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.     

Scaffold-based bone engineering by using genetically modified cells

The first generation of clinically applied tissue engineering concepts in the area of skin, cartilage and bone marrow regeneration was based on the isolation, expansion and implantation of cells from the patient's own tissue. Although successful in selective treatments, tissue engineering needs to overcome major challenges to allow widespread clinical application with predictable outcomes. One challenge is to present the cells in a matrix to the implantation site to allow the cells to survive the wound healing contraction forces, tissue remodeling in certain tissues such as bone and biomechanical loading. Hence, several tissue engineering strategies focus on the development of load-bearing scaffold/cell constructs. From a cell source point of view, bone engineers face challenges to isolate and expand cells with the highest potential to form osseous tissue along with harvesting tissue without extensive donor site morbidity. A major hurdle to tissue engineering is de-differentiation and limited ability to control cell phenotype following in vitro expansion. Due to early successes with genetic engineering, bone tissue engineers have used different strategies to genetically alter various types of mesenchymal cells to enhance the mineralization capacity of tissue-engineered scaffold/cell constructs. Although the development of multi-component scaffold/osteogenic cell constructs requires a combination of interdisciplinary research strategies, the following review is limited to describe the general aspects of bone engineering and to present overall directions of technology platforms, which include a genetic engineering component. This paper reviews the most recent work in the field and discusses the concepts developed and executed by a collaborative effort of the multi-disciplinary teams of the two authors.     

Adipose tissue engineering with cells in engineered matrices

Tissue engineering has shown promise for the development of constructs to facilitate large volume soft tissue augmentation in reconstructive and cosmetic plastic surgery. This article reviews the key progress to date in the field of adipose tissue engineering. In order to effectively design a soft tissue substitute, it is critical to understand the native tissue environment and function. As such, the basic physiology of adipose tissue is described and the process of adipogenesis is discussed. In this article, we have focused on tissue engineering using a cell-seeded scaffold approach, where engineered extracellular matrix substitutes are seeded with exogenous cells that may contribute to the regenerative response. The strengths and limitations of each of the possible cell sources for adipose tissue engineering, including adipose-derived stem cells, are detailed. We briefly highlight some of the results from the major studies to date, involving a range of synthetic and naturally derived scaffolds. While these studies have shown that adipose tissue regeneration is possible, more research is required to develop optimized constructs that will facilitate safe, predictable, and long-term augmentation in clinical applications.     

Adipose tissue engineering with cells in engineered matrices

Tissue engineering has shown promise for the development of constructs to facilitate large volume soft tissue augmentation in reconstructive and cosmetic plastic surgery. This article reviews the key progress to date in the field of adipose tissue engineering. In order to effectively design a soft tissue substitute, it is critical to understand the native tissue environment and function. As such, the basic physiology of adipose tissue is described and the process of adipogenesis is discussed. In this article, we have focused on tissue engineering using a cell-seeded scaffold approach, where engineered extracellular matrix substitutes are seeded with exogenous cells that may contribute to the regenerative response. The strengths and limitations of each of the possible cell sources for adipose tissue engineering, including adipose-derived stem cells, are detailed. We briefly highlight some of the results from the major studies to date, involving a range of synthetic and naturally derived scaffolds. While these studies have shown that adipose tissue regeneration is possible, more research is required to develop optimized constructs that will facilitate safe, predictable and long-term augmentation in clinical applications.Key words: tissue engineering, regenerative medicine, adipose tissue, adipose-derived stem cells, adipogenesis, cell culture, scaffolds, cell-biomaterial interactions     

Strategies for organ level tissue engineering

The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development, and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs – biomaterials, cells and bioactive molecules–and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to “scale up” every aspect of the research and development process.     

Strategies for organ level tissue engineering

The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue-engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs-biomaterials, cells and bioactive molecules-and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to "scale up" every aspect of the research and development process.     

Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering

Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.     

Engineering vascularized skeletal muscle tissue

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.     

Liver tissue engineering: promises and prospects of new technology

Today, many patients suffer from acute liver failure and hepatoma. This is an area of high unmet clinical need as these conditions are associated with very high mortality. There is an urgent need to develop techniques that will enable liver tissue engineering or generate a bioartificial liver, which will maintain or improve liver function or offer the possibility of liver replacement. Liver tissue engineering is an innovative way of constructing an implantable liver and has the potential to alleviate the shortage of organ donors for orthotopic liver transplantation. In this review we describe, from an engineering perspective, progress in the field of liver tissue engineering, including three main aspects involving cell sources, scaffolds and vascularization.     

