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There is increasing evidence that surface curvature at a near-cell-scale influences cell behaviour. Epithelial or endothelial cells lining small acinar or tubular body lumens, as those of the alveoli or blood vessels, experience such highly curved surfaces. In contrast, the most commonly used culture substrates for in vitro modelling of these human tissue barriers, ion track-etched membranes, offer only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically curved track-etched membranes, pre-serving the mainly spherical geometry of the cells' native microenvironment. The curved membranes were created by a combination of three-dimensional (3D) micro film (thermo)forming and ion track technology. We could successfully demonstrate the formation, the growth and a first characterization of confluent layers of lung epithelial cell lines and primary alveolar epithelial cells on membranes shaped into an array of hemispherical microwells. Besides their application in submerged culture, we could also demonstrate the compatibility of the bioinspired membranes for air-exposed culture. We observed a distinct cellular response to membrane curvature. Cells (or cell layers) on the curved membranes reveal significant differences compared to cells on flat membranes concerning membrane epithelialization, areal cell density of the formed epithelial layers, their cross-sectional morphology, and proliferation and apoptosis rates, and the same tight barrier function as on the flat membranes. The presented 3D membrane technology might pave the way for more predictive barrier in vitro models in future.

Microfluidic screening platforms offer new possibilities for performing in vitro cell-based assays with higher throughput and in a setting that has the potential to closely mimic the physiological microenvironment. Integrating functional biomaterials into such platforms is a promising approach to obtain a deeper insight into the interactions occurring at the cell-material interface. The success of such an approach is, however, largely dependent on the ability to miniaturize the biomaterials as well as on the choice of the assay used to study the cell-material interactions. In this work, we developed a microfluidic device, the main component of which is made of a widely used biocompatible polymer, polylactic acid (PLA). This device enabled cell culture under different fluidic regimes, including perfusion and diffusion. Through a combination of photolithography, two-photon polymerization and hot embossing, it was possible to microstructure the surface of the cell culture chamber of the device with highly defined geometrical features. Furthermore, using pyramids with different heights and wall microtopographies as an example, adhesion, morphology and distribution of human MG63 osteosarcoma cells were studied. The results showed that both the height of the topographical features and the microstructural properties of their walls affected cell spreading and distribution. This proof-of-concept study shows that the platform developed here is a useful tool for studying interactions between cells and clinically relevant biomaterials under controlled fluidic regimes.

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 SignificanceWe 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. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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 (R-q = 5.5 nm), the calcium phosphate pattern, consisting of dicalcium phosphate anhydrous before, and of beta-tricalcium phosphate after annealing, exhibited a less homogenous morphology and higher roughness (R-q = 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 mu m 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 main objective of this study was to evaluate the effectiveness of a mesenchymal stem cell (MSC)-seeded polyethylene-oxide-terephthalate/polybutylene-terephthalate (PEOT/PBT) scaffold for cartilage tissue repair in an osteochondral defect using a rabbit model. Material characterisation using scanning electron microscopy indicated that the scaffold had a 3D architecture characteristic of the additive manufacturing fabrication method, with a strut diameter of 296 +/- A 52 mu m and a pore size of 512 +/- A 22 mu m x 476 +/- A 25 mu m x 180 +/- A 30 mu m. In vitro optimisation revealed that the scaffold did not generate an adverse cell response, optimal cell loading conditions were achieved using 50 mu g/ml fibronectin and a cell seeding density of 25 x 10(6) cells/ml and glycosaminoglycan (GAG) accumulation after 28 days culture in the presence of TGF beta 3 indicated positive chondrogenesis. Cell-seeded scaffolds were implanted in osteochondral defects for 12 weeks, with cell-free scaffolds and empty defects employed as controls. On examination of toluidine blue staining for chondrogenesis and GAG accumulation, both the empty defect and the cell-seeded scaffold appeared to promote repair. However, the empty defect and the cell-free scaffold stained positive for collagen type I or fibrocartilage, while the cell-seeded scaffold stained positive for collagen type II indicative of hyaline cartilage and was statistically better than the cell-free scaffold in the blinded histological evaluation. In summary, MSCs in combination with a 3D PEOT/PBT scaffold created a reparative environment for cartilage repair. Science Foundation Ireland (SFI) Strategic Research Cluster (SRC), Grant No. SFI: 09/SRC B1794, Wellcome Trust Biomedical Vacation Scholarships Grant Number WTD004448, the European Union’s 7th Framework Programme under Grant Agreement No. HEALTH-2007-B- 223298 (PurStem) peer-reviewed

Calcium phosphates (CaPs) have a widespread use in bone repair and regeneration due to their osteoconductive and bioactive (bone bonding) nature. In the past decade, it has become apparent that a sub-group of CaPs with specific physico-chemical properties have the ability to induce bone formation in ectopic sites (ie, intramuscular or sub-cutaneously). In this chapter, we summarize the scientific evidence of CaP induced bone formation, describe material properties required for bone induction and discuss possible underlying mechanisms. In addition, we also provide data on the clinical efficacy of this unique group of CaPs.

