineering has expanded rapidly towards target applications of tissue repair and regeneration, whilst generating surprisingly novel models to study tissue modelling. However, clinical success in producing effective engineered tissues such as bone, skin, cartilage, and tendon, have been rare and limited. Problems tend to focus on how to stimulate the replacement of initial scaffold with mechanically functional, native extracellular matrix (principally collagen). Typical approaches have been to develop perfused and mechanically active bioreactors, with the use of native collagen itself as the initial scaffold, though the idea remains that cells do the fabrication (i.e. a cultivation process). We have developed a new, engineering approach, in which the final collagen template is fabricatedwithout cell involvement. The first part of this biomimetic engineering involves a plastic compression of cellular native collagen gels to form dense, strong, collagenous neotissues (in minutes). Further steps can be used to orientate and increase collagen fibril diameter, again by non-cell dependent engineering. This allows operator control of cell or matrix density and material properties (influencing biological half life and fate). In addition, this (non-cultivation) approach can incorporate techniques to generate localised 3D structures and zones at a meso-scale. In conclusion, the use of biomimetic engineering based on native collagen, rather than cell-cultivation approaches for bulk matrix fabrication, produces huge benefits. These include speed of fabrication (minutes instead of weeks and months), possibility of fine control of composition and 3D nano-micro scale structure and biomimetic complexity.     

Cell–scaffold mechanical interplay within engineered tissue

Effective tissue engineering requires appropriate selection of cells and scaffold, where the latter serves as a mechanical and biological support for cell growth and functionality. The optimal combination of cell source and scaffold properties can vary for each desired application. Such preconditions necessitate enhanced understanding of the interactions between cells and scaffold within engineered tissue. Several studies have examined the deforming effects cells induce in scaffolds via exertion of contractile forces. In contrast, other studies focus on the scaffold's biochemical and mechanical properties and their effects on cell behavior.This review summarizes the mechanical interplay between cells and scaffold within engineered tissue. We present evidence for contractile forces exerted by cells on three-dimensional (3D) scaffolds and discuss existing methods for their quantification. In addition, we address some theories related to the effects of scaffold stiffness and mechanical stimulation on cell behavior. Further understanding of the reciprocal effects between cells and scaffold will provide both enhanced knowledge regarding the expected properties of engineered tissue and more competent tissue regeneration techniques.     

Cell–scaffold interaction within engineered tissue

The structure of a tissue engineering scaffold plays an important role in modulating tissue growth. A novel gelatin–chitosan (Gel–Cs) scaffold with a unique structure produced by three-dimensional printing (3DP) technology combining with vacuum freeze-drying has been developed for tissue-engineering applications. The scaffold composed of overall construction, micro-pore, surface morphology, and effective mechanical property. Such a structure meets the essential design criteria of an ideal engineered scaffold. The favorable cell–matrix interaction supports the active biocompatibility of the structure. The structure is capable of supporting cell attachment and proliferation. Cells seeded into this structure tend to maintain phenotypic shape and secreted large amounts of extracellular matrix (ECM) and the cell growth decreased the mechanical properties of scaffold. This novel biodegradable scaffold has potential applications for tissue engineering based upon its unique structure, which acts to support cell growth.     

3D生物打印在软骨修复中的应用*

关节软骨损伤后的自我修复是医学界一直在研究和探讨的难题。3D生物打印技术可以精准的分配载细胞生物材料,构建复杂的三维活体组织,在优化软骨缺损修复组织的内部结构、机械性能以及生物相容性上有很大优势,因此近年来成为软骨修复组织工程领域的研究热点。重点介绍了软骨生物3D生物打印的最新进展,包括软骨生物打印“墨水”材料的选择、种子细胞的来源以及3D生物打印技术的发展。此外,还阐述了3D生物打印技术在组织工程学应用上的部分局限性,并对其在软骨修复领域的发展与应用进行了预测。     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

Decellularized omentum as novel biologic scaffold for reconstructive surgery and regenerative medicine

