Cell distribution in a scaffold with random architectures under the influence of fluid dynamics

Fluid dynamic environment and scaffold architectures have an important influence on cell growth and distribution inside the scaffold. A porous cylindrical scaffold with a central channel is seeded with the sheep mesenchymal stem cells (MSCs) in this study. Then the cell seeded scaffold is continuously perfused with alpha-MEM medium by a peristaltic pump for 7, 14, and 28 days. Histological study shows that the cell proliferation rates are different throughout the whole scaffolds. The different cell coverage is shown in various positions of the scaffold. A computational fluid dynamics (CFD) modeling is used to simulate the flow conditions within perfused cell-seeded scaffolds to give insight into the mechanisms of these cell growth phenomena. Relating the simulation results to perfusion experiments, the even fluid velocity (approximately 0.26-0.64 mm/s) and shear stress (approximately 0.0029-0.027 Pa) are found to correspond to increased cell proliferation within the cell-scaffold constructs. This method exhibits novel capabilities to compare results obtained for different perfusion rates or different scaffold microarchitectures and may allow specific fluid velocities and shear stresses to be determined that optimize the perfusion flow rate, porous scaffold architecture, and distribution of in vitro tissue growth.     

A comparative study of shear stresses in collagen-glycosaminoglycan and calcium phosphate scaffolds in bone tissue-engineering bioreactors

The increasing demand for bone grafts, combined with their limited availability and potential risks, has led to much new research in bone tissue engineering. Current strategies of bone tissue engineering commonly use cell-seeded scaffolds and flow perfusion bioreactors to stimulate the cells to produce bone tissue suitable for implantation into the patient's body. The aim of this study was to quantify and compare the wall shear stresses in two bone tissue engineering scaffold types (collagen-glycosaminoglycan (CG) and calcium phosphate) exposed to fluid flow in a perfusion bioreactor. Based on micro-computed tomography images, three-dimensional numerical computational fluid dynamics (CFD) models of the two scaffold types were developed to calculate the wall shear stresses within the scaffolds. For a given flow rate (normalized according to the cross-sectional area of the scaffolds), shear stress was 2.8 times as high in the CG as in the calcium-phosphate scaffold. This is due to the differences in scaffold geometry, particularly the pore size (CG pore size approximately 96 microm, calcium phosphate pore size approximately 350 microm). The numerically obtained results were compared with those from an analytical method that researchers use widely experimentalists to determine perfusion flow rates in bioreactors. Our CFD simulations revealed that the cells in both scaffold types were exposed to a wide range of wall shear stresses throughout the scaffolds and that the analytical method predicted shear stresses 12% to 21% greater than those predicted using the CFD method. This study demonstrated that the wall shear stresses in calcium phosphate scaffolds (745.2 mPa) are approximately 40 times as high as in CG scaffolds (19.4 mPa) when flow rates are applied that have been experimentally used to stimulate the release of prostaglandin E(2). These findings indicate the importance of using accurate computational models to estimate shear stress and determine experimental conditions in perfusion bioreactors for tissue engineering.     

Finite element study of scaffold architecture design and culture conditions for tissue engineering

Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.     

Effect of flow perfusion on the osteogenic differentiation of bone marrow stromal cells cultured on starch-based three-dimensional scaffolds

This study aims to investigate the effect of culturing conditions (static and flow perfusion) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells seeded on two novel scaffolds exhibiting distinct porous structures. Specifically, scaffolds based on SEVA-C (a blend of starch with ethylene vinyl alcohol) and SPCL (a blend of starch with polycaprolactone) were examined in static and flow perfusion culture. SEVA-C scaffolds were formed using an extrusion process, whereas SPCL scaffolds were obtained by a fiber bonding process. For this purpose, these scaffolds were seeded with marrow stromal cells harvested from femoras and tibias of Wistar rats and cultured in a flow perfusion bioreactor and in 6-well plates for 3, 7, and 15 days. The proliferation and alkaline phosphatase activity patterns were similar for both types of scaffolds and for both culture conditions. However, calcium content analysis revealed a significant enhancement of calcium deposition on both scaffold types cultured under flow perfusion. This observation was confirmed by Von Kossa-stained sections and tetracycline fluorescence. Histological analysis and confocal images of the cultured scaffolds showed a much better distribution of cells within the SPCL scaffolds than the SEVA-C scaffolds, which had limited pore interconnectivity, under flow perfusion conditions. In the scaffolds cultured under static conditions, only a surface layer of cells was observed. These results suggest that flow perfusion culture enhances the osteogenic differentiation of marrow stromal cells and improves their distribution in three-dimensional, starch-based scaffolds. They also indicate that scaffold architecture and especially pore interconnectivity affect the homogeneity of the formed tissue.     