Chondrogenesis and cartilage tissue engineering: the longer road to technology development

Joint injury and disease are painful and debilitating conditions affecting a substantial proportion of the population. The idea that damaged cartilage in articulating joints might be replaced seamlessly with tissue-engineered cartilage is of obvious commercial interest because the market for such treatments is large. Recently, a wealth of new information about the complex biology of chondrogenesis and cartilage has emerged from stem cell research, including increasing evidence of the role of physical stimuli in directing differentiation. The challenge for the next generation of tissue engineers is to identify the key elements in this new body of knowledge that can be applied to overcome current limitations affecting cartilage synthesis in vitro. Here we review the status of cartilage tissue engineering and examine the contribution of stem cell research to technology development for cartilage production.     

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.     

Fibrin gel enhanced trilayer structure in cell-cultured constructs

Efficient cell seeding and subsequent support from a substrate ensure optimal cell growth and neotissue development during tissue engineering, including heart valve tissue engineering. Fibrin gel as a cell carrier may provide high cell seeding efficiency and adhesion property, improved cellular interaction, and structural support to enhance cellular growth in trilayer polycaprolactone (PCL) substrates that mimic the structure of native heart valve leaflets. This cell carrier gel coupled with a trilayer PCL substrate may enable the production of native-like cell-cultured leaflet constructs suitable for heart valve tissue engineering. In this study, we seeded valvular interstitial cells onto trilayer PCL substrates with fibrin gel as a cell carrier and cultured them for 1 month in vitro to determine if this gel can improve cell proliferation and production of extracellular matrix within the trilayer cell-cultured constructs. We observed that the fibrin gel enhanced cellular proliferation, their vimentin expression, and collagen and glycosaminoglycan production, leading to improved structure and mechanical properties of the developing PCL cell-cultured constructs. Fibrin gel as a cell carrier significantly improved the orientations of the cells and their produced tissue materials within trilayer PCL substrates that mimic the structure of native heart valve leaflets and, thus, may be highly beneficial for developing functional tissue-engineered leaflet constructs.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.     

Numerical simulation of fluid field and in vitro three-dimensional fabrication of tissue-engineered bones in a rotating bioreactor and in vivo implantation for repairing segmental bone defects

In this paper, two-dimensional flow field simulation was conducted to determine shear stresses and velocity profiles for bone tissue engineering in a rotating wall vessel bioreactor (RWVB). In addition, in vitro three-dimensional fabrication of tissue-engineered bones was carried out in optimized bioreactor conditions, and in vivo implantation using fabricated bones was performed for segmental bone defects of Zelanian rabbits. The distribution of dynamic pressure, total pressure, shear stress, and velocity within the culture chamber was calculated for different scaffold locations. According to the simulation results, the dynamic pressure, velocity, and shear stress around the surface of cell-scaffold construction periodically changed at different locations of the RWVB, which could result in periodical stress stimulation for fabricated tissue constructs. However, overall shear stresses were relatively low, and the fluid velocities were uniform in the bioreactor. Our in vitro experiments showed that the number of cells cultured in the RWVB was five times higher than those cultured in a T-flask. The tissue-engineered bones grew very well in the RWVB. This study demonstrates that stress stimulation in an RWVB can be beneficial for cell/bio-derived bone constructs fabricated in an RWVB, with an application for repairing segmental bone defects.     

Seeing tissue as a 'phase of matter': exploring statistical mechanics for the cell

The field of tissue engineering aims to produce living, biological constructs which possess the appropriate spatial ordering of cells and their extra cellular matrix products. The complexity of a single cell and its interactions in a large collective have made development of useful models to assist in tissue culture difficult, and consequentially most tissue culture endeavors are limited to trial and error approaches. Some cell types display a natural tendency to spontaneously self-assemble into large domains of parallel-oriented cells. In this work, we show that these cell culture systems can be studied in the context of continuous disorder-order phase transformations. We suggest that collective ordering of the cells is controlled by the amount of noise in the walk of the individual cells (directional persistence) because undifferentiated mesenchymal stem cells display a seven-times higher directional persistence than mature fibroblasts and have a 24-times larger final-oriented domain size, an observation that corresponds with collective ordering in self-propelled particle systems. The study of cell culture systems using analogies derived from statistical mechanics yields simple, practical models offering insight into how a long-range order can be obtained in tissue-engineered constructs, providing a new paradigm for managing operations with large collectives of living cells.     