Homologous tissues, such as adipose tissue, may be an interesting source of acellular scaffolds, maintaining a complex physiological three-dimensional (3D) structure, to be recellularized with autologous cells. The aim of the present work is to evaluate the possibility of obtaining homologous acellular scaffolds from decellularization of the omentum, which is known to have a complex vascular network. Adult rat and human omenta were treated with an adapted decellularization protocol involving mechanical rupture (freeze-thaw cycles), enzymatic digestion (trypsin, lipase, deoxyribonuclease, ribonuclease) and lipid extraction (2-propanol). Histological staining confirmed the effectiveness of decellularization, resulting in cell-free scaffolds with no residual cells in the matrix. The complex 3D networks of collagen (azan-Mallory), elastic fibers (Van Gieson), reticular fibers and glycosaminoglycans (PAS) were maintained, whereas Oil Red and Sudan stains showed the loss of lipids in the decellularized tissue. The vascular structures in the tissue were still visible, with preservation of collagen and elastic wall components and loss of endothelial (anti-CD31 and -CD34 immunohistochemistry) and smooth muscle (anti-alpha smooth muscle actin) cells. Fat-rich and well vascularized omental tissue may be decellularized to obtain complex 3D scaffolds preserving tissue architecture potentially suitable for recellularization. Further analyses are necessary to verify the possibility of recolonization of the scaffold by adipose-derived stem cells in vitro and then in vivo after re implantation, as already known for homologus implants in regenerative processes.Key words: omentum, scaffold, decellularization, adipose tissue engineering, regenerative medicine, microvascularization     

Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design

The anterior cruciate ligament (ACL) is the most commonly injured intra-articular ligament of the knee. The insufficient vascularization of this tissue prevents it from healing completely after extreme tearing or rupture, creating a need for ACL grafts for reconstruction. The limitations of existing grafts have motivated the investigation of tissue-engineered ACL grafts. A successful tissue-engineered graft must possess mechanical properties similar to the ACL; to date no commercially available synthetic graft has achieved this. To accomplish this goal we have combined the techniques of polymer fiber braiding and twisting to design a novel poly L-lactic acid (PLLA) braid-twist scaffold for ACL tissue engineering. The scaffold is designed to accurately mimic the biomechanical profile and mechanical properties of the ACL. In this study, braid-twist scaffolds were constructed and compared to braided scaffolds and twisted fiber scaffolds. The addition of fiber twisting to the braided scaffold resulted in a significant increase in the ultimate tensile strength, an increase in ultimate strain, and an increase in the length of the toe region in these constructs over scaffolds that were braided. Based on the findings of this study, the braid-twist scaffold studied was found to be a promising construct for tissue engineering of the ACL.     

Design concepts and strategies for tissue engineering scaffolds

In the emerging field of tissue engineering and regenerative medicine, new viable and functional tissue is fabricated from living cells cultured on an artificial matrix in a simulated biological environment. It is evident that the specific requirements for the three main components, cells, scaffold materials, and the culture environment, are very different, depending on the type of cells and the organ-specific application. Identifying the variables within each of these components is a complex and challenging assignment, but there do exist general requirements for designing and fabricating tissue engineering scaffolds. Therefore, this review explores one of the three main components, namely, the key concepts, important parameters, and required characteristics related to the development and evaluation of tissue engineering scaffolds. An array of different design strategies will be discussed, which include mimicking the extra cellular matrix, responding to the need for mass transport, predicting the structural architecture, ensuring adequate initial mechanical integrity, modifying the surface chemistry and topography to provide cell signaling, and anticipating the material selection so as to predict the required rate of bioresorption. In addition, this review considers the major challenge of achieving adequate vascularization in tissue engineering constructs, without which no three-dimensional thick tissue such as the heart, liver, and kidney can remain viable.     

Synthetic tissue biology: tissue engineering meets synthetic biology

We propose the term "synthetic tissue biology" to describe the use of engineered tissues to form biological systems with metazoan-like complexity. The increasing maturity of tissue engineering is beginning to render this goal attainable. As in other synthetic biology approaches, the perspective is bottom-up; here, the premise is that complex functional phenotypes (on par with those in whole metazoan organisms) can be effected by engineering biology at the tissue level. To be successful, current efforts to understand and engineer multicellular systems must continue, and new efforts to integrate different tissues into a coherent structure will need to emerge. The fruits of this research may include improved understanding of how tissue systems can be integrated, as well as useful biomedical technologies not traditionally considered in tissue engineering, such as autonomous devices, sensors, and manufacturing.