fabrication-inverted-colloidal-crystal-poly-ethylene-glycol-scaffold

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Nanyang Technological University

Several new microorganisms have been isolated from soil samples with high epoxide hydrolase activity toward ethyl 3,4-epoxybutyrate. Screening was performed by enrichment culture on alkenes as sole carbon source, followed by chiral gas chromatography. Eight strains were discovered with enantioselectivity from moderate to high level and identified as bacterial and yeast species. Cells were cultivated under aerobic condition at 30 degrees C using glucose as carbon source and resting cells were used as biocatalysts for kinetic resolution of ethyl 3,4-epoxybutyrate. Among isolated microorganisms, Acinetobacter baumannii showed highest enantioselectivity for (S)-enantiomer, resulting in (R)-ethyl-3,4-epoxybutyrates (>99%ee, 46% yield). It is the first report on the fact that epoxide hydrolases originating from bacterial species of A. baumannii was applied to kinetic resolution of ethyl 3,4-epoxybutyrate in order to obtain enantiopure high-value-added (R)-ethyl-3,4-epoxybutyrate.

Production of (R)-ethyl-3,4-epoxybutyrate by newly isolated Acinetobacter baumannii containing epoxide hydrolase.

Epigallocatechin-3-gallate (EGCG), which is the main polyphenolic constituent of green tea, has emerged as a promising candidate for potential applications in selected anticancer therapeutics. Generally, tumor metastasis is known to be correlated with the alterations in cell adhesion and migration of normal cells. Nevertheless, the effect of EGCG on the biophysical responses of tumor cell adhering on extracellular matrix remains obscure. In this study, a thermosenstive poly(N-isopropylacrylamide) (PIPAAm) system was developed to elucidate the potential anti-tumor effect of EGCG on the deadhesion and migration of HepG2 cells. First, both XPS and ELISA validated the coating of laminin (LA) on PIPAAm. Second, a change of nanotopology of LA layer on PIPAAm across the lower solution critical temperature (LCST) was detected with AFM. HepG2 cells seeded on LA-coated PIPAAm surface was shown to go through deadhesion by lowering the temperature below the LCST. Interestingly, EGCG was shown to decelerate the thermally triggered deadhesion of HepG2 cell on LA coated PIPAAm. Moreover, the inhibition of cell deadhesion in EGCG treated cells was shown to be driven by actin remodeling. Interestingly, the modulation of cell deadhesion on LA coated PIPAAm by EGCG leads to the reduction of cell motility as shown by real-time cell migration assay. Overall, the use of PIPAAm system demonstrated the promise of EGCG as anticancer therapy through the suppression of cell deadhesion and migration.

Epigallocatechin-3-gallate induced modulation of cell deadhesion and migration on thermosensitive poly(N-isopropylacrylamide).

In cartilage tissue engineering, hydrogel is widely used as the scaffold for hosting and culturing chondrocyte suspension during neo-tissue formation. In order to develop cultured chondrocytes into a functional cartilage equivalent, the hydrogel must provide an ideal microenvironment for the rapidly proliferating chondrocytes. At the same time, the essential functions of chondrocytes, such as the secretion of type II collagen and glycosaminoglycans, must be maintained. In these studies, we quantitatively characterize the mechanobiology underlying a newly discovered "edge flourish" phenomenon of cultured chondrocytes within a three-dimensional agarose hydrogel, which may ultimately nurture scaffold-free cartilaginous tissue regeneration. First, real-time microscopy was used to track the spatiotemporal distributions of chondrocytes at different focal planes. The chondrocytes were observed to exhibit abundant neo-tissue outgrowth and significant cartilaginous phenotype at the edge of the hydrogel compared to those inside the hydrogel bulk. Secondly, the hydrogel surface stresses induced by the encapsulated chondrocytes were characterized quantitatively in real time using the finite-element method. Finally, the real-time three-dimensional matrix deformations of agarose hydrogel under the influence of chondrocytes were measured using a multiple-particle tracking assay. Our results indicate that the mechanism of the "edge flourish" phenomenon is induced by the oriented outgrowth of chondrocytic isogenous groups located at the edge of hydrogel. These isogenous groups exhibit directed outgrowth towards the surface of the hydrogel and eventually generate substantial surface tension on the interface of hydrogel and medium. Ultimately, the encapsulated chondrocytes closest to the hydrogel/medium interface will spontaneously sprout out of the hydrogel and form a layer of rich proliferative and chondrocytic extracellular matrix secreting chondrocytes at the surface of the hydrogel.

Biomechanical study of the edge outgrowth phenomenon of encapsulated chondrocytic isogenous groups in the surface layer of hydrogel scaffolds for cartilage tissue engineering.

