Exploring cellular adhesion and differentiation in a micro‐/nano‐hybrid polymer scaffold

Polymer scaffolds play an important role in three dimensional (3‐D) cell culture and tissue engineering. To best mimic the archiecture of natural extracellular matrix (ECM), a nano‐fibrous and micro‐porous combined (NFMP) scaffold was fabricated by combining phase separation and particulate leaching techniques. The NFMP scaffold possesses architectural features at two levels, including the micro‐scale pores and nano‐scale fibers. To evaluate the advantages of micro/nano combination, control scaffolds with only micro‐pores or nano‐fibers were fabricated. Cell grown in NFMP and control scaffolds were characterized with respect to morphology, proliferation rate, diffentiation and adhesion. The NFMP scaffold combined the advantages of micro‐ and nano‐scale structures. The NFMP scaffold nano‐fibers promoted neural differentiation and induced “3‐D matrix adhesion”, while the NFMP scaffold micro‐pores facilitated cell infiltration. This study represents a systematic comparison of cellular activities on micro‐only, nano‐only and micro/nano combined scaffolds, and demonstrates the unique advantages of the later. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Functional 3‐D cardiac co‐culture model using bioactive chitosan nanofiber scaffolds

The in vitro generation of a three‐dimensional (3‐D) myocardial tissue‐like construct employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration, drug testing, and tissue engineering applications. Despite significant progress in this field, current cardiac tissue models are not yet able to stably maintain functional characteristics of cardiomyocytes for long‐term culture and therapeutic purposes. The objective of this study was to fabricate bioactive 3‐D chitosan nanofiber scaffolds using an electrospinning technique and exploring its potential for long‐term cardiac function in the 3‐D co‐culture model. Chitosan is a natural polysaccharide biomaterial that is biocompatible, biodegradable, non‐toxic, and cost effective. Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the extracellular matrix (ECM) in vivo. The chitosan fibers were coated with fibronectin via adsorption in order to enhance cellular adhesion to the fibers and migration into the interfibrous milieu. Ventricular cardiomyocytes were harvested from neonatal rats and studied in various culture conditions (i.e., mono‐ and co‐cultures) for their viability and function. Cellular morphology and functionality were examined using immunofluorescent staining for alpha‐sarcomeric actin (SM‐actin) and gap junction protein, Connexin‐43 (Cx43). Scanning electron microscopy (SEM) and light microscopy were used to investigate cellular morphology, spatial organization, and contractions. Calcium indicator was used to monitor calcium ion flux of beating cardiomyocytes. The results demonstrate that the chitosan nanofibers retained their cylindrical morphology in long‐term cell cultures and exhibited good cellular attachment and spreading in the presence of adhesion molecule, fibronectin. Cardiomyocyte mono‐cultures resulted in loss of cardiomyocyte polarity and islands of non‐coherent contractions. However, the cardiomyocyte‐fibroblast co‐cultures resulted in polarized cardiomyocyte morphology and retained their morphology and function for long‐term culture. The Cx43 expression in the fibroblast co‐culture was higher than the cardiomyocytes mono‐culture and endothelial cells co‐culture. In addition, fibroblast co‐cultures demonstrated synchronized contractions involving large tissue‐like cellular networks. To our knowledge, this is the first attempt to test chitosan nanofiber scaffolds as a 3‐D cardiac co‐culture model. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiac structure and function. These studies will provide useful information to develop a strategy that allows us to generate engineered 3‐D cardiac tissue constructs using biocompatible and biodegradable chitosan nanofiber scaffolds for many tissue engineering applications. Biotechnol. Bioeng. 2013; 110: 637–647. © 2012 Wiley Periodicals, Inc.     

N‐isopropylacrylamide‐based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion

Human mesenchymal stem cells (hMSCs) are colony‐forming unit fibroblasts (CFU‐F) derived from adult bone marrow and have significant potential for many cell‐based tissue‐engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N‐isopropylacrylamide (NIPAM)‐based thermoresponsive polyelectrolyte multilayer (N‐PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N‐PEMU films were made via layer‐by‐layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU‐F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer‐by‐layer approach is an efficient way to form PNIPAM‐based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU‐forming ability and their multi‐lineage differentiation potential. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

New biomedical devices with selective peptide recognition properties. Part 1: Characterization and cytotoxicity of molecularly imprinted polymers

