Growth Behavior of Endothelial Cells According to Electrospun poly(D,L-Lactic-Co-Glycolic Acid) Fiber Diameter as a Tissue Engineering Scaffold

Investigating the effect of electrospun fiber diameter on endothelial cell proliferation provides an important guidance for the design of a fabric scaffold. In this study, we prepared biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) fibrous nonwoven mats with different fiber diameters ranged from 200 nm to 5 µm using the electrospinning technique. To control the fiber diameters of PLGA mats, 4 mixture solvents [hexafluoro-2-propanol, 2,2,2,-trifluoroethanol:dimethylformamide (9:1), 2,2,2,-trifluoroethanol:hexafluoro-2-propanol (9:1), chloroform] were used. Average diameters were 200 nm, 600 nm, 1.5 µm, and 5.0 µm, respectively. Stereoscopic structure and spatial characterization of fibrous PLGA mats were analyzed using atomic force microscopy and a porosimeter. The mechanical properties of PLGA mats were analyzed using a universal testing machine. The spreading behavior and infiltration of endothelial cells on PLGA mats were visualized by field emission scanning electron microscopy and hematoxylin and eosin staining. Cell proliferation on different PLGA fibers with different diameters was quantified using the MTT assay. Cells on 200 nm diameter PLGA mats showed rapid attachment and spreading. However, the cells did not penetrate the PLGA mat. Cells cultured on 600 nm and 1.5 µm diameter fibers could infiltrate the pores and cell proliferation was dramatically increased after 14 days. Secreted prostacyclin from endothelial cells on each mat was measured to examine the ability to inhibit platelet activation. This basic study on cell proliferation and fiber diameter with physical characterization provides a foundation for studies examining nonwoven fibrous PLGA mats as a tissue engineering scaffold.

Temperature-Responsive surface for novel co-culture systems of hepatocytes with endothelial cells: 2-D patterned and double layered co-cultures

We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintaed intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.