Design Of Tissue Engineering Scaffolds Research Articles

Synergic effects of crypt-like topography and ECM proteins on intestinal cell behavior in collagen based membranes

The basement membrane of small intestinal epithelium possesses complex topography at multiple scales ranging from the mesoscale to nanoscale. Specifically, intestinal crypt-villus units are comprised of hundred-micron-scale well-like invaginations and finger-like projections; intestinal cell phenotype is related to location on this crypt-villus unit. A biomimetic intestinal cell culture system composed of type I collagen based permeable cell culture membranes incorporating both micron-scale intestinal crypt-like topography and nanometer scale topography was fabricated. Membranes were pre-incubated with either laminin (Ln) or fibronectin (Fn), inoculated with intestinal epithelial Caco-2 cells and cultured for 1–21 days to study the relative significance of influence of crypt-like topography and biomimetic substrate chemistry on cell phenotype. Crypt-like topography inhibited Caco-2 differentiation during early culture, as evidenced by slower cell spreading and lower brush border enzyme activity. For example, alanine aminopeptidase activity was lower on Ln-coated patterned collagen (∼3.4 ± 0.24 mU/mg) compared to flat collagen (10.84 ± 0.55 mU/mg) at day 7. Caco-2 cultured on Fn-coated collagen started to spread earlier (1 day vs 3 days) and formed longer protrusions than on Ln-coated collagen. Pre-coating of Ln enhanced cell differentiation, as the maximum activity of a cell differentiation marker (alkaline phosphatase) was 2–3 times higher than on Fn-coated collagen, and maintained differentiated phenotype in long term (up to 21 days) culture. In general, compared to substrate topography, coating with ECM protein had more prominent and longer effect on cell behavior. Crypt-like topography affected Caco-2 spreading and differentiation during early culture, however the effect diminished as culture progressed. This information will benefit intestinal tissue engineering scaffold design, and modification of in vitro intestinal cell models.

Regulation of valvular interstitial cell calcification by adhesive peptide sequences

Knowledge of how valvular interstitial cells (VICs) interact with the extracellular matrix (ECM) would aid in not only better understanding the etiology of valvular disease but also constructing appropriate environments for valve tissue engineering. In this work, the calcification of VICs cultured on ECM coatings (fibronectin, fibrin, collagen, and laminin) or ECM-derived peptide sequences (RGDS, YIGSR, and DGEA) was quantified via several techniques. Neutralizing antibodies to specific adhesion receptors were also applied, followed by quantification of phenotypic markers related to valve calcification. The calcification of VICs varied with the ECM component or peptide that was presented on the culture substrate. VICs calcified the most on RGDS and least on YIGSR and DGEA, while blocking specific receptors revealed that disruption of VIC binding via the alpha(5)beta(1) integrin or the 67-kDa laminin receptor had a dramatic calcification-stimulating effect. Binding via the alpha(2)beta(1) integrin did not alter calcification or VIC phenotype. These findings were translated to 3D peptide-modified scaffold environments that demonstrated varying levels of disease expression by VICs. Thus, specific adhesion receptors play a significant role in mediating the interactions between VICs and ECM that lead to calcification, which provides important information regarding the mechanisms of valvular disease and scaffold design for valve tissue engineering.

Synthesis, degradation and biocompatibility of tyrosine-derived polycarbonate scaffolds

Polycarbonate terpolymers consisting of desaminotyrosyl-tyrosine alkyl esters (DTR), desaminotyrosyl-tyrosine (DT), and low molecular weight blocks of poly(ethylene glycol) (PEG) are a new class of polymers that have good engineering properties while also being resorbable in vivo. This study is the first evaluation of their (i) degradation behavior, (ii) in vitro cytotoxicity, and (iii) in vivo biocompatibility. Porous, tissue engineering scaffolds were prepared by a combination of solvent casting, porogen leaching and phase separation techniques. The scaffolds (>90% porosity) displayed (i) a bimodal pore distribution with micropores of less than 20 µm and macropores between 200 and 400 µm, (ii) a highly interconnected and open pore architecture, and (iii) a highly organized microstructure where the micropores are oriented and aligned along the walls of the macropores. Molecular weight (number average, Mn) and mass loss were determined in vitro (PBS at 37 °C) for up to 28 days. All three terpolymer compositions were fast degrading and retained only 10% of their initial molecular weight after 21 days, while mass loss during the 28 days was polymer composition-dependent. In vitro biocompatibility of the polymer scaffolds was determined up to 14 days by measuring metabolic activity of MC3T3.E1 (subclone 4) pre-osteoblasts. The outcome showed no statistical difference between cells cultured in monolayer and all tested polymer scaffolds. Robust cell attachment throughout the scaffold volume was observed by confocal microscopy and SEM. The biocompatibility of resorbing scaffolds was evaluated at 12 week in a critical sized defect (CSD) rabbit calvaria model and showed only a minimal inflammatory response. Overall, the results reported here illustrate the potential utility of tyrosine-derived polycarbonate terpolymers in the design of tissue engineering scaffolds.

