Microporous Honeycomb Films Support Enhanced Bone Formation In Vitro

Substrate topography influences cell adhesion, proliferation, and differentiation. In this study, poly (epsilon-caprolactone) (PCL) films with a well-defined honeycomb structure of porosity 3-4, 5-6, 10-11, or 15-16 mu m were contrasted with flat surfaces for their ability to support primary rat osteoblast adhesion and mineralized extracellular matrix deposition in vitro. Immunofluorescent visualization of vinculin and rhodamine phalloidin binding of actin were used to investigate cell adhesion and morphology. Localization of the alkaline phosphatase activity and Alizarin Red staining were performed to assess the osteoblast activity and deposition of a mineralized matrix. Scanning electron microscopy together with energy-dispersive X-ray spectroscopy was used to provide morphological analysis of cell-film interactions, the deposited matrix, and elemental analysis of the mineralized structures. After 24 h of culture, there were no differences in cell numbers on porous or flat PCL surfaces, but there were changes in cell morphology. Osteoblasts on honeycomb films had a smaller surface area and were less circular than cells on flat PCL. Analysis of cells cultured for 35 days under osteogenic conditions revealed that osteoblasts on all substrates acquired alkaline phosphatase activity, but levels of mineralized matrix were increased on films with 3-4-mu m pore sizes. The bone-like matrix with a Ca: P ratio of 1.69 +/- 0.08 could be identified in larger areas often aligning with substrate topography. In addition, smaller spherical deposits (0.5-1 mu m in diameter) with a Ca: P ratio of 1.3 +/- 0.08 were observed at the surface and particularly within the pores of the PCL film. Localization of vinculin showed significant decreases in the number of focal adhesion structures per unit cell area on 5-6, 10-11, and 15-16-mu m surfaces compared to flat PCL, while focal complexes with a smaller area (0-2 mu m(2)) were more abundant on 3-4 and 5-6-mu m surfaces. Observation of cell interaction with these surfaces identified cytoplasmic protrusions that extended into and sealed the pores of these PCL films creating an extracellular space in which, the conditions could influence the deposition and formation of the mineralized matrix.