A Glycosaminoglycan Based,Modular Tissue Scaffold System for Rapid Assembly of Perfusable,High Cell Density,Engineered Tissues

The limited ability to vascularize and perfuse thick, cell-laden tissue constructs has hindered efforts to engineer complex tissues and organs, including liver, heart and kidney. The emerging field of modular tissue engineering aims to address this limitation by fabricating constructs from the bottom up, with the objective of recreating native tissue architecture and promoting extensive vascularization. In this paper, we report the elements of a simple yet efficient method for fabricating vascularized tissue constructs by fusing biodegradable microcapsules with tunable interior environments. Parenchymal cells of various types, (i.e. trophoblasts, vascular smooth muscle cells, hepatocytes) were suspended in glycosaminoglycan (GAG) solutions (4%/1.5% chondroitin sulfate/carboxymethyl cellulose, or 1.5 wt% hyaluronan) and encapsulated by forming chitosan-GAG polyelectrolyte complex membranes around droplets of the cell suspension. The interior capsule environment could be further tuned by blending collagen with or suspending microcarriers in the GAG solution These capsule modules were seeded externally with vascular endothelial cells (VEC), and subsequently fused into tissue constructs possessing VEC-lined, inter-capsule channels. The microcapsules supported high density growth achieving clinically significant cell densities. Fusion of the endothelialized, capsules generated three dimensional constructs with an embedded network of interconnected channels that enabled long-term perfusion culture of the construct. A prototype, engineered liver tissue, formed by fusion of hepatocyte-containing capsules exhibited urea synthesis rates and albumin synthesis rates comparable to standard collagen sandwich hepatocyte cultures. The capsule based, modular approach described here has the potential to allow rapid assembly of tissue constructs with clinically significant cell densities, uniform cell distribution, and endothelialized, perfusable channels.     

Endothelial progenitor cells are integrated in newly formed capillaries and alter adjacent fibrovascular tissue after subcutaneous implantation in a fibrin matrix

Vascularization of bioartificial matrices is crucial for successful tissue engineering. Endothelial progenitor cells (EPC) have shown vascularization potential in ischemic conditions and may also support blood vessel formation in tissue-engineered matrices. The aim of our study was to investigate the impact of a well-characterized murine embryonal EPC line (T17b-EPC) on vascularization and fibrovascular granulation tissue formation after suspension in a fibrine matrix followed by subcutaneous implantation in a separation chamber in rats. EPC were fluorescently labelled in vitro prior to implantation. After 3, 7 or 14 days, animals were killed followed by explantation and histological analysis of the constructs. Before the end of the experiment, Bandeirea Simplicifolia lectin was intravenously injected to mark the vascular ingrowth into the implanted constructs. The transplanted cells were histologically detected at all time-points and located almost exclusively within the fibrin matrix at day 3 but the number of cells in the clot continuously decreased over day 7 to day 14. Conversely, cells were detected within the newly formed granulation tissue in increasing numbers from day 3 over day 7 to day 14. Transplanted cells were also found in the intermuscular septa. Cell viability was confirmed by use of an EPC clone expressing β-galactosidase. Fluorescence microscopy demonstrated integration of the transplanted cells in newly formed blood vessels within the fibrovascular granulation tissue adjacent to the fibrin clot. Presence of cells in the fibrin clot lead to thicker granulation tissue and an increased blood vessel diameter compared to cell-free controls. Organ standard controls showed presence of the transplanted cells in spleens at day 14 after transplantation. In summary, EPC exhibited biological activity after subcutaneous implantation in a fibrin matrix by migration from the fibrin clot into the granulation tissue and along intermuscular septae, undergoing differentiation into mature endothelial cells and integration into newly formed blood vessels and altering fibrovascular granulation tissue development. EPC may hold promise to modulate blood vessel formation in bioartificial matrices.     

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.     

Role of growth factors and endothelial cells in therapeutic angiogenesis and tissue engineering

To achieve the goals of engineering large complex tissues, and possibly internal organs, vascularization of the regenerating tissue is essential. To maintain the initial volume after implantation of regenerated tissue, improved vascularization is considered to be important. Recent advances in understanding the process of blood vessel growth has offered significant tools for the neovascularization of bioengineered tissues and therapeutic angiogenesis. Several angiogenic growth factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) were used for vascularization of ischemic tissues. Other approaches such as prevascularization of the scaffold, prior to cell seeding, and incorporation of endothelial cells in the bioengineered tissue showed encouraging results. In this article, we will review recent advances in angiogenic growth factors, and discuss the role of these growth factors and endothelial cells in therapeutic angiogenesis and tissue engineering.     

细胞三维培养：组织工程的关键技术突破口

组织工程是有望从根本上解决组织,器官缺损或失能的医学难题的一门新兴边缘学科,组织,器官发育的细胞和分子机制的进一步揭示和体外构建工程组织,器官的细胞培养技术的进步将使组织工程在新的千年成为广泛应用的新的治疗模式。细胞三维培养要成为体外构建工程组织,器官的成熟技术体系需先解决以下问题;（1）细胞;（2）生物材料;（3）培养基;（4）培养装置;（5）异型细胞的共培养;（6）细胞三维培养物血管化。