Biomimetic Stratified Scaffold Design for Ligament-to-Bone Interface Tissue Engineering

The emphasis in the field of orthopaedic tissue engineering is on imparting biomimetic functionality to tissue engineered bone or soft tissue grafts and enabling their translation to the clinic. A significant challenge in achieving extended graft functionality is engineering the biological fixation of these grafts with each other as well as with the host environment. Biological fixation will require re-establishment of the structure-function relationship inherent at the native soft tissue-to-bone interface on these tissue engineered grafts. To this end, strategic biomimicry must be incorporated into advanced scaffold design. To facilitate integration between distinct tissue types (e.g., bone with soft tissues such as cartilage, ligament, or tendon), a stratified or multi-phasic scaffold with distinct yet continuous tissue regions is required to pre-engineer the interface between bone and soft tissues. Using the ACL-to-bone interface as a model system, this review outlines the strategies for stratified scaffold design for interface tissue engineering, focusing on identifying the relevant design parameters derived from an understanding of the structure-function relationship inherent at the soft-to-hard tissue interface. The design approach centers on first addressing the challenge of soft tissue-to-bone integration ex vivo, and then subsequently focusing on the relatively less difficult task of bone-to-bone integration in vivo. In addition, we will review stratified scaffold design aimed at exercising spatial control over heterotypic cellular interactions, which are critical for facilitating the formation and maintenance of distinct yet continuous multi-tissue regions. Finally, potential challenges and future directions in this emerging area of advanced scaffold design will be discussed.

Design of porous polymeric scaffolds by gas foaming of heterogeneous blends

One of the challenges in tissue engineering scaffold design is the realization of structures with a pre-defined multi-scaled porous network. Along this line, this study aimed at the design of porous scaffolds with controlled porosity and pore size distribution from blends of poly(epsilon-caprolactone) (PCL) and thermoplastic gelatin (TG), a thermoplastic natural material obtained by de novo thermoplasticization of gelatin. PCL/TG blends with composition in the range from 40/60 to 60/40 (w/w) were prepared by melt mixing process. The multi-phase microstructures of these blends were analyzed by scanning electron microscopy and dynamic mechanical analysis. Furthermore, in order to prepare open porous scaffolds for cell culture and tissue replacement, the TG and PCL were selectively extracted from the blends by the appropriate combination of solvent and extraction parameters. Finally, with the proposed combination of gas foaming and selective polymer extraction technologies, PCL and TG porous materials with multi-scaled and highly interconnected porosities were designed as novel scaffolds for new-tissue regeneration.

The correlation of pore morphology, interconnectivity and physical properties of 3D ceramic scaffolds with bone ingrowth

In the design of tissue engineering scaffolds, design parameters including pore size, shape and interconnectivity, mechanical properties and transport properties should be optimized to maximize successful inducement of bone ingrowth. In this paper we describe a 3D micro-CT and pore partitioning study to derive pore scale parameters including pore radius distribution, accessible radius, throat radius, and connectivity over the pore space of the tissue engineered constructs. These pore scale descriptors are correlated to bone ingrowth into the scaffolds. Quantitative and visual comparisons show a strong correlation between the local accessible pore radius and bone ingrowth; for well connected samples a cutoff accessible pore radius of ∼100 μ m is observed for ingrowth. The elastic properties of different types of scaffolds are simulated and can be described by standard cellular solids theory: ( E/ E 0) = ( ρ/ ρ s) n . Hydraulic conductance and diffusive properties are calculated; results are consistent with the concept of a threshold conductance for bone ingrowth. Simple simulations of local flow velocity and local shear stress show no correlation to in vivo bone ingrowth patterns. These results demonstrate a potential for 3D imaging and analysis to define relevant pore scale morphological and physical properties within scaffolds and to provide evidence for correlations between pore scale descriptors, physical properties and bone ingrowth.