Molecular imprinting is a technique for the synthesis of polymers capable to bind target molecules selectively. The imprinting of large proteins, such as cell adhesion proteins or cell receptors, opens the way to important and innovative biomedical applications. However, such molecules can incur into important conformational changes during the preparation of the imprinted polymer impairing the specificity of the recognition cavities. The "epitope approach" can overcome this limit by adopting, as template, a short peptide sequence representative of an accessible fragment of a larger protein. The resulting imprinted polymer can recognize both the template and the whole molecule thanks to the specific cavities for the epitope. In this work two molecularly imprinted polymer formulations (a macroporous monolith and nanospheres) were obtained using the protected peptide Z-Thr-Ala-Ala-OMe, as template, and Z-Thr-Ile-Leu-OMe, as analogue for the selectivity evaluation, methacrylic acid, as functional monomer, and trimethylolpropane trimethacrylate and pentaerythritol triacrylate (PETRA), as cross-linkers. Polymers were synthesized by precipitation polymerization and characterized by standard techniques. Polymerization and rebinding solutions were analyzed by high performance liquid chromatography. The highly cross-linked polymers retained about 70% of the total template amount, against (20% for the less cross-linked ones). The extracted template amount and the rebinding capacity decreased with the cross-linking degree, while the selectivity showed the opposite behaviour. The PETRA cross-linked polymers showed the best recognition (MIP 2-, alpha=1.71) and selectivity (MIP 2+, alpha'=5.58) capabilities. The cytotoxicity tests showed normal adhesion and proliferation of fibroblasts cultured in the medium that was put in contact with the imprinted polymers.     

Molecularly imprinted polymeric membranes

Molecularly imprinted polymeric membranes have been emerged since 1990. Among various kinds of molecular imprinting studies, the application of molecular imprinting to membrane separation is still a novel investigation. In the present review paper, molecularly imprinted polymeric membranes are summarized and examined. The application of molecular imprinting to membrane separation shortly leads to high performance separation membranes.     

A cell leakproof PLGA‐collagen hybrid scaffold for cartilage tissue engineering

A cell leakproof porous poly(DL ‐lactic‐co‐glycolic acid) (PLGA)‐collagen hybrid scaffold was prepared by wrapping the surfaces of a collagen sponge except the top surface for cell seeding with a bi‐layered PLGA mesh. The PLGA‐collagen hybrid scaffold had a structure consisting of a central collagen sponge formed inside a bi‐layered PLGA mesh cup. The hybrid scaffold showed high mechanical strength. The cell seeding efficiency was 90.0% when human mesenchymal stem cells (MSCs) were seeded in the hybrid scaffold. The central collagen sponge provided enough space for cell loading and supported cell adhesion, while the bi‐layered PLGA mesh cup protected against cell leakage and provided high mechanical strength for the collagen sponge to maintain its shape during cell culture. The MSCs in the hybrid scaffolds showed round cell morphology after 4 weeks culture in chondrogenic induction medium. Immunostaining demonstrated that type II collagen and cartilaginous proteoglycan were detected in the extracellular matrices. Gene expression analyses by real‐time PCR showed that the genes encoding type II collagen, aggrecan, and SOX9 were upregulated. These results indicated that the MSCs differentiated and formed cartilage‐like tissue when being cultured in the cell leakproof PLGA‐collagen hybrid scaffold. The cell leakproof PLGA‐collagen hybrid scaffolds should be useful for applications in cartilage tissue engineering. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Down‐regulation of Notch‐1 and Jagged‐1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,mTOR, and NF‐κB signaling pathways

Notch signaling is involved in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, and survival. Notch‐1 over‐expression has been reported in prostate cancer metastases. Likewise, Notch ligand Jagged‐1 was found to be over‐expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues, suggesting the biological significance of Notch signaling in prostate cancer progression. However, the mechanistic role of Notch signaling and the consequence of its down‐regulation in prostate cancer have not been fully elucidated. Using multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, real‐time RT‐PCR, Western blotting, migration, invasion assay and ELISA, we found that down‐regulation of Notch‐1 or Jagged‐1 was mechanistically associated with inhibition of cell growth, migration, invasion and induction of apoptosis in prostate cancer cells, which was mediated via inactivation of Akt, mTOR, and NF‐κB signaling. Consistent with these results, we found that the down‐regulation of Notch‐1 or Jagged‐1 led to decreased expression and the activity of NF‐κB downstream genes such as MMP‐9, VEGF, and uPA, contributing to the inhibition of cell migration and invasion. Taken together, we conclude that the down‐regulation of Notch‐1 or Jagged‐1 mediated inhibition of cell growth, migration and invasion, and the induction of apoptosis was in part due to inactivation of Akt, mTOR, and NF‐κB signaling pathways. Our results further suggest that inactivation of Notch signaling pathways by innovative strategies could be a potential targeted approach for the treatment of metastatic prostate cancer. J. Cell. Biochem. 109: 726–736, 2010. © 2010 Wiley‐Liss, Inc.     