The past half century has seen explosive growth in the use of medical implants. Orthopedic, cardiac, oral, maxillofacial and plastic surgeons are examples of medical specialists treating millions of patients each year by implanting devices varying from pace makers, artificial hip joints, breast and dental implants, to implantable hearing aids. All such medical implants make use of special materials, known as biomaterials, defined as “materials intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ, or function of the body” [D.F. Williams, The Williams Dictionnary of Biomaterials, Liverpool University Press, Liverpool, 1999]. While the priority for the first generation of biomaterials was inertness with living tissues, the field is shifting towards biologically active systems in order to improve their performance and to expand their use. Biomaterials can be combined as scaffolds with cells (i.e. tissue engineering), growth factors or genetic material in order to trigger tissue regeneration. In addition, recent reports have shown the possibility to design biomaterials that can activate cellular processes and tissue formation solely by their intrinsic physicochemical and three dimensional spatial properties. This article reviews the recent developments in the design of biomaterials that integrate our understanding of cellular and molecular mechanisms with materials science. After an overview of the physicochemical and biological processes occurring at the interface between the biomaterials and biological milieu, we will address the biological principles contributing to the design and engineering of advanced biomaterials for application towards recent therapeutic strategies for tissue regeneration. Finally, future directions for the design of advanced biomaterials will be discussed.

Solid Free-Form Fabrication (SFF) technologies allow the fabrication of anatomical 3D scaffolds from computer tomography (CT) or magnetic resonance imaging (MRI) patients’ dataset. These structures can be designed and fabricated with a variable, interconnected and accessible porous network, resulting in modulable mechanical properties, permeability, and architecture that can be tailored to mimic a specific tissue to replace or regenerate. In this study, we evaluated whether anatomical meniscal 3D scaffolds with matching mechanical properties and architecture are beneficial for meniscus replacement as compared to meniscectomy. After acquiring CT and MRI of porcine menisci, 3D fiber-deposited (3DF) scaffolds were fabricated with different architectures by varying the deposition pattern of the fibers comprising the final structure. The mechanical behaviour of 3DF scaffolds with different architectures and of porcine menisci was measured by static and dynamic mechanical analysis and the effect of these tissue engineering templates on articular cartilage was assessed by finite element analysis (FEA) and compared to healthy conditions or to meniscectomy. Results show that 3DF anatomical menisci scaffolds can be fabricated with pore different architectures and with mechanical properties matching those of natural menisci. FEA predicted a beneficial effect of meniscus replacement with 3D scaffolds in different mechanical loading conditions as compared to meniscectomy. No influence of the internal scaffold architecture was found on articular cartilage damage. Although FEA predictions should be further confirmed by in vitro and in vivo experiments, this study highlights meniscus replacement by SFF anatomical scaffolds as a potential alternative to meniscectomy.

Many studies have shown the influence of soluble factors and material properties on the differentiation capacity of mesenchymal stromal cells (MSCs) cultured as monolayers. These types of two-dimensional (2D) studies can be used as simplified models to understand cell processes related to stem cell sensing and mechano-transduction in a three-dimensional (3D) context. For several other mechanisms such as cell-cell signaling, cell proliferation and cell morphology, it is well-known that cells behave differently on a planar surface compared to cells in 3D environments. In classical tissue engineering approaches, a combination of cells, 3D scaffolds and soluble factors are considered as the key ingredients for the generation of mechanically stable 3D tissue constructs. However, when MSCs are used for tissue engineering strategies, little is known about the maintenance of their differentiation potential in 3D scaffolds after the removal of differentiation soluble factors. In this study, the differentiation potential of human MSCs (hMSCs) into the chondrogenic and osteogenic lineages on two distinct 3D scaffolds, additive manufactured electrospun scaffolds, was assessed and compared to conventional 2D culture. Human MSCs cultured in the presence of soluble factors in 3D showed to differentiate to the same extent as hMSCs cultured as 2D monolayers or as scaffold-free pellets, indicating that the two scaffolds do not play a consistent role in the differentiation process. In the case of phenotypic changes, the achieved differentiated phenotype was not maintained after the removal of soluble factors, suggesting that the plasticity of hMSCs is retained in 3D cell culture systems. This finding can have implications for future tissue engineering approaches in which the validation of hMSC differentiation on 3D scaffolds will not be sufficient to ensure the maintenance of the functionality of the cells in the absence of appropriate differentiation signals.

