Fabrication of Coated Polycaprolactone Scaffolds and Their Effects on Murine Embryonic Stem Cells
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Fabrication of Coated Polycaprolactone Scaffolds and Their Effects on Murine Embryonic Stem Cells Michael H. Tollon1, Takashi Hamazaki2, Bradley J. Willenberg3, Christopher Batich1, 3, Naohiro Terada2 1 Materials Science and Engineering, 2Pathology, Immunology, and Laboratory Medicine, 3 Biomedical Engineering University of Florida, Gainesville, FL 32611, U.S.A. ABSTRACT In the past decade, tissue engineering has become a great interest in materials science research. Embryonic stem (ES) cell transplantation has become one of the most researched therapies for restoring tissue and organ function. Many studies have investigated the use of porous biodegradable scaffolds to promote cell adhesion, growth, proliferation, differentiation, and to help steer the course of tissue development. Research has shown that extracellular matrices and the basement membranes affect various cell types and cellular behaviors. However, the effects of these materials on ES cell behavior are currently understudied and poorly understood. In this study, the synthetic biodegradable polymer polycaprolactone (PCL) was chosen to create an interconnected, fibrous foam structure. A phase separated scaffold method was developed and the product made was coated with various extracellular materials. When the phase separated PCL scaffolds were coated with Matrigel and gelatin solutions, murine ES (mES) cells attached, spread, and differentiated within the scaffolds. There was little growth on the uncoated material. Coating effects on mES cells were analyzed using flow cytometry, reverse-transcriptase polymerase chain reaction and scanning electron microscopy. It was found that coating the scaffold with different extracellular matrices affects mES cell morphology and differentiation. Matrigel coating causes expression of neural proteins and gelatin produces a hepatocytelike cell.
INTRODUCTION Tissue engineering aims to restore, maintain and/or improve tissue function(s). Biomaterial scaffolds are fundamental components of many tissue engineering strategies. Currently, attempts are being made to utilize degradable polymeric scaffolds loaded with embryonic stem (ES) cells to affect neo-tissue development both in-vitro and in-vivo [1, 2]. Specific scaffold factors influencing ES cell differentiation fate are less known however. We investigated the influence of extracellular matrix scaffold coatings on murine ES (mES) cell morphology, proliferation, and differentiation.
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MATERIALS AND METHODS Poly(ε-caprolactone) (PCL) (Mw ~ 120,000) was obtained from Sigma Aldrich. Field emission scanning electron microscopy (FESEM) was preformed with a JEOL 6335F at 10kV. All fluorescence microscopy was conducted using an Olympus IX70. Flow cytometry was performed with a FACS Sort instrument equipped with CellQuest Acquisition software (BD Biosciences). Fabrication Briefly, a 5% (wt/vol) PCL solution was mixed with deionized (DI) water to generate a fibrous web. Four webs were then layered to create a 3-D scaffold. Scaffolds were sectioned into 2.5cm x 2
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