Fabrication techniques of biomimetic scaffolds in three‐dimensional cell culture: A review

In the last four decades, several researchers worldwide have routinely and meticulously exercised cell culture experiments in two‐dimensional (2D) platforms. Using traditionally existing 2D models, the therapeutic efficacy of drugs has been inappropriately validated due to the failure in generating the precise therapeutic response. Fortunately, a 3D model addresses the foregoing limitations by recapitulating the in vivo environment. In this context, one has to contemplate the design of an appropriate scaffold for favoring the organization of cell microenvironment. Instituting pertinent model on the platter will pave way for a precise mimicking of in vivo conditions. It is because animal cells in scaffolds oblige spontaneous formation of 3D colonies that molecularly, phenotypically, and histologically resemble the native environment. The 3D culture provides insight into the biochemical aspects of cell–cell communication, plasticity, cell division, cytoskeletal reorganization, signaling mechanisms, differentiation, and cell death. Focusing on these criteria, this paper discusses in detail, the diversification of polymeric scaffolds based on their available resources. The paper also reviews the well‐founded and latest techniques of scaffold fabrication, and their applications pertaining to tissue engineering, drug screening, and tumor model development.     

Fabrication of a cell-adhesive protein imprinting surface with an artificial cell membrane structure for cell capturing

We proposed a new molecular imprinting procedure based on molecular integration for the purpose of cell capture. We selected the cell-adhesive protein fibronectin (FN) as the imprinting protein for preparing templates and evaluated selective cell adhesion on the FN imprinting substrate. Silica beads with a diameter of 15 μm were used as the stamp matrix and FN molecules were adsorbed as a monolayer. The FN recognition sites were constructed by integrating a surfactant as the ligand and immobilizing it with new biocompatible photoreactive phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units. As control substrates, imprinting procedures were carried out using albumin (BSA imprinting substrate) and without imprinting protein (non-imprinting substrate). The binding of FN from the cell culture medium with the fetal calf serum was achieved on the FN imprinting substrate, and induced the cell adhesion. On the other hand, on the non-imprinted and BSA imprinting substrates, the FN scarcely bound from the cell culture medium, and subsequent cell adhesion could not be observed on the substrate. These results indicate that the FN binding sites were well constructed by arranging the ligand surfactant to a suitable position and immobilized by the photoreactive MPC polymer. The MPC polymer prevented the nonspecific adsorption of proteins from the cell culture medium. We concluded that this procedure is convenient and can be potentially used for the preparation of surfaces for cell engineering devices.     

Osteostimulation scaffolds of stem cells: BMP‐7‐derived peptide‐decorated alginate porous scaffolds promote the aggregation and osteo‐differentiation of human mesenchymal stem cells

The scaffolds for stem cell‐based bone tissue engineering should hold the ability to guide stem cells osteo‐differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide‐1 (BFP‐1), derived from bone morphogenetic protein‐7, was grafted to alginate polymer chains to prepare peptide‐decorated alginate porous scaffolds (pep‐APS) for promoting osteo‐differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep‐APS exhibited porous structure with about 90% porosity (pore size 100–300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep‐APS were enhanced in vitro. Moreover, pep‐APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo‐related genes expression was obviously up‐regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep‐APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.     

Three‐dimensional cell culture microarray for high‐throughput studies of stem cell fate

We have developed a novel three‐dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high‐throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox‐1, whose levels were also measured in situ using a GFP reporter system. In addition, the high‐throughput capacity of the platform was tested using a dual‐slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor‐4 (FGF‐4) on the pluripotency of mouse ES cells. This high‐throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. Biotechnol. Bioeng. 2010; 106: 106–118. © 2010 Wiley Periodicals, Inc.     

