Cell aggregation enhances bone formation by human mesenchymal stromal cells.

The amount of bone generated using current tissue engineering approaches is insufficient for many clinical applications. Previous in vitro studies suggest that culturing cells as 3D aggregates can enhance their osteogenic potential, but the effect on bone formation in vivo is unknown. Here, we use agarose wells to generate uniformly sized mesenchymal stromal cell (MSC) aggregates. When combined with calcium phosphate ceramic particles and a gel prepared from human platelet-rich plasma, we generated a tissue engineered construct which significantly improved in vivo bone forming capacity as compared to the conventional system of using single cells seeded directly on the ceramic surface. Histology demonstrated the reproducibility of this system, which was tested using cells from four different donors. In vitro studies established that MSC aggregation results in an up-regulation of osteogenic transcripts. And finally, the in vivo performance of the constructs was significantly diminished when unaggregated cells were used, indicating that cell aggregation is a potent trigger of in vivo bone formation by MSCs. Cell aggregation could thus be used to improve bone tissue engineering strategies.

Modeling mechanical signals on the surface of µCT and CAD based rapid prototype scaffold models to predict (early stage) tissue development.

In the field of tissue engineering, mechano-regulation theories have been applied to help predict tissue development in tissue engineering scaffolds in the past. For this, finite element models (FEMs) were used to predict the distribution of strains within a scaffold. However, the strains reported in these studies are volumetric strains of the material or strains developed in the extracellular matrix occupying the pore space. The initial phase of cell attachment and growth on the biomaterial surface has thus far been neglected. In this study, we present a model that determines the magnitude of biomechanical signals on the biomaterial surface, enabling us to predict cell differentiation stimulus values at this initial stage. Results showed that magnitudes of the 2D strain--termed surface strain--were lower when compared to the 3D volumetric strain or the conventional octahedral shear strain as used in current mechano-regulation theories. Results of both µCT and CAD derived FEMs from the same scaffold were compared. Strain and fluid shear stress distributions, and subsequently the cell differentiation stimulus, were highly dependent on the pore shape. CAD models were not able to capture the distributions seen in the µCT FEM. The calculated mechanical stimuli could be combined with current mechanobiological models resulting in a tool to predict cell differentiation in the initial phase of tissue engineering. Although experimental data is still necessary to properly link mechanical signals to cell behavior in this specific setting, this model is an important step towards optimizing scaffold architecture and/or stimulation regimes.

Gene expression profiling of dedifferentiated human articular chondrocytes in monolayer culture.

OBJECTIVE: When primary chondrocytes are cultured in monolayer, they undergo dedifferentiation during which they lose their phenotype and their capacity to form cartilage. Dedifferentiation is an obstacle for cell therapy for cartilage degeneration. In this study, we aimed to systemically evaluate the changes in gene expression during dedifferentiation of human articular chondrocytes to identify underlying mechanisms. METHODS: RNA was isolated from monolayer-cultured primary human articular chondrocytes at serial passages. Gene expression was analyzed by microarray. Based on the microarray analysis, relevant genes and pathways were identified. Their functions in chondrocyte dedifferentiation were further investigated. RESULTS: In vitro expanded human chondrocytes showed progressive changes in gene expression. Strikingly, an overall decrease in total gene expression was detected, which was both gradual and cumulative. DNA methylation was in part responsible for the expression downregulation of a number of genes. Genes involved in many pathways such as the extracellular-signal-regulated kinase (ERK) and Bone morphogenetic protein (BMP) pathways exhibited significant changes in expression. Inhibition of ERK pathway did not show dramatic effects in counteracting dedifferentiation process. BMP-2 was able to decelerate the dedifferentiation and reinforce the maintenance of chondrocyte phenotype in monolayer culture. CONCLUSION: Our study not only improves our knowledge of the intricate signaling network regulating maintenance of chondrocyte phenotype, but also contributes to improved chondrocyte expansion and chondrogenic performance for cell therapy.

Gremlin 1, frizzled-related protein, and Dkk-1 are key regulators of human articular cartilage homeostasis.