In clinical islet transplantation, allogeneic islets of Langerhans are transplanted into the portal vein of patients with type 1 diabetes, enabling the restoration of normoglycemia. After intra-hepatic transplantation several factors are involved in the decay in islet mass and function mainly caused by an immediate blood mediated inflammatory response, lack of vascularization, and allo- and autoimmunity. Bioengineered scaffolds can potentially provide an alternative extra-hepatic transplantation site for islets by improving nutrient diffusion and blood supply to the scaffold. This would ultimately result in enhanced islet viability and functionality compared to conventional intra portal transplantation. In this regard, the biomaterial choice, the three-dimensional (3D) shape and scaffold porosity are key parameters for an optimal construct design and, ultimately, transplantation outcome. We used 3D bioplotting for the fabrication of a 3D alginate-based porous scaffold as an extra-hepatic islet delivery system. In 3D-plotted alginate scaffolds the surface to volume ratio, and thus oxygen and nutrient transport, is increased compared to conventional bulk hydrogels. Several alginate mixtures have been tested for INS1E ?-cell viability. Alginate/gelatin mixtures resulted in high plotting performances, and satisfactory handling properties. INS1E ?-cells, human and mouse islets were successfully embedded in 3D-plotted constructs without affecting their morphology and viability, while preventing their aggregation. 3D plotted scaffolds could help in creating an alternative extra-hepatic transplantation site. In contrast to microcapsule embedding, in 3D plotted scaffold islets are confined in one location and blood vessels can grow into the pores of the construct, in closer contact to the embedded tissue. Once revascularization has occurred, the functionality is fully restored upon degradation of the scaffold.

A highly porous Ti6Al4V with interconnected porous structure has been developed in our previous study. By using a so-called "Multiple coating" technique, the porous Ti6Al4V can be tailored to resemble cancellous bone in terms of porous structure and mechanical properties. A thin layer of Ti6Al4V slurry was coated on the struts of base porous Ti6Al4V to improve the pore structure. After two additional coating, pore sizes ranged from 100 microm to 700 microm, and the porosity was decreased from approximately 90% to approximately 75%, while the compressive strength was increased from 10.3 +/- 3.3 MPa to 59.4 +/- 20.3 MPa and the Young's modulus increased from 0.8 +/- 0.3 GPa to 1.8 +/- 0.3 GPa. The pore size and porosity are similar to that of cancellous bone, meanwhile the compressive strength is higher than that of cancellous bone, and the Young's modulus is between that of cancellous bone and cortical bone. Porosity, pore size and mechanical properties can be controlled by the parameters in such multiple coating processes. Therefore the porous Ti6Al4V with the characteristics of cancellous bone is expected to be a promising biomaterial for biomedical applications.

In this study, we investigated the influence of octacalcium phosphate (OCP) coating on osteoinductive behaviour of the biomaterials.Porous titanium alloy (Ti6Al4V), hydroxyapatite (HA), biphasic calcium phosphate (BCP) and polyethylene glyco terephtalate/polybuthylene terephtalate (PEGT-PBT) copolymer, all uncoated and coated with biomimetically produced OCP, were implanted in back muscles of 10 goats for 6 and 12 weeks. Uncoated Ti6Al4V and HA did not show any bone formation after intramuscular implantation. All OCP coated implants, except PEGT-PBT did induce bone in the soft tissue. The reason for the non-inductive behaviour of the copolymer is probably its softness, that makes it impossible to maintain its porous shape after implantation. Both uncoated and OCP coated BCP induced bone. However, the amount of animals in which the bone was induced was higher in the coated BCP implants in comparison to the uncoated ones.Osteoinductive potential of biomaterials is influenced by various material characteristics, such as chemical composition, crystallinity, macro- and microstructure.OCP coating has a positive effect on osteoinductivity of the biomaterials. The combination of the advantages of biomimetic coating method above traditional methods, and a good osteoinductivity of OCP coating that is produced by using this method, opens new possibilities for designing more advanced orthopaedic implants. (C) 2004 Kluwer Academic Publishers.