Molecularly imprinted polymers—A closer look at the control polymer used in determining the imprinting effect: A mini review

Molecular recognition displayed by naturally occurring receptors has continued to inspire new innovations aimed at developing systems that can mimic this natural phenomenon. Since 1930s, a technology called molecular imprinting for producing biomimetic receptors has been in place. In this technology, tailor made binding sites that selectively bind a given target analyte (also called template) are incorporated in a polymer matrix by polymerizing functional monomers and cross‐linking monomers around a target analyte followed by removal of the analyte to leave behind cavities specific to the analyte. The success of the imprinting process is defined by two main figures of merit, that is, the imprinting factor, and selectivity, which are determined by comparing the amount of target analyte or structural analogue bound by the molecularly imprinted polymer (MIP) and the nonimprinted polymer (NIP). NIP is a control synthesized alongside the MIP but in the absence of the template. However, questions arise on whether these figures of merit are reliable measures of the imprinting effect because of the significant differences between the MIP and the NIP in terms of their physical and chemical characteristics. Therefore, this review critically looks into this subject, with a view of defining the best approaches for determining the imprinting effect.     

Bioassembly of three‐dimensional embryonic stem cell‐scaffold complexes using compressed gases

Tissues are composed of multiple cell types in a well‐organized three‐dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue‐engineered constructs should have well‐defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue‐ and organ‐specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two‐dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well‐defined microstructure were constructed into a multilayer cell‐scaffold complex using low pressure carbon dioxide (CO₂) and nitrogen (N₂). The mouse ES cells in the assembled constructs were viable, retained the ES cell‐specific gene expression of Oct‐4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO₂ was replaced with N₂. The compressed gas‐assisted bioassembly of stem cell‐polymer constructs opens up a new avenue for tissue engineering and cell therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Comparison of molecular mechanisms mediating cell contact phenomena in model developmental systems: an exploration of universality

Are there universal molecular mechanisms associated with cell contact phenomena during metazoan ontogenesis? Comparison of adhesion systems in disparate model systems indicates the existence of unifying principles. Requirements for multicellularity are (a) the construction of three‐dimensional structures involving a crucial balance between adhesiveness and motility; and (b) the establishment of integration at molecular, cellular, tissue, and organismal levels of organization. Mechanisms for (i) cell–cell and cell–substrate adhesion, (if) cell movement, (Hi) cell‐cell communication, (iv) cellular responses, (v) regulation of these processes, and (vi) their integration with patterning, growth, and other developmental processes are all crucial to metazoan development, and must have been present for the emergence and radiation of Metazoa. The principal unifying themes of this review are the dynamics and regulation of cell contact phenomena. Our knowledge of the dynamic molecular mechanisms underlying cell contact phenomena remains fragmentary. Here we examine the molecular bases of cell contact phenomena using extant model developmental systems (representing a wide range of phyla) including the simplest i.e. sponges, and the eukaryotic protist Dictyostelium discoideum, the more complex Drosophila melanogaster, and vertebrate systems. We discuss cell contact phenomena in a broad developmental context. The molecular language of cell contact phenomena is complex; it involves a plethora of structurally and functionally diverse molecules, and diverse modes of intermolecular interactions mediated by protein and/or carbohydrate moieties. Reasons for this are presumably the necessity for a high degree of specificity of inter‐molecular interactions, the requirement for a multitude of different signals, and the apparent requirement for an increasingly large repertoire of cell contact molecules in more complex developmental systems, such as the developing vertebrate nervous system. However, comparison of molecular models for dynamic adhesion in sponges and in vertebrates indicates that, in spite of significant differences in the details of the way specific cell–cell adhesion is mediated, similar principles are involved in the mechanisms employed by members of disparate phyla. Universal requirements are likely to include (a) rapidly reversible intermolecular interactions; (b) low‐affinity intermolecular interactions with fast on–off rates; (c) the compounding of multiple intermolecular interactions; (d) associated regulatory signalling systems. The apparent widespread employment of molecular mechanisms involving cadherin‐like cell adhesion molecules suggests the fundamental importance of cadherin function during development, particularly in epithelial morphogenesis, cell sorting, and segregation of cells.     

Molecularly imprinted cryogels for human interferon‐alpha purification from human gingival fibroblast culture

Interferons are important proteins for the immune system because of their antiviral, anti‐proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α‐2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN‐α‐MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN‐α‐MIP cryogel was found as 254.8 × 10⁴ IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption–desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α‐2b on HeLa cell line was found between the values 3.45 × 10⁸ IU/mg and 3.75 × 10⁸ IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α‐2b. Copyright © 2013 John Wiley & Sons, Ltd.     