APPLICATION OF BIOMECHANICS TO TISSUE ENGINEERING: A PERSONAL VIEW

Cellular biomechanics is an area of study that is receiving more attention as time progresses. The response of cells to their mechanical environment, including biomechanical stimuli, has far-reaching ramifications for the area of tissue engineering, especially for tissues designed to withstand mechanical loading (e.g. bone, cartilage, tendons and ligaments, and arteries). The effects of mechanical stimuli on cells are only recently being examined, and the potential role of mechanical stimuli in tissue engineering is still one that is largely ignored in the design of tissue engineering scaffolds. The relationship of mechanical properties of scaffolds or of mechanical stimuli to cell behavior is complex, but vital to the development of the field. Also, understanding the complex interplay of form and environment on cells involves an increase in our knowledge of how cells react to their total environment including mechanical stimuli and material properties. In order to improve tissue engineering outcomes, a nexus must be developed between the mechanical, biochemical, and biological studies of cellular behavior, in the context of extremely complex systems.

The effect of geometry on three-dimensional tissue growth

Tissue formation is determined by uncountable biochemical signals between cells; in addition, physical parameters have been shown to exhibit significant effects on the level of the single cell. Beyond the cell, however, there is still no quantitative understanding of how geometry affects tissue growth, which is of much significance for bone healing and tissue engineering. In this paper, it is shown that the local growth rate of tissue formed by osteoblasts is strongly influenced by the geometrical features of channels in an artificial three-dimensional matrix. Curvature-driven effects and mechanical forces within the tissue may explain the growth patterns as demonstrated by numerical simulation and confocal laser scanning microscopy. This implies that cells within the tissue surface are able to sense and react to radii of curvature much larger than the size of the cells themselves. This has important implications towards the understanding of bone remodelling and defect healing as well as towards scaffold design in bone tissue engineering.

Design of Tissue Engineering Scaffolds as Delivery Devices for Mechanical and Mechanically Modulated Signals

New approaches to tissue engineering aim to exploit endogenous strategies such as those occurring in prenatal development and recapitulated during postnatal healing. Defining tissue template specifications to mimic the environment of the condensed mesenchyme during development allows for exploitation of tissue scaffolds as delivery devices for extrinsic cues, including biochemical and mechanical signals, to drive the fate of mesenchymal stem cells seeded within. Although a variety of biochemical signals that modulate stem cell fate have been identified, the mechanical signals conducive to guiding pluripotent cells toward specific lineages are less well characterized. Furthermore, not only is spatial and temporal control of mechanical stimuli to cells challenging, but also tissue template geometries vary with time due to tissue ingrowth and/or scaffold degradation. Hence, a case study was carried out to analyze flow regimes in a testbed scaffold as a first step toward optimizing scaffold architecture. A pressure gradient was applied to produce local (nm-micron) flow fields conducive to migration, adhesion, proliferation, and differentiation of cells seeded within, as well as global flow parameters (micron-mm), including flow velocity and permeability, to enhance directed cell infiltration and augment mass transport. Iterative occlusion of flow channel dimensions was carried out to predict virtually the effect of temporal geometric variation (e.g., due to tissue development and growth) on delivery of local and global mechanical signals. Thereafter, insights from the case study were generalized to present an optimization scheme for future development of scaffolds to be implemented in vitro or in vivo. Although it is likely that manufacture and testing will be required to finalize design specifications, it is expected that the use of the rational design optimization will reduce the number of iterations required to determine final prototype geometries and flow conditions. As the range of mechanical signals conducive to guiding cell fate in situ is further elucidated, these refined design criteria can be integrated into the general optimization rubric, providing a technological platform to exploit nature's endogenous tissue engineering strategies for targeted tissue generation in the lab or the clinic.

Silk Fibroin Microfluidic Devices.

Silk Fibroin Microfluidic Devices.

