Novel chitosan-polycaprolactone blends as potential scaffold and carrier for corneal endothelial transplantation.

PURPOSE: The aim of this prospective study was to evaluate whether blending two kinds of biomaterials, chitosan and polycaprolactone (PCL), can be used as scaffold and carrier for growth and differentiation of corneal endothelial cells (CECs). METHODS: A transparent, biocompatible carrier with cultured CECs on scaffold would be a perfect replacement graft. In the initial part of experiment, for essential and biocompatible test, chitosan and PCL were evaluated respectively and blended in various proportions by coating. In the later part of this study, for evaluation of potential application, homogenous solutions of 25%, 50%, and 75% PCL compositions were attempted to structure blend membranes. RESULTS: Chitosan, PCL 25, PCL 50, and PCL 75 blends could maintain transparency of culturing substrata. BCECs were found to be reached confluence successfully after 7 days on PCL 25, PCL 50, and PCL 75. The expression of tight junction and extracellular matrix protein were observed as well. Alternatively, only PCL 25 could make blend membrane with enough strength during preparation for carrier in culture. On this blend membrane, the growth pattern and phenotype of BCECs could be observed well. CONCLUSIONS: A ratio of 75:25 (chitosan:PCL) blends showed enough mechanical properties as well as suitable support for cellular activity in cultivating BCECs. Thus, a novel methodology of biodegradable carrier from chitosan and PCL has potential to be a good replacement scaffold for raising CECs for clinical transplantation.

Polyvinylidene fluoride for proliferation and preservation of bovine corneal endothelial cells by enhancing type IV collagen production and deposition.

In this study, biomaterials with different hydrophobic properties including polyvinyl alcohol (PVA), poly(ethylene-co-vinyl alcohol) (EVAL), tissue culture polystyrene (TCPS), and polyvinylidene fluoride (PVDF) were examined in the bovine corneal endothelial cells (BCECs) culture system to elucidate their possible impact on clinical demand and scientific interest. It was found that BCECs were inhibited to attach onto the PVA surface. Conversely, relatively more hydrophobic biomaterials EVAL, TCPS, and PVDF successfully initiate BCEC adhesion. Compared to EVAL, cultured BCECs on TCPS and PVDF exhibited higher viability. Furthermore, fibroblastic transformation on EVAL and TCPS was observed at day 17, but BCECs maintained typical hexagonal shape on the PVDF surface at day 21. This phenomenon can be rescued by previously coating type IV collagen on TCPS but not on EVAL. In addition, when BCECs were cultured on PVDF, the expressions of gap junction connexin-43, differentiation marker N-cadherin, and tight junction ZO-1 were well-developed, resembling the physiological phenotypes. After examining the type IV collagen expression by Western blot analysis and protein absorption test, a possible explanation for the better proliferation and preservation of BCECs on the PVDF substrate is that PVDF is a bioactive substratum which enables BCECs to synthesize and reserve more extracellular matrix type IV collagen, paving an important way to provide a more preferential environment for BCEC cultures. Accordingly, promoting CEC growth effects after cell-biomaterial association may be applied to the tissue engineering of corneal endothelium.

The behaviors of long-term cryopreserved human hepatocytes on different biomaterials.

Isolated human hepatocytes have been extensively investigated due to clinical demand and scientific interest. With a limited supply of available liver tissue and the rapid loss of liver-specific functions in vitro, cryopreservation of human hepatocytes serves as an alternative way to maintain availability of hepatocytes. The purpose of this study was to evaluate the biological behaviors of long-term (more than 4 years) cryopreserved human hepatocytes on biomaterials, including polyvinyl alcohol (PVA), poly(ethylene-co-vinyl alcohol), polyvinylidene fluoride, commercial tissue culture polystyrene (TCPS), and collagen-coated TCPS. Cell attachment was observed by scanning electron microscopy and quantified by lactate dehydrogenase assay. Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction activity. Cell functions were determined by albumin secretion and urea synthesis. Results indicated that human hepatocytes could be cryopreserved for more than 4 years without losing liver-specific functions. Also, PVA was proposed to serve as an appropriate and promising substrate for culturing long-term cryopreserved hepatocytes, maintaining high cell attachment and high level of liver-specific functions.

Electrophoretic behaviors of human hepatoma HepG2 cells.

The electrophoretic mobility of HepG2 cells was measured and a charge-regulated model was proposed to simulate the results obtained. Here, a cell was simulated by a rigid core and an ion-penetrable membrane layer containing both acidic and basic functional groups. The influences of the key parameters, including the pH, the ionic strength, the thickness of the membrane layer of a cell, the density and the dissociation constant of the dissociable functional groups in the membrane layer, and the binding constant of divalent cations on the electrophoretic mobility of a cell were investigated. In particular, the role of the buffer used in the experiment was discussed; this effect was neglected in almost all the relevant theoretical analyses in the literature. We showed that the binding ability of divalent cations to the dissociated functional groups in the membrane layer of a cell ranks as Ca(2+)>Mg(2+)>hexamethonium.

The alteration of cell membrane charge after cultured on polymer membranes.

In this work, cell electrophoresis, measuring the electrophoretic mobility of cells, was used to investigate the variation of surface charge property of cells after cultured on different polymer membranes. HepG2 cell line, derived from a well-differentiated, human hepatoma, was used as a model cell. The polymer biomaterials used in this study included polyvinyl alcohol (PVA), poly(ethylene-co-vinyl alcohol) (EVAL), and polyvinylidene fluoride (PVDF). For cells cultured in the presence of serum, cell mobility after being cultured on PVA substrates was considerably higher than that on EVAL or PVDF substrates. This effect was completely suppressed by cycloheximide (CHX) in the serum-free medium. Taken together, the cell surface charge property can be altered after cells cultured on different polymer substrates. The precise mechanism by which the variation of electrophoretic mobility of cultured cells is unknown, but it is reasonable to assume that the polymer substrates could influence the absorption of serum proteins on cell membrane surface to change cell electrophoretic mobility and, simultaneously, to regulate adhesion, growth and function of cultured cells.