Amino acid sequence preferences to control cell‐specific organization of endothelial cells,smooth muscle cells,and fibroblasts

Effective surface modification with biocompatible molecules is known to be effective in reducing the life‐threatening risks related to artificial cardiovascular implants. In recent strategies in regenerative medicine, the enhancement and support of natural repair systems at the site of injury by designed biocompatible molecules have succeeded in rapid and effective injury repair. Therefore, such a strategy could also be effective for rapid endothelialization of cardiovascular implants to lower the risk of thrombosis and stenosis. To achieve this enhancement of the natural repair system, a biomimetic molecule that mimics proper cellular organization at the implant location is required. In spite of the fact that many reported peptides have cell‐attracting properties on material surfaces, there have been few peptides that could control cell‐specific adhesion. For the advanced cardiovascular implants, peptides that can mimic the natural mechanism that controls cell‐specific organization have been strongly anticipated. To obtain such peptides, we hypothesized the cellular bias toward certain varieties of amino acids and examined the cell preference (in terms of adhesion, proliferation, and protein attraction) of varieties and of repeat length on SPOT peptide arrays. To investigate the role of specific peptides in controlling the organization of various cardiovascular‐related cells, we compared endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs). A clear, cell‐specific preference was found for amino acids (longer than 5‐mer) using three types of cells, and the combinational effect of the physicochemical properties of the residues was analyzed to interpret the mechanism. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A brief review of extrusion‐based tissue scaffold bio‐printing

Extrusion‐based bio‐printing has great potential as a technique for manipulating biomaterials and living cells to create three‐dimensional (3D) scaffolds for damaged tissue repair and function restoration. Over the last two decades, advances in both engineering techniques and life sciences have evolved extrusion‐based bio‐printing from a simple technique to one able to create diverse tissue scaffolds from a wide range of biomaterials and cell types. However, the complexities associated with synthesis of materials for bio‐printing and manipulation of multiple materials and cells in bio‐printing pose many challenges for scaffold fabrication. This paper presents an overview of extrusion‐based bio‐printing for scaffold fabrication, focusing on the prior‐printing considerations (such as scaffold design and materials/cell synthesis), working principles, comparison to other techniques, and to‐date achievements. This paper also briefly reviews the recent development of strategies with regard to hydrogel synthesis, multi‐materials/cells manipulation, and process‐induced cell damage in extrusion‐based bio‐printing. The key issue and challenges for extrusion‐based bio‐printing are also identified and discussed along with recommendations for future, aimed at developing novel biomaterials and bio‐printing systems, creating patterned vascular networks within scaffolds, and preserving the cell viability and functions in scaffold bio‐printing. The address of these challenges will significantly enhance the capability of extrusion‐based bio‐printing.     

Two‐Photon polymerization for microfabrication of three‐dimensional scaffolds for tissue engineering application

In natural tissues cells are embedded in a three‐dimensional fibrous network of biopolymers like collagen, hyaluronic acid etc. This extracellular matrix (ECM) influences the cell fate, the differentiation status, metabolic processes and provides structural integrity. For a three‐dimensional or physiological cell cultivation that are required in biomedical applications (e.g. tissue engineering, BioMEMS) scaffolds are needed. These scaffolds mimic the ECM according to their biocompatibility which comprises aspects of surface compatibility and importantly for tissue engineering applications aspects of structural compatibility. We have evaluated scaffold design parameters for the three‐dimensional cultivation of chondrocytes for the tissue engineering of artificial cartilage. Two‐photon polymerization is a powerful technique for fabrication of polymeric three‐dimensional micro‐ and submicro‐structures. The photoinitiation system for two‐photon polymerization is excited by simultaneous absorption of two photons leading to chemical polymerization reactions. Due to a tight confinement of the excitation volume around the focal point, this method can produce micrometer sized objects maintaining a high spatial resolution down to 100 nm. Two‐photon processes require very high photon densities which are provided by pulsed femtosecond lasers. The potential of this approach for microfabrication of scaffolds for tissue engineering is demonstrated by investigation of the cell response to microstructures with complex three‐dimensional geometry and feature sizes in the range of few micrometers.     

Integrin α8β1 regulates adhesion,migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown. Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.