Effects of the architecture of tissue engineering scaffolds on cell seeding and culturing

The advance of rapid prototyping techniques has significantly improved control over the pore network architecture of tissue engineering scaffolds. In this work, we have assessed the influence of scaffold pore architecture on cell seeding and static culturing, by comparing a computer designed gyroid architecture fabricated by stereolithography with a random pore architecture resulting from salt leaching. The scaffold types showed comparable porosity and pore size values, but the gyroid type showed a more than 10-fold higher permeability due to the absence of size-limiting pore interconnections. The higher permeability significantly improved the wetting properties of the hydrophobic scaffolds and increased the settling speed of cells upon static seeding of immortalised mesenchymal stem cells. After dynamic seeding followed by 5 days of static culture gyroid scaffolds showed large cell populations in the centre of the scaffold, while salt-leached scaffolds were covered with a cell sheet on the outside and no cells were found in the scaffold centre. It was shown that interconnectivity of the pores and permeability of the scaffold prolonged the time of static culture before overgrowth of cells at the scaffold periphery occurred. Furthermore, novel scaffold designs are proposed to further improve the transport of oxygen and nutrients throughout the scaffolds and to create tissue engineering grafts with a designed, pre-fabricated vasculature.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Deformation simulation of cells seeded on a collagen-GAG scaffold in a flow perfusion bioreactor using a sequential 3D CFD-elastostatics model

Tissue-engineered bone shows promise in meeting the huge demand for bone grafts caused by up to 4 million bone replacement procedures per year, worldwide. State-of-the-art bone tissue engineering strategies use flow perfusion bioreactors to apply biophysical stimuli to cells seeded on scaffolds and to grow tissue suitable for implantation into the patient's body. The aim of this study was to quantify the deformation of cells seeded on a collagen-GAG scaffold which was perfused by culture medium inside a flow perfusion bioreactor. Using a μCT scan of an unseeded collagen-GAG scaffold, a sequential 3D CFD-deformation model was developed. The wall shear stress and the hydrostatic wall pressure acting on the cells were computed through the use of a CFD simulation and fed into a linear elastostatics model in order to calculate the deformation of the cells. The model used numerically seeded cells of two common morphologies where cells are either attached flatly on the scaffold wall or bridging two struts of the scaffold. Our study showed that the displacement of the cells is primarily determined by the cell morphology. Although cells of both attachment profiles were subjected to the same mechanical load, cells bridging two struts experienced a deformation up to 500 times higher than cells only attached to one strut. As the scaffold's pore size determines both the mechanical load and the type of attachment, the design of an optimal scaffold must take into account the interplay of these two features and requires a design process that optimizes both parameters at the same time.     

Flow Perfusion Improves Seeding of Tissue Engineering Scaffolds with Different Architectures

Engineered bone grafts have been generated in static and dynamic systems by seeding and culturing osteoblastic cells on 3-D scaffolds. Seeding determines initial cellularity and cell spatial distribution throughout the scaffold, and affects cell–matrix interactions. Static seeding often yields low seeding efficiencies and poor cell distributions; thus creating a need for techniques that can improve these parameters. We have evaluated the effect of oscillating flow perfusion on seeding efficiency and spatial distribution of MC3T3-E1 pre-osteoblastic cells in fibrous polystyrene matrices (20, 35 and 50-μm fibers) and foams prepared by salt leaching, using as controls statically seeded scaffolds. An additional control was investigated where static seeding was followed by unidirectional perfusion. Oscillating perfusion resulted in the most efficient technique by yielding higher seeding efficiencies, more homogeneous distribution and stronger cell–matrix interactions. Cell surface density increased with inoculation cell number and then reached a maximum, but significant detachment occurred at greater flow rates. Oxygen plasma treatment of the fibers greatly improved seeding efficiency. Having similar porosity and dimensions, fibrous matrices yielded higher cell surface densities than foams. Fluorescence microscopy and histological analyses in polystyrene and PLLA scaffolds demonstrated that perfusion seeding produced more homogeneous cell distribution, with fibrous matrices presenting greater uniformity than the foams.     

Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique

In this study, we present and characterize a fiber deposition technique for producing three-dimensional poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT/PBT) block co-polymer scaffolds with a 100% interconnecting pore network for engineering of articular cartilage. The technique allowed us to "design-in" desired scaffold characteristics layer by layer by accurately controlling the deposition of molten co-polymer fibers from a pressure-driven syringe onto a computer controlled x-y-z table. By varying PEGT/PBT composition, porosity and pore geometry, 3D-deposited scaffolds were produced with a range of mechanical properties. The equilibrium modulus and dynamic stiffness ranged between 0.05-2.5 and 0.16-4.33 MPa, respectively, and were similar to native articular cartilage explants (0.27 and 4.10 MPa, respectively). 3D-deposited scaffolds seeded with bovine articular chondrocytes supported a homogeneous cell distribution and subsequent cartilage-like tissue formation following in vitro culture as well as subcutaneous implantation in nude mice. This was demonstrated by the presence of articular cartilage extra cellular matrix constituents (glycosaminoglycan and type II collagen) throughout the interconnected pore volume. Similar results were achieved with respect to the attachment of expanded human articular chondrocytes, resulting in a homogeneous distribution of viable cells after 5 days dynamic seeding. The processing methods and model scaffolds developed in this study provide a useful method to further investigate the effects of scaffold composition and pore architecture on articular cartilage tissue formation.     

振荡流动对组织工程用灌注式生物 反应器中剪切力分布影响

目的 考察振荡流动以及三维支架孔径和孔隙率对生物反应器内流速和剪切力分布的影响,并根据理论计算结果为脱细胞骨三维支架和灌注式生物反应器制备提出优化方法。方法 针对实验室前期制备的骨组织工程用脱细胞骨三维支架和灌注式生物反应器,将脱细胞骨三维支架简化为各向同性的多孔介质,对生物反应器内的流速和剪切力分布进行理论建模。结果 振荡流作用时,多孔支架材料内速度和达西剪切力呈现一致的变化规律,不同半径处流速和达西剪切力差异减小,有利于在骨组织工程中对种子细胞进行均匀三维培养。提高入口灌流速度可提高平均达西剪切力;增加多孔支架孔径或孔隙率对支架内流速峰值影响不大,但会显著降低平均达西剪切力;提高入口振荡流动振荡频率可降低支架内流速最大峰值,显著减小不同半径处流速的差异。结论 适宜的振荡流易产生利于骨组织工程干细胞所需剪切力,研究结果有望为优化骨组织工程中种子细胞的三维培养方法提供理论指导。     

Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering

We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young's moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis.     

Flow perfusion culture of human fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.     

Three-dimensional plotted scaffolds with controlled pore size gradients: Effect of scaffold geometry on mechanical performance and cell seeding efficiency

Scaffolds produced by rapid prototyping (RP) techniques have proved their value for tissue engineering applications, due to their ability to produce predetermined forms and structures featuring fully interconnected pore architectures. Nevertheless, low cell seeding efficiency and non-uniform distribution of cells remain major limitations when using such types of scaffold. This can be mainly attributed to the inadequate pore architecture of scaffolds produced by RP and the limited efficiency of cell seeding techniques normally adopted. In this study we aimed at producing scaffolds with pore size gradients to enhance cell seeding efficiency and control the spatial organization of cells within the scaffold. Scaffolds based on blends of starch with poly(ε-caprolactone) featuring both homogeneously spaced pores (based on pore sizes of 0.75 and 0.1 mm) and pore size gradients (based on pore sizes of 0.1-0.75-0.1 and 0.75-0.1-0.75 mm) were designed and produced by three-dimensional plotting. The mechanical performance of the scaffolds was characterized using dynamic mechanical analysis (DMA) and conventional compression testing under wet conditions and subsequently characterized using scanning electron microscopy and micro-computed tomography. Osteoblast-like cells were seeded onto such scaffolds to investigate cell seeding efficiency and the ability to control the zonal distribution of cells upon seeding. Scaffolds featuring continuous pore size gradients were originally produced. These scaffolds were shown to have intermediate mechanical and morphological properties compared with homogenous pore size scaffolds. The pore size gradient scaffolds improved seeding efficiency from ～35% in homogeneous scaffolds to ～70% under static culture conditions. Fluorescence images of cross-sections of the scaffolds revealed that scaffolds with pore size gradients induce a more homogeneous distribution of cells within the scaffold.     