OBJECTIVE: The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation. METHODS: Whole-genome gene-expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA. RESULTS: Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled-related protein (FRP), and Dkk-1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long-bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single-nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population-based cohorts. CONCLUSION: Taken together, our study identified Gremlin 1, FRP, and Dkk-1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

Scaffolds with a standardized macro-architecture fabricated from several calcium phosphate ceramics using an indirect rapid prototyping technique.

Calcium phosphate ceramics, commonly applied as bone graft substitutes, are a natural choice of scaffolding material for bone tissue engineering. Evidence shows that the chemical composition, macroporosity and microporosity of these ceramics influences their behavior as bone graft substitutes and bone tissue engineering scaffolds but little has been done to optimize these parameters. One method of optimization is to place focus on a particular parameter by normalizing the influence, as much as possible, of confounding parameters. This is difficult to accomplish with traditional fabrication techniques. In this study we describe a design based rapid prototyping method of manufacturing scaffolds with virtually identical macroporous architectures from different calcium phosphate ceramic compositions. Beta-tricalcium phosphate, hydroxyapatite (at two sintering temperatures) and biphasic calcium phosphate scaffolds were manufactured. The macro- and micro-architectures of the scaffolds were characterized as well as the influence of the manufacturing method on the chemistries of the calcium phosphate compositions. The structural characteristics of the resulting scaffolds were remarkably similar. The manufacturing process had little influence on the composition of the materials except for the consistent but small addition of, or increase in, a beta-tricalcium phosphate phase. Among other applications, scaffolds produced by the method described provide a means of examining the influence of different calcium phosphate compositions while confidently excluding the influence of the macroporous structure of the scaffolds.

Engineering vascularised tissues in vitro.

Tissue engineering aims at replacing or regenerating tissues lost due to diseases or traumas (Langer and Vacanti, 1993). However, mimicking in vitro the physiological complexity of vascularized tissue is a major obstacle, which possibly contributes to impaired healing in vivo. In higher organisms, native features including the vascular network, the lymphatic networks and interstitial flow promote both mass transport and organ development. Attempts to mimic those features in engineered tissues will lead to more clinically relevant cell-based therapies. Aside from current strategies promoting angiogenesis from the host, an alternative concept termed prevascularization is emerging. It aims at creating a biological vasculature inside an engineered tissue prior to implantation. This vasculature can rapidly anastamose with the host and enhances tissue survival and differentiation. Interestingly, growing evidence supports a role of the vasculature in regulating pattern formation and tissue differentiation. Thus, prevascularized tissues also benefit from an intrinsic contribution of their vascular system to their development. From those early attempts are emerging a body of principles and strategies to grow and maintain, in vitro, those self-assembled biological vascular networks. This could lead to the generation of engineered tissues of more physiologically relevant complexity and improved regenerative potential.

Design of biphasic polymeric 3-dimensional fiber deposited scaffolds for cartilage tissue engineering applications.

This report describes a novel system to create rapid prototyped 3-dimensional (3D) fibrous scaffolds with a shell-core fiber architecture in which the core polymer supplies the mechanical properties and the shell polymer acts as a coating providing the desired physicochemical surface properties. Poly[(ethylene oxide) terephthalate-co-poly(butylene) terephthalate] (PEOT/PBT) 3D fiber deposited (3DF) scaffolds were fabricated and examined for articular cartilage tissue regeneration. The shell polymer contained a higher molecular weight of the initial poly(ethylene glycol) (PEG) segments used in the copolymerization and a higher weight percentage of the PEOT domains compared with the core polymer. The 3DF scaffolds entirely produced with the shell or with the core polymers were also considered. After 3 weeks of culture, scaffolds were homogeneously filled with cartilage tissue, as assessed by scanning electron microscopy. Although comparable amounts of entrapped chondrocytes and of extracellular matrix formation were found for all analyzed scaffolds, chondrocytes maintained their rounded shape and aggregated during the culture period on shell-core 3DF scaffolds, suggesting a proper cell differentiation into articular cartilage. This finding was also observed in the 3DF scaffolds fabricated with the shell composition only. In contrast, cells spread and attached on scaffolds made simply with the core polymer, implying a lower degree of differentiation into articular cartilaginous tissue. Furthermore, the shell-core scaffolds displayed an improved dynamic stiffness as a result of a "prestress" action of the shell polymer on the core one. In addition, the dynamic stiffness of the constructs increased compared with the stiffness of the bare scaffolds before culture. These findings suggest that shell-core 3DF PEOT/PBT scaffolds with desired mechanical and surface properties are a promising solution for improved cartilage tissue engineering.