Repair of articular cartilage defects using tissue engineered constructs composed of a scaffold and cultured autologous cells holds promise for future treatments. However, nutrient limitation (e.g. oxygen) has been suggested as a cause of the onset of chondrogenesis solely within the peripheral boundaries of larger constructs. In the present study, oxygen gradients were evaluated by microelectrode measurements in two porous polyethylene glycol terephthalate/polybutylene terephthalate (PEGT/PBT) scaffold architectures, a compression-molded and particle-leached sponge (CM) and a 3D-deposited fiber (3DF) scaffold. During the first 14 days in vitro, gradients intensified, after which a gradual decrease of the gradients was observed in vitro. In vivo, however, gradients changed instantly and became less pronounced. Although similar gradients were observed regardless of scaffold type, significantly more cells were present in the center of 3DF constructs after 2 weeks of in vivo culture. Our results stress the importance of a rationally designed scaffold for tissue-engineering applications. Organized structures, such as the 3DF PEGT/PBT polymer scaffolds, offer possibilities for regulation of nutrient supply and, therefore, hold promise for clinical approaches for cartilage repair. (C) 2004 Elsevier Ltd. All rights reserved.

Regenerating or replacing bone, chondral and osteochondral defects, is an active field in tissue engineering. A general strategy is to use a temporary scaffold in which cells are seeded onto the scaffold prior to implantation or attracted into the scaffold from surrounding tissues in the implantation site to form the desired tissue. Several biomaterials have been used for the fabrication of scaffolds, including polycaprolactone (PCL) which is often used for musculoskeletal tissue engineering. The effect of the PCL scaffold architecture on the cell behavior has been investigated. However, the mechanical properties of the bulk material were not taken into account in these studies. PCL is available in a range of molecular weights, resulting in a range of bulk mechanical properties. Since bulk material stiffness is able to direct cell differentiation, it is likely that the molecular weight of PCL may influence cell behavior. Here, we investigated the bulk material properties of both low and high molecular weight PCL scaffolds fabricated through additive manufacturing. The low molecular weight PCL showed a lower bulk material stiffness. During in vitro cell culture, this resulted in a stronger tendency for hypertrophic chondrogenic differentiation compared to the high molecular weight PCL. This study shows that apart from the polymer chemistry and scaffold architecture, the bulk mechanical properties of the polymer used is an important parameter in scaffold fabrication. This is an important finding for the optimization of osteochondral tissue engineering.

Background: There is little information on specific interactions between dermal fibroblasts and epidermal keratinocytes. The use of engineered skin equivalents consisting of organotypic cocultures of keratinocytes and fibroblasts offers an attractive approach for such studies. - Objectives: To examine the role fibroblasts play in generation and maintenance of reconstructed epidermis. - Methods: Human keratinocytes were seeded on collagen matrices populated with increasing numbers of fibroblasts and cultured for 2 weeks at the air–liquid interface. - Results: In the absence of fibroblasts, stratified epidermis with only three or four viable cell layers was formed. In the presence of fibroblasts, keratinocyte proliferation was stimulated and epidermal morphology was improved. Epidermal morphogenesis was also markedly improved in epidermis generated in organotypic keratinocyte monocultures grown in medium derived from dermal equivalents or from organotypic keratinocyte–fibroblast cocultures. These observations clearly indicate the proliferation-stimulating activity of soluble factors released from fibroblasts. Under all experimental conditions, onset of keratinocyte differentiation was shown by the expression of keratin 10 in all suprabasal cell layers. With increasing numbers of fibroblasts incorporated into the collagen matrix, the expression of markers associated with keratinocyte activation, e.g. keratins 6, 16 and 17 and the cornified envelope precursor SKALP decreased, and involucrin localization shifted toward the granulosum layer. This fibroblast-mediated effect was even more pronounced when the fibroblasts were precultured in the collagen matrices for 1 week instead of overnight. The basement membrane proteins collagen VII and laminin 5 were present at the epithelial–matrix border. The expression of integrin α6β4 and of E-cadherin was comparable with that seen in native skin and was not significantly modulated by fibroblasts. Under all experimental conditions the expression of integrin subunits α2, α3 and β1 was upregulated, indicating keratinocyte activation. - Conclusions: Our results illustrate that numbers of fibroblasts in the collagen matrix and their functional state is a critical factor for establishment of normal epidermal morphogenesis.