Anchorage-dependent cells such as smooth muscle cells (SMCs) rely on the transmission of actomyosin-generated traction forces to adhere and migrate on the extracellular matrix. The cellular traction forces exerted by SMCs on substrate can be measured from the deformation of substrate with embedded fluorescent markers. With the synchronous use of phase-contrast and fluorescent microscopy, the deformation of polyacrylamide (PAM) gel substrate can be quantitatively determined using particle image velocimetry. This displacement map is then input as boundary conditions for the stress analysis on PAM gel by the finite-element method. In addition to optical microscopy, atomic force microscopy was also used to characterize the PAM substrate using the contact mode, from which the elasticity of PAM can be quantified using Hertzian theory. This provides baseline information for the stress analysis of PAM gel deformation. The material model introduced for the computational part is the Mooney-Rivlin constitutive law because of its long proven usefulness in predicting polymers' mechanical behaviour. Numerical results showed that adhesive stresses are high around the cell edges, which is in accordance with the general phenomena of cellular focal adhesion. Further calculations on the total traction forces indicate a slightly contact-dominated regime for a broad range of Mooney-Rivlin stiffnesses.

Experimental and numerical determination of cellular traction force on polymeric hydrogels.

Thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) with a particular lower critical solution temperature (LCST) have been applied for the non-invasive harvesting of confluent cell layer. Until now, the effect of adhesive ligand on the biophysical responses of cells during cell layer harvesting from PIPAAm has not been elucidated. In this study, the deadhesion kinetics of smooth muscle cells (SMC) on various adhesive ligands immobilized on PIPAAm were investigated. Firstly, the formation of elastin (EL), laminin (LA), hyaluronic acid (HA) and collagen (CL) coating on PIPAAm surfaces were validated with XPS, microBCA assay and AFM. It was shown that EL was most effective in driving cell retraction on PIPAAm surface. Moreover, the highest rate of initial SMC deadhesion on EL-PIPAAm was driven by the formation of stress fibers. Interestingly, HA was most effective in preventing initial SMC detachment from PIPAAm surface in comparison with EL, LA and CL. Also, the adhesion energy of SMC on HA-PIPAAm remained constant, which was two times and six times higher than that on CL-PIPAAm and EL-PIPAAm, respectively from 20 min onward. Overall, the results reported herein pave the way for the engineering of the invasive regeneration/recovery of cells/tissue with adhesive ligand.

Effect of adhesive ligand on cell deadhesion kinetics on poly(N-isopropylacrylamide).

During the past two decades, novel biomaterial scaffold for cell attachment and culture has been developed for applications in tissue engineering, biosensing and regeneration medicine. Tissue engineering of blood vessels remains a challenge owing to the complex three-layer histology involved. In order to engineer functional blood vessels, it is essential to recapitulate the characteristics of vascular smooth muscle cells (SMCs) inside the tunica media, which is known to be critical for vasoconstriction and vasodilation of the circulatory system. Until now, there has been a lack of understanding on the mechanotransduction of the SMC layer during the transformation from viable synthetic to quiescent contractile phenotypes. In this study, microfabricated arrays of discontinuous microwalls coated with fluorescence microbeads were developed to probe the mechanotransduction of the SMC layer. First, the system was exploited for stimulating the formation of a highly aligned orientation of SMCs in native tunica medium. Second, atomic force microscopy in combination with regression analysis was applied to measure the elastic modulus of a polyacrylamide gel layer coated on the discontinuous microwall arrays. Third, the conventional traction force assay for single cell measurement was extended for applications in three-dimensional cell aggregates. Then, the biophysical effects of discontinuous microwalls on the mechanotransduction of the SMC layer undergoing cell alignment were probed. Generally, the cooperative multiple cell-cell and cell-microwall interactions were accessed quantitatively by the newly developed assay with the aid of finite-element modelling. The results show that the traction forces of highly aligned cells lying in the middle region between two opposing microwalls were significantly lower than those lying adjacent to the microwalls. Moreover, the spatial distributions of Von Mises stress during the cell alignment process were dependent on the collective cell layer orientation. Immunostaining of the SMC sheet further demonstrated that the collective mechanotransduction induced by three-dimensional topographic cues was correlated with the reduction of actin and vinculin expression. In addition, the online two-dimensional LC-MS/MS analysis verified the modulation of focal adhesion formation under the influence of microwalls through the regulation in the expression of three key cytoskeletal proteins.

Article	Year
Production of (R)-ethyl-3,4-epoxybutyrate by newly isolated Acinetobacter baumannii containing epoxide hydrolase. Applied microbiology and biotechnology\| PubMed ID: 18301887	2008
Epigallocatechin-3-gallate induced modulation of cell deadhesion and migration on thermosensitive poly(N-isopropylacrylamide). Journal of biomedical materials research. Part A\| PubMed ID: 21661094	2011
Biomechanical study of the edge outgrowth phenomenon of encapsulated chondrocytic isogenous groups in the surface layer of hydrogel scaffolds for cartilage tissue engineering. Acta biomaterialia\| PubMed ID: 21906699	2012
Experimental and numerical determination of cellular traction force on polymeric hydrogels. Interface focus\| PubMed ID: 23050082	2011
Effect of adhesive ligand on cell deadhesion kinetics on poly(N-isopropylacrylamide). Bio-medical materials and engineering\| PubMed ID: 24642971	2014
Collective cell traction force analysis on aligned smooth muscle cell sheet between three-dimensional microwalls. Interface focus\| PubMed ID: 24748953	2014

Soon Seng Ng

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