3D screening device for the evaluation of cell response to different electrospun microtopographies.

Micro- and nano-topographies of scaffold surfaces play a pivotal role in tissue engineering applications, influencing cell behavior such as adhesion, orientation, alignment, morphology and proliferation. In this study, a novel microfabrication method based on the combination of soft-lithography and electrospinning for the production of micro-patterned electrospun scaffolds was proposed. Subsequently, a 3D screening device for electrospun meshes with different micro-topographies was designed, fabricated and biologically validated. Results indicated that the use of defined patterns could induce specific morphological variations in human mesenchymal stem cell cytoskeletal organization, which could be related to differential activity of signaling pathways. STATEMENT OF SIGNIFICANCE: We introduce a novel and time saving method to fabricate 3D micropatterns with controlled micro-architectures on electrospun meshes using a custom made collector and a PDMS mold with the desired topography. A possible application of this fabrication technique is represented by a 3D screening system for patterned electrospun meshes that allows the screening of different scaffold/electrospun parameters on cell activity. In addition, what we have developed in this study could be modularly applied to existing platforms. Considering the different patterned geometries, the cell morphological data indicated a change in the cytoskeletal organization with a close correspondence to the patterns, as shown by phenoplot and boxplot analysis, and might hint at the differential activity of cell signaling. The 3D screening system proposed in this study could be used to evaluate topographies favoring cell alignment, proliferation and functional performance, and has the potential to be upscaled for high-throughput.

3D fiber-deposited scaffolds for tissue engineering: influence of pores geometry and architecture on dynamic mechanical properties.

One of the main issues in tissue engineering is the fabrication of scaffolds that closely mimic the biomechanical properties of the tissues to be regenerated. Conventional fabrication techniques are not sufficiently suitable to control scaffold structure to modulate mechanical properties. Within novel scaffold fabrication processes 3D fiber deposition (3DF) showed great potential for tissue engineering applications because of the precision in making reproducible 3D scaffolds, characterized by 100% interconnected pores with different shapes and sizes. Evidently, these features also affect mechanical properties. Therefore, in this study we considered the influence of different structures on dynamic mechanical properties of 3DF scaffolds. Pores were varied in size and shape, by changing fibre diameter, spacing and orientation, and layer thickness. With increasing porosity, dynamic mechanical analysis (DMA) revealed a decrease in elastic properties such as dynamic stiffness and equilibrium modulus, and an increase of the viscous parameters like damping factor and creep unrecovered strain. Furthermore, the Poisson's ratio was measured, and the shear modulus computed from it. Scaffolds showed an adaptable degree of compressibility between sponges and incompressible materials. As comparison, bovine cartilage was tested and its properties fell in the fabricated scaffolds range. This investigation showed that viscoelastic properties of 3DF scaffolds could be modulated to accomplish mechanical requirements for tailored tissue engineered applications.

Online measurement of oxygen consumption by goat bone marrow stromal cells in a combined cell-seeding and proliferation perfusion bioreactor.