The properties of a series of multiblock copolymers, based on hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(butylene terephthalate) (PBT) blocks were investigated with respect to their application as a matrix for controlled release of proteins. The degree of swelling, Q, of the copolymers increased with increasing PEG content and with increasing molecular weight of the PEG segment. Within the composition range tested, Q varied from 1.26 for polymers with PEG segments of 600 g/mol and a PBT content of 60 weight.% up to 3.64 for polymers with PEG segments of 4000 g/mol and a PEG/PBT weight ratio of 80:20. Equilibrium stress (compression)-strain measurements were performed in order to estimate mesh sizes. The mesh size of the copolymers ranged from 38 to 93 A, which was experimentally confirmed by diffusion of vitamin B(12) (hydrodynamic diameter d(h)=16.6 A), lysozyme (d(h)=41 A) and bovine serum albumin (d(h)=72 A). The in vitro degradation of PEG/PBT copolymers with a PEG block length of 1000 g/mol and PEG/PBT weight ratios of 70:30, 60:40 and 40:60 was studied. Matrices with increasing PEG contents exhibited a faster weight loss in phosphate-buffered saline (pH 7.4) at 37 degrees C. Over a degradation period of 54 days, M(n) decreased by about 35-45%, while the composition of the matrices, determined by NMR, remained almost constant.

In our earlier study, we showed that the surface hydroxyl groups of hydroxyapatite have the ability to react with organic isocyanate groups. In this study, the feasibility of grafting poly(methyl methacrylate) (PMMA), poly(n-butyl methacrylate) (PBMA), and Poly(hydroxyethyl methacrylate) [poly(HEMA)] by using the reaction of isocyanate groups with the hydroxyl groups on the surface of HA was investigated. Double bonds were introduced to the surface of HA via the coupling reaction of isocyanateoethyl methacrylate (ICEM) with HA, or through hexamethylene diisocyanate (HMDI) with hydroxyethyl methacrylate (HEMA) and HA, followed by radical polymerization in MMA, BMA, or HEMA. Infrared spectra indicated the existence of polymers on the surfaces of HA. Thermogravimetric analysis also confirmed the presence of grafted polymers on the surface of HA powder particles (20-26 wt%). The polymers gave typical PMMA, PBMA, or poly(HEMA) infrared spectra, with the exception of amide bands, a result of the coupling reaction of ICEM or HMDI with hydroxy groups of HA or HEMA. Therefore it is concluded that the polymers were chemically bonded to the surface of HA through the isocyanate groups of ICEM or HMDI.

Plasma treatment is a method to modify the physicochemical properties of biomaterials, which consequently may affect interactions with cells. Based on the rationale that physical cues on the surface of culture substrates and implants, such as surface roughness, have proven to alter cell behaviour, we used electrospinning to fabricate fibrous three-dimensional scaffolds made of a poly (ethylene oxide terephthalate)/poly (butylene terephthalate) copolymer to mimic the physical microenvironment of extracellular matrix and applied radio-frequency oxygen plasma treatment to create nanoscale roughness. Scanning electron microscopy (SEM) analysis revealed a fibre diameter of 5.49 +/- 0.96 mu m for as-spun meshes. Atomic force microscopy (AFM) measurements determined an exponential increase of surface roughness with plasma treatment time. An increase in hydrophilicity after plasma treatment was observed, which was associated with higher oxygen content in plasma treated scaffolds compared to untreated ones. A more pronounced adsorption of bovine serum albumin occurred on scaffolds treated with plasma for 15 and 30 min compared to untreated fibres. Clinically relevant human mesenchymal stromal cells (hMSCs) were cultured on untreated, 15 and 30 min treated scaffolds. SEM analysis confirmed cell attachment and a pronounced spindle-like morphology on all scaffolds. No significant differences were observed between different scaffolds regarding the amount of DNA, metabolic activity and alkaline phosphatase (ALP) activity after 7 days of culture. The amount of ALP positive cells increased between 7 and 21 days of culture on both untreated and 30 min treated meshes. In addition, ALP staining of cells on plasma treated meshes appeared more pronounced than on untreated meshes after 21 days of culture. Quantitative polymerase chain reaction showed significant upregulation of bone sialoprotein and osteonectin expression on oxygen plasma treated fibres compared to untreated fibres in basic culture medium after 7 days of culture, while no differences were observed in the expression of other osteogenic markers. At 21 days, no osteocalcin protein could be detected by ELISA at any of the substrates. In conclusion, this study shows that oxygen plasma treatment can successfully be applied to modify the nanoscale surface properties of polymeric electrospun fibre meshes, which in turn may positively affect osteogenic differentiation of hMSCs.