Micro-Computed Tomography Based Computational Fluid Dynamics for the Determination of Shear Stresses in Scaffolds Within a Perfusion Bioreactor

Perfusion bioreactors are known to exert shear stresses on cultured cells, leading to cell differentiation and enhanced extracellular matrix deposition on scaffolds. The influence of the scaffold’s porous microstructure is investigated for a polycaprolactone (PCL) scaffold with a regular microarchitecture and a silk fibroin (SF) scaffold with an irregular network of interconnected pores. Their complex 3D geometries are imaged by micro-computed tomography and used in direct pore-level simulations of the entire scaffold–bioreactor system to numerically solve the governing mass and momentum conservation equations for fluid flow through porous media. The velocity field and wall shear stress distribution are determined for both scaffolds. The PCL scaffold exhibited an asymmetric distribution with peak and plateau, while the SF scaffold exhibited a homogenous distribution and conditioned the flow more efficiently than the PCL scaffold. The methodology guides the design and optimization of the scaffold geometry.     

Development of a compliant and cytocompatible micro-fibrous polyethylene terephthalate vascular scaffold

Bioengineering approaches have been intensively applied to create small diameter vascular grafts using artificial materials. However, a fully successful, high performing and anti-thrombogenic structure has not been achieved yet. In this study, we have designed and fabricated a novel non-woven fibrous vascular graft with biomechanical properties closely resembling those of native vessels. Vascular cell growth, preservation of cell phenotype, retention of vasoactive properties, as well as the effect of gelatin coating on the cellular interaction with the scaffolds under static and shear stress conditions were investigated. The non-woven fibrous scaffolds were made from melt blown polyethylene terephthalate fiber webs stacked by means of a consolidation technique. The scaffold variables were fiber diameter distribution and the number of consolidated web stacks. SEM analysis confirmed various fiber diameter and pore size ranges corresponding to the different conditions. The scaffolds showed burst pressure values of ～1500 mmHg and compliance (8.4 ± 1.0 × 10(-2) % mmHg(-1) ) very similar to those of native arteries (～8 × 10(-2) % mmHg(-1) ). The structure with the smallest fiber diameter range (1-5 μm) and pore size range (1-20 μm) was the most suitable for the growth of human brain endothelial cells and aortic smooth muscle cells. The cells maintained their specific cell phenotype, expressed collagen and elastin and produced cAMP in response to α-calcitonin gene-related peptide. However, under shear stress conditions (0.9 dyne cm(-2) ), only 30% of the cells were retained in both uncoated and gelatin-coated scaffolds indicating the need for improving the cell retention capacity of these structures, which is our future research direction. This study indicates that the biomechanical and biocompatible properties of this novel vascular scaffold are promising for the development of a vascular graft with similar characteristics to those of native vessels.     

Phase separation,pore structure,and properties of nanofibrous gelatin scaffolds

The development of three-dimensional (3D) biomimetic scaffolds which provide an optimal environment for cells adhesion, proliferation and differentiation, and guide new tissue formation has been one of the major goals in tissue engineering. In this work, a processing technique has been developed to create 3D nanofibrous gelatin (NF-gelatin) scaffolds, which mimic both the physical architecture and the chemical composition of natural collagen. Gelatin matrices with nanofibrous architecture were first created by using a thermally induced phase separation (TIPS) technique. Macroporous NF-gelatin scaffolds were fabricated by combining the TIPS technique with a porogen-leaching process. The processing parameters were systematically investigated in relation to the fiber diameter, fiber length, surface area, porosity, pore size, interpore connectivity, pore wall architecture, and mechanical properties of the NF-gelatin scaffolds. The resulting NF-gelatin scaffolds possess high surface areas (>32 m²/g), high porosities (>96%), well-connected macropores, and nanofibrous pore wall structures. The technique advantageously controls macropore shape and size by paraffin spheres, interpore connectivity by assembly conditions (time and temperature of heat treatment), pore wall morphology by phase separation and post-treatment parameters, and mechanical properties by polymer concentration and crosslinking density. Compared to commercial gelatin foam (Gelfoam^®), the NF-gelatin scaffold showed much better dimensional stability in a tissue culture environment. The NF-gelatin scaffolds, therefore, are excellent scaffolds for tissue engineering.     

