Design criteria for a printed tissue engineering construct: A mathematical homogenization approach

Cartilage tissue repair procedures currently under development aim to create a construct in which patient-derived cells are seeded and expanded ex vivo before implantation back into the body. The key challenge is producing physiologically realistic constructs that mimic real tissue structure and function. One option with vast potential is to print strands of material in a 3D structure called a scaffold that imitates the real tissue structure; the strands are composed of gel seeded with cells and so provide a template for cartilaginous tissue growth. The scaffold is placed in the construct and pumped with nutrient-rich culture medium to supply nutrients to the cells and remove waste products, thus promoting tissue growth.In this paper we use asymptotic homogenization to determine the effective flow and transport properties of such a printed scaffold system. These properties are used to predict the distribution of nutrient/waste products through the construct, and to specify design criteria for the scaffold that will optimize the growth of functional tissue.     

Improved infiltration of stem cells on electrospun nanofibers

Nanofibrous scaffolds have been recently used in the field of tissue engineering because of their nano-size structure which promotes cell attachment, function, proliferation and infiltration. In this study, nanofibrous polyethersulfone (PES) scaffolds was prepared via electrospinning. The scaffolds were surface modified by plasma treatment and collagen grafting. The surface changes then investigated by contact angle measurements and FTIR-ATR. The results proved grafting of the collagen on nanofibers surface and increased hydrophilicity after plasma treatment and collagen grafting. The cell interaction study was done using stem cells because of their ability to differentiate to different kinds of cell lines. The cells had normal morphology on nanofibers and showed very high infiltration through collagen grafted PES nanofibers. This infiltration capability is very useful and needed to make 3D scaffolds in tissue engineering.     

The famous versus the inconvenient - or the dawn and the rise of 3D-culture systems

One of the greatest impacts on in vitro cell biology was the introduction of three-dimensional (3D) culture systems more than six decades ago and this era may be called the dawn of 3D-tissue culture. Although the advantages were obvious, this field of research was a "sleeping beauty" until the 1970s when multicellular spheroids were discovered as ideal tumor models. With this rebirth, organotypical culture systems became valuable tools and this trend continues to increase. While in the beginning, simple approaches, such as aggregation culture techniques, were favored due to their simplicity and convenience, now more sophisticated systems are used and are still being developed. One of the boosts in the development of new culture techniques arises from elaborate manufacturing and surface modification techniques, especially micro and nano system technologies that have either improved dramatically or have evolved very recently. With the help of these tools, it will soon be possible to generate even more sophisticated and more organotypic-like culture systems. Since 3D perfused or superfused systems are much more complex to set up and maintain compared to use of petri dishes and culture flasks, the added value of 3D approaches still needs to be demonstrated.     

JNK-binding protein 1 regulates NF-κB activation through TRAF2 and TAK1

The mitogen-activated protein kinase (MAPK) cascades, including c-Jun N-terminal kinase (JNK), are composed of a MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Previously, we reported that JNK-binding protein 1 (JNKBP1) enhances JNK activation induced by the TGF-β-activated kinase1 (TAK1) MAPKKK in transfected cells. We have investigated whether JNKBP1 functions as an adaptor protein for nuclear factor (NF)-κB activation mediated by TAK1 in COS-7 cells. Co-expression experiments showed that JNKBP1 interacted with not only TAK1, but also with its upstream regulators, TNF-receptor associated factors 2 and 6 (TRAF2 and TRAF6). An endogenous interaction between JNKBP1 and TRAF2 or TAK1 was confirmed by immunoprecipitation analysis. We also found that JNKBP1 could enhance the NF-κB activation induced by TAK1 and TRAF2, and could promote TRAF2 polyubiquitination. These results suggest a scaffolding role for JNKBP1 in the TRAF2-TAK1-NF-κB signaling pathway.     

Cell–scaffold mechanical interplay within engineered tissue

Effective tissue engineering requires appropriate selection of cells and scaffold, where the latter serves as a mechanical and biological support for cell growth and functionality. The optimal combination of cell source and scaffold properties can vary for each desired application. Such preconditions necessitate enhanced understanding of the interactions between cells and scaffold within engineered tissue. Several studies have examined the deforming effects cells induce in scaffolds via exertion of contractile forces. In contrast, other studies focus on the scaffold's biochemical and mechanical properties and their effects on cell behavior.This review summarizes the mechanical interplay between cells and scaffold within engineered tissue. We present evidence for contractile forces exerted by cells on three-dimensional (3D) scaffolds and discuss existing methods for their quantification. In addition, we address some theories related to the effects of scaffold stiffness and mechanical stimulation on cell behavior. Further understanding of the reciprocal effects between cells and scaffold will provide both enhanced knowledge regarding the expected properties of engineered tissue and more competent tissue regeneration techniques.     