The field of biomaterials research is witnessing a steady rise in high-throughput screening approaches, comprising arrays of materials of different physico-chemical composition in a chip format. Even though the cell arrays provide many benefits in terms of throughput, they also bring new challenges. One of them is the establishment of robust homogeneous cell seeding techniques and strong control over cell culture, especially for long time periods. To meet these demands, seeding cells with low variation per tester area is required, in addition to robust cell culture parameters. In this study, we describe the development of a modular chip carrier which represents an important step in standardizing cell seeding and cell culture conditions in array formats. Our carrier allows flexible and controlled cell seeding and subsequent cell culture using dynamic perfusion. To demonstrate the application of our device, we successfully cultured and evaluated C2C12 premyoblast cell viability under dynamic conditions for a period of 5 days using an automated pipeline for image acquisition and analysis. In addition, using computational fluid dynamics, lactate and BMP-2 as model molecules, we estimated that there is good exchange of nutrients and metabolites with the flowing medium, whereas no cross-talk between adjacent TestUnits should be expected. Moreover, the shear stresses to the cells can be tailored uniformly over the entire chip area. Based on these findings, we believe our chip carrier may be a versatile tool for high-throughput cell experiments in biomaterials sciences.

Knowledge of materials associated with osteoinduction gained in the last two decades has created the possibility of replacing autologous bone, the gold standard bone grafting material, with these materials in clinics. This chapter presents the current status regarding the phenomenon and the material factors associated with osteoinduction. Meanwhile, the possible mechanism behind bone induction in the presence of materials is discussed and the possible clinical significance is described. Future directions of this research field are also suggested.

Sufficient nutrient and oxygen transport is a potent modulator of cell proliferation in in vitro tissue-engineered constructs. The lack of oxygen and culture medium can create a potentially lethal environment and limit cellular metabolic activity and growth. Diffusion through scaffold and multi-cellular tissue typically limits transport in vitro, leading to potential hypoxic regions and reduction in the viable tissue thickness. For the in vitro generation of clinically relevant tissue-engineered grafts, current nutrient diffusion limitations should be addressed. Major approaches to overcoming these include culture with bioreactors, scaffolds with artificial microvasculature, oxygen carriers and pre-vascularization of the engineered tissues. This study focuses on the development and utilization of a new perfusion culture system to provide adequate nutrient delivery to cells within large three-dimensional (3D) scaffolds. Perfusion of oxygenated culture medium through porous hollow fiber (HF) integrated within 3D free form fabricated (FFF) scaffolds is proposed. Mouse pre-myoblast (C2C12) cells cultured on scaffolds of poly(ethylene-oxide-terephthalate)–poly(butylene-terephthalate) block copolymer (300PEOT55PBT45) integrated with porous HF membranes of modified poly(ether-sulfone) (mPES, Gambro GmbH) is used as a model system. Various parameters such as fiber transport properties, fiber spacing within a scaffold and medium flow conditions are optimized. The results show that four HF membranes integrated with the scaffold significantly improve the cell density and cell distribution. This study provides a basis for the development of a new HF perfusion culture methodology to overcome the limitations of nutrient diffusion in the culture of large 3D tissue constructs.

The shear stresses in printed scaffold systems for tissue engineering depend on the flow properties and void volume in the scaffold. In this work, computational fluid dynamics (CFD) is used to simulate flow fields within porous scaffolds used for cell growth. From these models the shear stresses acting on the scaffold fibres are calculated. The results led to the conclusion that the Darcian (k 1) permeability constant is a good predictor for the shear stresses in scaffold systems for tissue engineering. This permeability constant is easy to calculate from the distance between and thickness of the fibres used in a 3D printed scaffold. As a consequence computational effort and specialists for CFD can be circumvented by using this permeability constant to predict the shear stresses. If the permeability constant is below a critical value, cell growth within the specific scaffold design may cause a significant increase in shear stress. Such a design should therefore be avoided when the shear stress experienced by the cells should remain in the same order of magnitude.

Trauma or degenerative diseases of the joints are common clinical problems resulting in high morbidity. Although various orthopedic treatments have been developed and evaluated, the low repair capacities of articular cartilage renders functional results unsatisfactory in the long term. Over the last decade, a different approach (tissue engineering) has emerged that aims not only to repair impaired cartilage, but also to fully regenerate it, by combining cells, biomaterials mimicking extracellular matrix (scaffolds) and regulatory signals. The latter is of high importance as growth factors have the potency to induce, support or enhance the growth and differentiation of various cell types towards the chondrogenic lineage. Therefore, the controlled release of different growth factors from scaffolds appears to have great potential to orchestrate tissue repair effectively.This review aims to highlight considerations and limitations of the design, materials and processing methods available to create scaffolds, in relation to the suitability to incorporate and release growth factors in a safe and defined manner. Furthermore, the current state of the art of signalling molecules release from scaffolds and the impact on cartilage regeneration in vitro and in vivo is reported and critically discussed.The strict aspects of biomaterials, scaffolds and growth factor release from scaffolds for cartilage tissue engineering applications are considered.Engineering defined scaffolds that deliver growth factors in a controlled way is a task seldom attained. If growth factor delivery appears to be beneficial overall, the optimal delivery conditions for cartilage reconstruction should be more thoroughly investigated.