Delivery of non-viral gene carriers from sphere-templated fibrin scaffolds for sustained transgene expression

Delivery of safe and controlled levels of biomimetic cues to govern host response and reorganization is a fundamental component in the design of tissue engineering scaffolds. Non-viral gene delivery is an approach that exploits the cell machinery to produce proteins while avoiding genomic DNA incorporation. We describe a method to integrate polymeric non-viral gene carriers (polyplexes) within a novel three-dimensional, sphere-templated fibrin scaffold suitable for soft tissue engineering applications. After seeding the scaffolds with NIH-3T3 fibroblasts, different transgene expression profiles were achieved based on the spatial distribution of polyplexes within the scaffold. Scaffolds with polyplexes coated onto the surface of inter-connected pores showed peak transfection at day 5 and linear expression through 15 days. Scaffolds with polyplexes embedded within the fibrils of the biopolymer showed peak expression at 7–9 days and showed linear expression for 21–29 days, depending on the polymer:DNA ratio. Surface-coated polyplexes achieved one order of magnitude greater expression than polyplexes embedded within the scaffold. The integrated material formulations developed in this work provide a useful technology for tissue engineering applications by demonstrating the ability to provide long-term biomimetic cues through non-viral gene delivery.

Design and Fabrication of Bone Tissue Engineering Scaffolds via Rapid Prototyping and CAD

It is an important aspect that rapid prototyping technique can be used into tissue engineering. Biomimetic porous scaffolds made of calcium phosphate mineral are promising structures to develop bone substitute. Bone tissue engineering scaffold characteristics and properties are often considered to be critical factors in their design and fabrication. In this paper according to their demand, some scaffolds were introduced and designed with Computer Aided Design (CAD) software. In order to fabricate the scaffolds, biomaterials, β-tricalcium phosphate (β-TCP), and polymeric blends were used on a selective laser sintering (SLS) system, a kind of rapid prototyping machine, to produce specimens. Then the specimens were treated by high temperature to assess their suitability on SLS processing.

Approaches to heart valve tissue engineering scaffold design

Heart valve disease is a significant cause of mortality worldwide. However, to date, a nonthrombogenic, noncalcific prosthetic, which maintains normal valve mechanical properties and hemodynamic flow, and exhibits sufficient fatigue properties has not been designed. Current prosthetic designs have not been optimized and are unsuitable treatment for congenital heart defects. Research is therefore moving towards the development of a tissue engineered heart valve equivalent. Two approaches may be used in the creation of a tissue engineered heart valve, the traditional approach, which involves seeding a scaffold in vitro, in the presence of specific signals prior to implantation, and the guided tissue regeneration approach, which relies on autologous reseeding in vivo. Regardless of the approach taken, the design of a scaffold capable of supporting the growth of cells and extracellular matrix generation and capable of withstanding the unrelenting cardiovascular environment while forming a tight seal during closure, is critical to the success of the tissue engineered construct. This paper focuses on the quest to design, such a scaffold.

Computational design of tissue engineering scaffolds

Tissue engineering utilizes porous biomaterial scaffolds to deliver biological factors that accelerate tissue healing. These two functions require scaffolds be designed for mechanical loading and mass transport. The purpose of this paper was to apply both ad hoc and topology optimization homogenization based design approaches to create scaffold architectures, and to determine how these architectures compare to theoretical bounds on effective stiffness. Open cell scaffold architectures demonstrated a wide range of permeability, but were all below isotropic effective stiffness bounds. Wavy fiber architectures provide a means to approach the lower bounds. Using image-based techniques, designed architectures may be incorporated in anatomic shapes.

Current development of scaffold design and fabrication in bone tissue engineering

组织工程骨是由人工支架材料体外复合扩增的成骨细胞或其前体细胞及生物活性分子构成,高孔隙支架材料在细胞黏附、增殖和新骨组织形成过程中起到最重要的作用.过去的十年,产生和发展了许多合成生物可降解支架材料的技术,骨组织工程研究取得了可喜的成绩.本文主要是回顾这些技术在支架材料的设计、制做工艺,以及各种技术不同参数对支架材料(孔隙率、孔径大小、孔隙连通率等)的影响,阐述不同技术的发展过程的优缺点。

Erratum: Porous scaffold design for tissue engineering

Nature Materials 4, 518–524 (2005) doi: 10.1038/nmat1421 In this Progress Article, the caption for Figure 6 was incorrect; the correct wording is below. Figure 6 Cartilage regeneration by chondrocyte delivery on designed Bioplotter-fabricated PEG/PBT scaffolds is superior to PEG/PBT scaffolds made by porogen leaching.