Influence of the porosity of starch-based fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal cells cultured in a flow perfusion bioreactor

This study investigates the influence of the porosity of fiber mesh scaffolds obtained from a blend of starch and poly(epsilon-caprolactone) on the proliferation and osteogenic differentiation of marrow stromal cells cultured under static and flow perfusion conditions. For this purpose, biodegradable scaffolds were fabricated by a fiber bonding method into mesh structures with two different porosities-- 50 and 75%. These scaffolds were then seeded with marrow stromal cells harvested from Wistar rats and cultured in a flow perfusion bioreactor or in 6-well plates for up to 15 days. Scaffolds of 75% porosity demonstrated significantly enhanced cell proliferation under both static and flow perfusion culture conditions. The expression of alkaline phosphatase activity was higher in flow cultures, but only for cells cultured onto the higher porosity scaffolds. Calcium deposition patterns were similar for both scaffolds, showing a significant enhancement of calcium deposition on cellscaffold constructs cultured under flow perfusion, as compared to static cultures. Calcium deposition was higher in scaffolds of 75% porosity, but this difference was not statistically significant. Observation by scanning electron microscopy showed the formation of pore-like structures within the extracellular matrix deposited on the higher porosity scaffolds. Fourier transformed infrared spectroscopy with attenuated total reflectance and thin-film X-ray diffraction analysis of the cell-scaffold constructs after 15 days of culture in a flow perfusion bioreactor revealed the presence of a mineralized matrix similar to bone. These findings indicate that starch-based scaffolds, in conjunction with fluid flow bioreactor culture, minimize diffusion constraints and provide mechanical stimulation to the marrow stromal cells, leading to enhancement of differentiation toward development of bone-like mineralized tissue. These results also demonstrate that the scaffold structure, namely, the porosity, influences the sequential development of osteoblastic cells and, in combination with the culture conditions, may affect the functionality of tissues formed in vitro.     

A mesofluidics-based test platform for systematic development of scaffolds for in situ cardiovascular tissue engineering

Recently, in situ tissue engineering has emerged as a new approach to obtain autologous, living replacement tissues with off-the-shelf availability. The method is based on the use of an instructive biodegradable scaffold that is capable of repopulation with host cells in situ and subsequent tissue formation. This approach imposes high demands on scaffold properties. For cardiovascular grafts, the repopulation with endogenous cells from the circulation is further hypothesized to be influenced by the hemodynamic environment of the scaffold. To systematically study the effect of scaffold properties on the response of circulating cells, we aimed to develop a mesofluidics-based in vitro test platform that enables on-stage investigation of the interaction of circulating cells with three-dimensional (3D) synthetic scaffolds under physiologic hemodynamic conditions. The test platform consists of a custom-developed cross-flow chamber that houses small-scale 3D scaffolds. The cross-flow chamber is incorporated into a flow-loop to drive a cell suspension along the scaffold with physiological wall shear stress and perfusion pressure. The fluidics system is validated numerically and experimentally using a computational fluid dynamics model and real-time microbead tracing studies, demonstrating a fully developed flow profile with a homogeneous shear stress distribution over the scaffold. Wall shear stresses and pressure can be controlled independently, well within the target physiological range (0-8 Pa and 0-100 mmHg, respectively). Bench-top evaluation is performed using electrospun poly(?-caprolactone) scaffolds with varying fiber diameter, exposed to a suspension of human peripheral blood mononuclear cells in pulsatile flow for 72 h. Cell adhesion and infiltration are monitored using time-lapsed confocal laser scanning microscopy. In conclusion, we have successfully developed a mesofluidics platform to study cell-scaffold interactions under hemodynamic conditions in vitro. This platform not only enables us to systematically screen and develop potential scaffolds for future in situ cardiovascular tissue engineering approaches, but also acts as a tool to further elucidate processes as observed in vivo.     

Effect of 3D-microstructure of bioabsorbable PGA:TMC scaffolds on the growth of chondrogenic cells

Various biomaterial scaffolds have been investigated for cartilage tissue engineering, although little attention has been paid to the effect of scaffold microstructure on tissue growth. Non-woven, fibrous, bioabsorbable scaffolds constructed from a copolymer of glycolide and trimethylene carbonate with varying levels of porosity and pore size were seeded with mesenchymal stroma cells with a chondrogenic lineage. Scaffolds and media were evaluated for both cell and extracellular matrix organization and content after up to 28 days of culture in a spinner flask. Analysis of DNA and glycosaminoglycan contents showed that the most porous of the three scaffold types, with a porosity of 81% and a porometry determined mean flow pore diameter of 54 microm, supported the most rapid proliferation of cells and accumulation of extracellular matrix. Analysis of the high porosity scaffold system, using Western Blot and immunohistochemistry confirmed the presence of collagen type II and absence of collagen type I, and demonstrated cells with a chondrocyte morphology with aggrecan and collagen II accumulation attached to the scaffolds. It was concluded that the 3D-microstructural characteristics of the scaffold (interconnecting porosity and pore size) play an important role in proliferation and phenotype of chondrogenic cells and accumulation of extracellular matrix molecules.