In an effort to produce clinically useful volumes of tissue engineered bone products, a direct perfusion bioreactor system was developed. Perfusion flow rate, flow direction, and the position of the bioreactor are factors that influenced the amounts and homogeneity of the cells seeded on the scaffold surface. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cm(3)) of small-sized macroporous biphasic calcium phosphate (BCP) scaffolds (2-6 mm). Cell load and cell distribution were shown using Methylene Blue block staining, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate the viability of the cells. Although cells were not distributed homogenously after cell seeding, the scaffolds were covered with a viable, homogeneous cell layer after 25 days of cultivation. The hybrid structures became interconnected, and a dense layer of extracellular matrix formed on and in the scaffolds. Online oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption could possibly be used to estimate GBMSC population doubling times during growth in this bioreactor system. On the basis of our results, we conclude that a direct perfusion bioreactor system is capable of seeding and proliferating GBMSCs on BCP ceramic scaffolds that can be monitored online during cultivation.

A comparison of bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) implanted in muscle and bone of dogs at different time periods.

Physicochemical modification could implement synthetic materials into osteoinductive materials, which induce bone formation in nonosseous tissues. We hereby studied the relevance between the osteogenic capacities of osteoinductive materials in nonosseous tissues and in osseous sites. Biphasic calcium phosphate ceramic (BCP) and hydroxyapatite ceramic (HA) were implanted in femoral muscles and femoral cortical bone of dogs for 7, 14, 21, 30, 45, 60, 90, 180, and 360 days, respectively. Two dogs were used in each time point. In each dog, four cylinders (phi5x6 mm) per material were implanted in femoral muscles and 2 cylinders (phi5x6 mm) per material in femoral cortical bone. The harvested samples were processed for both histological and histomorphometric analyses. Bone was observed in BCP implanted in femoral muscles since day 30, while in HA since day 45. Quantitatively, more bone was formed in BCP than in HA at each time point after day 30 (p<0.05). The earlier and more bone formed in BCP than in HA suggests BCP a higher osteoinductive potential than HA in muscle. In femoral cortical bone defects, a bridge of bone in the defect with BCP was observed at day 21, while with HA at day 30. At days 14, 21, and 30, significantly more bone was formed in BCP than in HA (p<0.05). The results herein show that osteogenic capacities of osteoinductive materials in nonosseous tissues and osseous sites are correlated: the higher the osteoinductive potential of the material, the faster the bone repair.

Dynamic mechanical properties of 3D fiber-deposited PEOT/PBT scaffolds: an experimental and numerical analysis.

Mechanical properties of three-dimensional (3D) scaffolds can be appropriately modulated through novel fabrication techniques like 3D fiber deposition (3DF), by varying scaffold's pore size and shape. Dynamic stiffness, in particular, can be considered as an important property to optimize the scaffold structure for its ultimate in vivo application to regenerate a natural tissue. Experimental data from dynamic mechanical analysis (DMA) reveal a dependence of the dynamic stiffness of the scaffold on the intrinsic mechanical and physicochemical properties of the material used, and on the overall porosity and architecture of the construct. The aim of this study was to assess the relationship between the aforementioned parameters, through a mathematical model, which was derived from the experimental mechanical data. As an example of how mechanical properties can be tailored to match the natural tissue to be replaced, articular bovine cartilage and porcine knee meniscus cartilage dynamic stiffness were measured and related to the modeled 3DF scaffolds dynamic stiffness. The dynamic stiffness of 3DF scaffolds from poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) copolymers was measured with DMA. With increasing porosity, the dynamic stiffness was found to decrease in an exponential manner. The influence of the scaffold architecture (or pore shape) and of the molecular network properties of the copolymers was expressed as a scaffold characteristic coefficient alpha, which modulates the porosity effect. This model was validated through an FEA numerical simulation performed on the structures that were experimentally tested. The relative deviation between the experimental and the finite element model was less than 15% for all of the constructs with a dynamic stiffness higher than 1 MPa. Therefore, we conclude that the mathematical model introduced can be used to predict the dynamic stiffness of a porous PEOT/PBT scaffold, and to choose the biomechanically optimal structure for tissue engineering applications.

Micro-aggregates do not influence bone marrow stromal cell chondrogenesis.

Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC-based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro-aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFß3 added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFß, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro-aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro-aggregates, under all TGFß conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro-aggregates. Copyright © 2014 John Wiley & Sons, Ltd.