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.

3D porous Ti6Al4V scaffolds were successfully directly fabricated by a rapid prototyping technology: 3D fibre deposition. In this study, the rheological properties of Ti6Al4V slurry was studied and the flow rate was analyzed at various pressures and nozzle diameters. Scaffolds with different fibre diameter and porosity were fabricated. ESEM observation and mechanical tests were performed on the obtained porous Ti6Al4V scaffolds with regard to the porous structure and mechanical properties. The results show that these scaffolds have 3D interconnected porous structure and a compressive strength which depends on porosity at constant fibre diameters and on the fibre diameter at constant porosity. These Ti6Al4V scaffolds are expected to be constructs for biomedical applications.

A highly porous Ti6Al4V could be produced with a porous polymeric sponge and Ti6Al4V slurry. However, the rheological properties of Ti6Al4V slurry appeared to be the key issue in the preparation of porous Ti6Al4V. In this study, factors having influence on the rheological properties of Ti6Al4V slurry were addressed in detail. Ti6Al4V powders, organic thickening agents (binders), dispersants, concentration of powder and pH values were optimised with regard to the rheological properties of Ti6Al4V slurry. The results show that Ti6Al4V powder with a mean diameter of 45 microm and spherical shape is beneficial for the preparation of Ti6Al4V slurry. Meanwhile binders with two ingredients, which decompose at different temperatures, have the advantage to keep the shape after debinding. The optimised procedure, based on the findings, made it possible to produce highly porous Ti6Al4V with reticulate porous structure. Porous Ti6Al4V produced by this way is expected to be a promising biomaterial for tissue engineering scaffolds and orthopaedic implant applications.

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

Functional cartilage implants for orthopedic surgery or in vitro tissue evaluation can be created from expanded chondrocytes and biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems results in their dedifferentiation. The hallmark of this process is the switch of collagen synthesis from type II to type I. The aim of this study was to evaluate the postexpansion chondrogenic potential of microcarrier-expanded bovine articular chondrocytes in pellet cultures. A selection of microcarriers was screened for initial attachment of chondrocytes. On the basis of those results and additional selection criteria related to clinical application, Cytodex-1 microcarriers were selected for further investigation. Comparable doubling times were obtained in T-flask and microcarrier cultures. During propagation on Cytodex-1 microcarriers, cells acquired a spherical-like morphology and the presence of collagen type II was detected. Both observations are indicative of a differentiated chondrocyte. Pellet cultures of microcarrier-expanded cells showed cartilage-like morphology and staining for proteoglycans and collagen type II after 14 days. In contrast, pellets of T-flask-expanded cells had a fibrous appearance and showed abundant staining only for collagen type I. Therefore, culture of chondrocytes on microcarriers may offer useful and cost-effective cell expansion opportunities in the field of cartilage tissue engineering.

Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems often results in dedifferentiation. This investigation focuses on the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers. Redifferentiation was evaluated in vitro via pellet cultures in three different culture media. Furthermore, the chondrogenic potential of expanded cells seeded in polyethylene glycol terephthalate/ polybuthylene terephthalate (PEGT/PBT) scaffolds, cultured for 14 days in vitro, and subsequently implanted subcutaneously in nude mice, was assessed. Chondrocytes remained viable during microcarrier culture and yielded doubling times (1.07+/-0.14 days) comparable to T-flask expansion (1.20+/-0.36 days). Safranin-O staining from pellet culture in different media demonstrated that production of GAG per cell was enhanced by microcarrier expansion. Chondrocyte-polymer constructs with cells expanded on microcarriers contained significantly more proteoglycans after subcutaneous implantation (288.5+/-29.2 microg) than those with T-flask-expanded cells (164.0+/-28.7 microg). Total collagen content was similar between the two groups. This study suggests that macroporous gelatin microcarriers are effective matrices for nasal chondrocyte expansion, while maintaining the ability of chondrocyte differentiation. Although the exact mechanism by which chondrocyte redifferentiation is induced through microcarrier expansion has not yet been elucidated, this technique shows promise for cartilage tissue engineering approaches.

