Three Dimensional OCT in the Engineering of Tissue Constructs: A Potentially Powerful Tool for Assessing Optimal Scaffold Structure

Abstract

Optical Coherence Tomography (OCT) provides detailed, real-time information on the structure and composition of constructs used in tissue engineering. The focus of this work is the OCT three-dimensional assessment of scaffolding architecture and distribution of cells on it. PLGA scaffolds were imaged in two and three-dimensions, both seeded and unseeded with cells. Then two types of scaffolds were reconstructed in three dimensions. Both scaffolding types were examined at three different seeding densities. The importance of three-dimensional assessments was evident, particularly with respect to porosity and identification of asymmetrical cell distribution.

Fig. 1

This image demonstrates a schematic of the OCT system used to generate images of the seeded and unseeded scaffolding. Imaging was performed at 10 frames per second that allowed rapid three-dimensional high-resolution reconstruction. The light source operates at a central frequency of 1300 nanometers that corresponds to an axial resolution of 10 microns measured from the point spread function off a mirror. The lateral resolution is approximately 25 microns. The scan rate is up to 3000 lines per second with a dynamic range of 100 decibels. The power on the sample was 10 milliwatts. An imaging catheter was used that was 0.019 inches in diameter with a focal line length of 2.0 millimeters. Recorded data was reconstructed in 3-D with Image J.

Fig. 2

Photographs of unseeded PLGA (a) and Helistat scaffolding (b). The porosity can be noted.

Fig. 3

Figures 3a and b show two dimensional images of PLGA scaffolding unseeded and seeded, respectively. The scaffolds were seeded with human embryonic kidney cells (HEK-293). It can be seen that in two dimensions little detail is noted in the image and it is difficult, if not impossible, to assess both porosity and the degree of cell growth.

Fig. 4

This figure shows unseeded (a), low density cell seeding (b), and high density cell seeding in PGLA scaffolding imaged in three dimensions. In 4a one rotational frame of the three dimensional scaffolding image is shown. We can see that pore size and distribution is well delineated compared to Fig. (3). In Fig. (3b) we see cell distribution in the scaffolding and the loss of porosity (arrow). This sample was seeded with 1,000,000 HEK-293 cells. In Fig. (3c) the scaffolding has been seeded with 2 million cells. In the periphery of the scaffolding there has been an almost complete loss in pores while the area most distant to the center still shows strong evidence of porosity, emphasizing asymmetrical distribution.

Fig. 5

Three-dimensional imaging of the Helistat^® scaffolding. In 5a a Helistat^® scaffolding that consists of collagen type one, is shown in an unseeded sample. In image b, the scaffolding sample has been seeded with 1,000,000 cells (slightly higher due to the looseness of the collagen) and a reduction in the porosity is noted. However, it can be seen that the largest concentrations of cells occur on the outside of the Helistat^® sponge with relatively little present in the interior. In image 5c, it is noted that where 2,000,000 cells are used to seed the graft the density has increased, however, the center of the graft again still remains relatively unseeded.