Increased cell seeding efficiency in bioplotted three-dimensional PEOT/PBT scaffolds.

In regenerative medicine studies, cell seeding efficiency is not only optimized by changing the chemistry of the biomaterials used as cell culture substrates, but also by altering scaffold geometry, culture and seeding conditions. In this study, the importance of seeding parameters, such as initial cell number, seeding volume, seeding concentration and seeding condition is shown. Human mesenchymal stem cells (hMSCs) were seeded into cylindrically shaped 4 × 3 mm polymeric scaffolds, fabricated by fused deposition modelling. The initial cell number ranged from 5 × 10(4) to 8 × 10(5) cells, in volumes varying from 50 µl to 400 µl. To study the effect of seeding conditions, a dynamic system, by means of an agitation plate, was compared with static culture for both scaffolds placed in a well plate or in a confined agarose moulded well. Cell seeding efficiency decreased when seeded with high initial cell numbers, whereas 2 × 10(5) cells seemed to be an optimal initial cell number in the scaffolds used here. The influence of seeding volume was shown to be dependent on the initial cell number used. By optimizing seeding parameters for each specific culture system, a more efficient use of donor cells can be achieved. Copyright © 2013 John Wiley & Sons, Ltd.

Triphasic scaffolds for the regeneration of the bone-ligament interface.

A triphasic scaffold (TPS) for the regeneration of the bone-ligament interface was fabricated combining a 3D fiber deposited polycaprolactone structure and a polylactic co-glycolic acid electrospun. The scaffold presented a gradient of physical and mechanical properties which elicited different biological responses from human mesenchymal stem cells. Biological test were performed on the whole TPS and on scaffolds comprised of each single part of the TPS, considered as the controls. The TPS showed an increase of the metabolic activity with culturing time that seemed to be an average of the controls at each time point. The importance of differentiation media for bone and ligament regeneration was further investigated. Metabolic activity analysis on the different areas of the TPS showed a similar trend after 7 days in both differentiation media. Total alkaline phosphatase (ALP) activity analysis showed a statistically higher activity of the TPS in mineralization medium compared to the controls. A different glycosaminoglycans amount between the TPS and its controls was detected, displaying a similar trend with respect to ALP activity. Results clearly indicated that the integration of electrospinning and additive manufacturing represents a promising approach for the fabrication of scaffolds for the regeneration of tissue interfaces, such as the bone-to-ligament one, because it allows mimicking the structural environment combining different biomaterials at different scales.

3D high throughput screening and profiling of embryoid bodies in thermoformed microwell plates.

3D organoids using stem cells to study development and disease are now widespread. These models are powerful to mimic in vivo situations but are currently associated with high variability and low throughput. For biomedical research, platforms are thus necessary to increase reproducibility and allow high-throughput screens (HTS). Here, we introduce a microwell platform, integrated in standard culture plates, for functional HTS. Using micro-thermoforming, we form round-bottom microwell arrays from optically clear cyclic olefin polymer films, and assemble them with bottom-less 96-well plates. We show that embryonic stem cells aggregate faster and more reproducibly (centricity, circularity) as compared to a state-of-the-art microwell array. We then run a screen of a chemical library to direct differentiation into primitive endoderm (PrE) and, using on-chip high content imaging (HCI), we identify molecules, including regulators of the cAMP pathway, regulating tissue size, morphology and PrE gene activity. We propose that this platform will benefit to the systematic study of organogenesis in vitro.

Surface micropatterning with zirconia and calcium phosphate ceramics by micromoulding in capillaries.