Protein release from a series of biodegradable poly(ether ester) multiblock copolymers, based on poly(ethylene glycol) (PEG) and poly(butylene terephthalate) (PBT) was investigated. Lysozyme-containing PEG/PBT films and microspheres were prepared using an emulsion technique. Proteins were effectively encapsulated and dense polymer matrices were formed. The swelling in water of PEG/PBT films reached equilibrium within 3 days. The degree of swelling increased with increasing PEG content and with increasing molecular weight of the PEG segment. The release rate of lysozyme from PEG/PBT films could be tailored very precisely by controlling the copolymer composition. Release rates increased with increasing PEG/PBT weight ratio and increasing molecular weight of the PEG segment. For films prepared from block copolymers with PEG blocks of 4000 g/mol, first-order lysozyme release was observed. For matrices prepared from polymers with PEG segments of 1000 and 600 g/mol, the lysozyme release profile followed near zero-order kinetics. A mathematical description of the release mechanism was developed which takes into account the effect of polymer hydrolytic degradation on solute diffusion. The model was found to be consistent with the experimental observations. Finally, determination of the activity of released protein showed that lysozyme was not damaged during the formulation, storage and release periods.

Protein-containing films and microspheres, based on poly(ethylene glycol)-poly(butylene terephthalate) (PEG-PBT) multiblock copolymers, were prepared from water-in-oil (w/o) emulsions. The properties of the matrices could be controlled by the water-to-polymer ratio (w/p) in the w/o emulsion. A linear increase in water uptake of the matrices was observed with increasing emulsion w/p. This could be explained by an increase in the number of dispersed water-rich domains in the polymer matrix. At low volume fraction of the dispersed phase (epsilon), lysozyme release was mainly dependent on the permeability of the swollen polymer bulk. Above a critical volume fraction (the percolation threshold epsilon(c)), the dispersed water-rich phase formed an interconnected network, which largely enhanced the permeability of the matrix. Determination of the permeability of PEG-PBT matrices for vitamin B(12) as a function of epsilon confirmed the formation of such an interconnected network. This interconnected network could be used to achieve controlled release of a large protein (bovine serum albumin, BSA) from PEG-PBT films and microspheres. Due to its hydrodynamic diameter, BSA was screened by the polymer network when epsilon was low. However above epsilon(c), the fraction released BSA increased with increasing volume fraction of the dispersed phase. A very rapid BSA release could be obtained, with the majority of the incorporated BSA released within 1 day, as well as a slow and continuous release, lasting for over 150 days. When BSA-containing microspheres were prepared with a volume fraction just below the percolation threshold, a delayed release was observed. This was attributed to the effect of polymer degradation on matrix permeability.

The surface grafting reactions of a series of isocyanates with hydroxyapatite particles at different temperatures were studied by Infrared spectrophotometry (IR) and thermal gravimetric analysis (TGA). The study results show that both hexamethylene diisocyanate (HMDI) and isocyanatoethyl methacrylate (ICEM) react readily with HA while ethyl isocyanate acetate (EIA) and butyl isocyanate (BIC) have lower reactivity towards HA particles. It also has been found that the reaction of ICEM with HA follows a second-order reaction mechanism, despite the heterogeneous nature of the reaction, while the reaction of HMDI with HA does not due to the complexity of the reaction. Based on this study, it is concluded that ICEM and HMDI are suitable agents for the coupling of polymers due to their reactivity towards HA.

Summary 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.

Objective: Recent animal studies suggest that activation of Wnt/β-catenin signaling in articular chondrocytes might be a driving factor in the pathogenesis of osteoarthritis (OA) by stimulating amongst others the expression of matrix metalloproteinases (MMPs). This study aimed to investigate the role of Wnt/β-catenin signaling in IL-1β-induced MMP expression in human chondrocytes. Methods: Primary cultures of human, mouse and bovine articular chondrocytes as well as human mesenchymal stem cells (hMSCs) and mouse embryonic fibroblasts (MEFs) were used. Multiple strategies for activation and inhibition of signaling pathways were used. Reporter assays and co-immunoprecipitations were used to study the interaction between β-catenin and NF-κB. Results: In contrast to animal chondrocytes, in human chondrocytes Wnt/β-catenin is a potent inhibitor of MMP1, -3 and -13 expression and generic MMP activity both in basal conditions and after IL-1β stimulation. This effect is independent of TCF/LEF transcription factors but is due to an inhibitory protein-protein interaction between β-catenin and NF-κB. Furthermore we show that IL-1β indirectly activates β-catenin signaling by inducing canonical Wnt7B expression and by inhibiting the expression of canonical Wnt antagonists. Conclusion: Our data reveal an unexpected anti-catabolic role of Wnt/β-catenin signaling in human chondrocytes by counteracting NF-κB-mediated MMP expression induced by IL-1β in a negative feedback loop.