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SIMULTANEOUS OPTICAL COHERENCE AND CONFOCAL FLUORESCENCE MICROSCOPY IMAGING OF POLY(e-CAPROLACTONE) TISSUE ENGINEERING SCAFFOLDS

Forrest A. Landis, Joy P. Dunkers, and Marc T. Cicerone

Polymers Division, Materials Science and Engineering Laboratory

A microscopic technique has been developed which simultaneously utilizes optical coherence microscopy (OCM) to probe the morphology of polymeric tissue engineering scaffolds and confocal fluorescence microscopy (CFM) to examine the infiltration and proliferation of cells in the scaffold. OCM/CFM imaging has advantages over current techniques as it provides for a non-invasive, insitu examination of the cells within the scaffold in addition to a characterization of the porous, three-dimensional structure of the membrane. In this study, an immiscible blend of a 50/50 mixture (by mass) of poly(ethylene oxide) (PEO) and poly(e-caprolactone) (PCL) was prepared by melt extrusion. The extruded sample was pressed into a thick film and held in the melt at 80 oC to allow for coarsening of the phase-separated domains before cooling to room temperature and crystallization of the blend. The water-soluble PEO was washed from the blend by soaking in water to leave a porous, bicontinuous, and biodegradable PCL membrane with pore diameters in the range of 50 mm to 300 mm. The PCL based scaffold was cultured with fetal chick osteoblasts for 10 weeks and stained with nuclear fast red dye. The OCM images showed dark regions indicating pores within the sample that formed when the PEO was washed out of the blend. When the same area of the scaffold was examined using CFM, bright regions were observed at the edges of the pores which are due to the fluorescence of the stained cells. The CFM image indicates that the cells are penetrating into the membrane and tend to adhere to the pore walls. This OCM/CFM technique has also been utilized to develop three-dimensional renderings of the scaffold morphology by scanning progressively deeper into the sample and combining these depth slices into a composite image.

Name: Forrest Landis
Division: Polymers Division (854)
Laboratory: MSEL
Room and Building address: B122/224
Mail Stop: 8543
Telephone: 301 975-8110
FAX: 301 975-4977
email: forrest.landis@nist.gov

Sigma Xi member?: No

Best Category: Biology and Biotechnology