An increasing demand exists for biomaterials that are able to actively participate in the process of repair and regeneration of damaged or diseased organs and tissues. Patterning of surfaces of biomaterials with distinct chemical or physical cues is an attractive way to obtain spatial control over their interactions with the biological system. In the current study, micromoulding in capillaries method was used to pattern silicon substrates with bioinert yttria-stabilised zirconia or with bioactive calcium phosphate ceramics, both widely used biomaterials in orthopaedics and dentistry. Micrometer-scale patterns consisted of parallel lines with varying width and spacing. Both ceramics were successfully deposited on the substrate in a pattern defined by the mould. While the yttria-stabilised zirconia pattern was highly homogenous and smooth (Rq = 5.5 nm), the calcium phosphate pattern, consisting of dicalcium phosphate anhydrous before, and of ß-tricalcium phosphate after annealing, exhibited a less homogenous morphology and higher roughness (Rq = 893 nm). Both materials allowed attachment and proliferation of the MG-63 osteosarcoma cell line, independent of the pattern used. While a preferential orientation of cells in the direction of the pattern lines was observed for all patterns, this effect was more pronounced on the lines with a width of up to 20 µm on both yttria-stabilised zirconia and calcium phosphate ceramics, as compared to wider patterns. Furthermore, the cells retained an elongated morphology for a longer period of time on narrow patterns. Micromoulding in capillaries appeared to be a suitable method to pattern both types of ceramics, however further optimisation is needed to improve homogeneity and obtain better control over the chemical phase and crystalline structure of calcium phosphate patterns.

The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage.

A highly interconnecting and accessible pore network has been suggested as one of a number of prerequisites in the design of scaffolds for tissue engineering. In the present study, two processing techniques, compression-molding/particulate-leaching (CM), and 3D fiber deposition (3DF), were used to develop porous scaffolds from biodegradable poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) co-polymers with varying pore architectures. Three-dimensional micro-computed tomography (microCT) was used to characterize scaffold architectures and scaffolds were seeded with articular chondrocytes to evaluate tissue formation. Scaffold porosity ranged between 75% and 80%. Average pore size of tortuous CM scaffolds (182 microm) was lower than those of organized 3DF scaffolds (525 microm). The weight ratio of glycosaminoglycans (GAG)/DNA, as a measure of cartilage-like tissue formation, did not change after 14 days of culture whereas, following subcutaneous implantation, GAG/DNA increased significantly and was significantly higher in 3DF constructs than in CM constructs, whilst collagen type II was present within both constructs. In conclusion, 3DF PEGT/PBT scaffolds create an environment in vivo that enhances cartilaginous matrix deposition and hold particular promise for treatment of articular cartilage defects.

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs.

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.

Three-dimensional fiber-deposited PEOT/PBT copolymer scaffolds for tissue engineering: influence of porosity, molecular network mesh size, and swelling in aqueous media on dynamic mechanical properties.

Among novel scaffold fabrication techniques, 3D fiber deposition (3DF) has recently emerged as a means to fabricate well-defined and custom-made scaffolds for tissue regeneration, with 100% interconnected pores. The mechanical behavior of these constructs is dependent not only on different three-dimensional architectural and geometric features, but also on the intrinsic chemical properties of the material used. These affect the mechanics of the solid material and eventually of 3D porous constructs derived from them. For instance, poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymers are known to have mechanical properties, depending on the PEOT/PBT weight ratio in block form and on the molecular weight of the initial poly(ethylene glycol) (PEG) blocks. These differences are enhanced even more by their different swelling properties in aqueous media. Therefore, this article examines the influence of copolymer compositions in terms of their swelling on dynamic mechanical properties of solid material and porous 3DF scaffolds. The molecular weight of the starting PEG blocks used in the copolymer synthesis varied from 300 to 1000 g/mol. The PEOT/PBT weight ratio in the blocks used varied from 55/45 to 80/20. This corresponded to an increase of the swelling ratio Q from 1.06 to 2.46, and of the mesh size xi from approximately 9 Angstrom to approximately 47 Angstrom. With increased swelling, dynamic mechanical analysis (DMA) revealed a decrease in elastic response and an increase of viscoelasticity. Thus, by coupling structural and chemical characteristics, the viscoelastic properties of PEOT/PBT 3DF scaffolds may be fine tuned to achieve mechanical requirements for a variety of engineered tissues. Ultimately, the combination of 3DF and DMA may be useful to validate the hypothesis that mimicking the biomechanical behavior of a specific tissue for its optimal replacement is an important issue for at least some tissue-engineering applications.