Design and analysis of tissue engineering scaffolds that mimic soft tissue mechanical anisotropy

Tissue engineered constructs must exhibit tissue-like functional properties, including mechanical behavior comparable to the native tissues they are intended to replace. Moreover, the ability to reversibly undergo large strains can help to promote and guide tissue growth. Electrospun poly (ester urethane) ureas (ES-PEUU) are elastomeric and allow for the control of fiber diameter, porosity, and degradation rate. ES-PEUU scaffolds can be fabricated to have a well-aligned fiber network, which is important for applications involving mechanically anisotropic soft tissues. We have developed ES-PEUU scaffolds under variable speed conditions and modeled the effects of fiber orientation on the macro-mechanical properties of the scaffold. To illustrate the ability to simulate native tissue mechanical behavior, we demonstrated that the high velocity spun scaffolds exhibited highly anisotropic mechanical properties closely resembling the native pulmonary heart valve leaflet. Moreover, use of the present fiber-level structural constitutive model allows for the determination of electrospinning conditions to tailor ES-PEUU scaffolds for specific soft tissue applications. The results of this study will help to provide the basis for rationally designed mechanically anisotropic soft tissue engineered implants.

Characterizing nanoscale topography of the aortic heart valve basement membrane for tissue engineering heart valve scaffold design.

A fully effective prosthetic heart valve has not yet been developed. A successful tissue-engineered valve prosthetic must contain a scaffold that fully supports valve endothelial cell function. Recently, topographic features of scaffolds have been shown to influence the behavior of a variety of cell types and should be considered in rational scaffold design and fabrication. The basement membrane of the aortic valve endothelium provides important parameters for tissue engineering scaffold design. This study presents a quantitative characterization of the topographic features of the native aortic valve endothelial basement membrane; topographical features were measured, and quantitative data were generated using scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and light microscopy. Optimal conditions for basement membrane isolation were established. Histological, immunohistochemical, and TEM analyses following decellularization confirmed basement membrane integrity. SEM and AFM photomicrographs of isolated basement membrane were captured and quantitatively analyzed. The basement membrane of the aortic valve has a rich, felt-like, 3-D nanoscale topography, consisting of pores, fibers, and elevations. All features measured were in the sub-100 nm range. No statistical difference was found between the fibrosal and ventricular surfaces of the cusp. These data provide a rational starting point for the design of extracellular scaffolds with nanoscale topographic features that mimic those found in the native aortic heart valve basement membrane.

Fabrication of polymeric scaffolds with a controlled distribution of pores

The design of tissue engineering scaffolds must take into account many factors including successful vascularisation and the growth of cells. Research has looked at refining scaffold architecture to promote more directed growth of tissues through well-defined anisotropy in the pore structure. In many cases it is also desirable to incorporate therapeutic ingredients, such as growth factors, into the scaffold so that their release occurs as the scaffold degrades. Therefore, scaffold fabrication techniques must be found to precisely control, not only the overall porosity of scaffolds, but also the pore size, shape and spatial distribution. This work describes the use of a regularly shaped porogen, sugar spheres, to manufacture polymeric scaffolds. Results show that pre-assembling the spheres created scaffolds with a constant porosity of 60%, but with varying pores sizes from 200-800 microm, leading to a variation in the surface area and likely degradation rate of the scaffolds. Employing different polymer impregnation techniques tailored the number of pores present with a diameter of less than 100 microm to suit different functions, and altering the packing structure of the sugar spheres created scaffolds with novel layered porosity. Replacing sugar spheres with sugar strands formed scaffolds with pores aligned in one direction.

Heparin Modulates Human Osteosarcoma (MG-63) Activities on Collagen-Coated Substrata

Collagen, the major extracellular matrix component, is a popular biomaterial due to its excellent biocompatibility. Collagen is widely used in sutures, corneal bandages, drug delivery gels, and as wound dressings, as well as in a newly emerging interdisciplinary biomedical field, tissue engineering. A better understanding of the interaction between cells and collagen substrata combining other ECM components should allow for a more rational scaffold design for tissue engineering. In this study, we explored the effects of heparin on the behavior of osteoblast-like cell lines (MG-63) on collagen-based substrates. Collagen mixture (0.1mg/mL) with heparin at different concentrations (0.02mg/mL, 0.1mg/mL, and 0.5mg/mL) was coated onto tissue culture polystyrene plates. We found that low concentration of heparin (0.02mg/mL) enhanced cell adhesion and spreading on collagen-coated plates, but did not influence cell growth and alkaline phosphatase activity. On the other hand, high doses of heparin (0.1 and 0.5mg/mL) decreased cell adhesion and spreading, as well as cell proliferation. Our results suggest that a small amount of heparin might support osteoblast attachment and spreading although high heparin concentration is not suitable for cell culture.