Scaffold Proteins IRSp53 and Spinophilin Regulate Localized Rac Activation by T-lymphocyte Invasion and Metastasis Protein 1 (TIAM1)

The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors.     

Chondrogenesis of articular chondrocytes in hydroxyapatite/chitin/chitosan scaffolds supplemented with pituitary extract

This study analyzes the effects of pituitary extract supplement on the cultivation of bovine knee chondrocytes (BKCs) in three‐dimensional chitin/chitosan biomaterials. Transforming growth factor‐β1 (TGF‐β1) in the supplement was identified by Western blot near 23 kDa, and the immunoassayed concentration of TGF‐β1 in the supplement was about 33 ng/mL. The typical pore diameter of the chitin/chitosan scaffolds was 250 μm, indicating an apposite void space for chondrocyte growth. The characteristic width of needlelike hydroxyapatite crystals was 85 nm after chemical co‐precipitation of hydroxyapatite on the pore surfaces of the scaffolds. Over 4‐wk cultivation, the amounts of proliferated BKCs, secreted glycosaminoglycans and produced collagen were improved with the concentration of TGF‐β1 in culture medium. In addition, the cultivated constructs revealed mature neocartilage with lacunas enclosing BKCs, demonstrating chondrogenesis. Pituitary extract supplement was more efficient in the synthesis of extracellular matrix than pure TGF‐β1. Hence, an appropriate addition of the supplement can enhance the formation of cartilaginous components in the scaffolds to regenerate articular cartilage.     

Visualization of the chromosome scaffold and intermediates of loop domain compaction in extracted mitotic cells

A novel extraction protocol for cells cultured on coverslips is described. Observations of the extraction process in a perfusion chamber reveal that cells of all mitotic stages are not detached from coverslips during extraction, and all stages can be recognized using phase contrast images. We studied the extracted cell morphology and distribution of a major scaffold component - topoisomerase IIalpha, in extracted metaphase and anaphase cells. An extraction using 2M NaCl leads to destruction of chromosomes at the light microscope level. Immunogold studies demonstrate that the only residual structure observed is an axial chromosome scaffold that contains topoisomerase IIalpha. In contrast, mitotic chromosomes are swelled only partially after an extraction using dextran sulphate and heparin, and it appears that this treatment does not lead to total destruction of loop domains. In this case, the chromosome scaffold and numerous structures resembling small rosettes are revealed inside extracted cells. The rosettes observed condense after addition of Mg2+-ions and do not contain topoisomerase IIalpha suggesting that these structures correspond to intermediates of loop domain compaction. We propose a model of chromosome structure in which the loop domains are condensed into highly regular structures with rosette organization.     

Evolution of Metazoan Cell Junction Proteins: The Scaffold Protein MAGI and the Transmembrane Receptor Tetraspanin in the Demosponge Suberites domuncula

Until recently the positioning of the sponges (phylum Porifera) within the metazoan systematics was hampered by the lack of molecular evidence for the existence of junctional structures in the surface cell layers. In this study two genes related to the tight junctions are characterized from the demosponge Suberites domuncula: tetraspanin (SDTM4SF), a cell surface receptor, and MAGI (SDMAGI), a MAGUK (membrane-associated guanylate kinase homologue) protein. Especially the MAGI protein is known in other metazoan animal phyla to exist exclusively in tight junctions. The characteristic domains of MAGI proteins (six PDZ domains, two WW domains, and a truncated guanylate kinase motif) are conserved in the sponge protein. The functional analysis of SDMAGI done by in situ hybridization shows its expression in the surface epithelial layers (exopinacoderm and endopinacoderm). Northern blot studies reveal that expression of SDMAGI and SDTM4SF increases after formation of the pinacoderm layer in the animals as well as in primmorphs. These results support earlier notions that sponges contain junctional structures. We conclude that sponges contain epithelia whose cells are organized by cell junctions.The sequence from Suberites domuncula reported here, the protein membrane-associated guanylate kinase with an inverted arrangement (MAGI), is deposited in the EMBL/GenBank database under accession number AJ580406.