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Technical Highlights in Heath Care
 

Scaffold Structure and Cell Function Through Multi-modal Imaging and Quantitative Visualization
 
Non-destructive, in vitro evaluation of tissue engineered medical products (TEMPs) will shorten their development time. Imaging techniques such as collinear optical coherence and confocal fluorescence microscopies are being used to address the challenges of imaging these systems. An equally important component of the work is the image quantification as a vehicle to evaluate the voluminous imaging data.
 

Joy P. Dunkers and Marcus T. Cicerone
Tissue engineered medical products (TEMPs) are often three-dimensional (3D) hybrid materials consisting of a porous scaffold upon which the tissue is grown. While it is generally understood that a complex interaction of many variables influences the success of the TEMPs, the precise nature of these interactions has yet to be worked out in many instances. A significant difficulty in furthering the understanding of the interaction between these factors and cell behavior is the lack of a high-resolution imaging technique that can penetrate deeply and non-destructively into the scaffold. We have developed an approach that uses advanced optical imaging to non-invasively monitor the developing tissue. However, before any assessment of the tissue viability can be made, the volumes of imaging data must be rigorously analyzed. Therefore, an equally important component of this effort is image visualization and quantification. Progress in these areas is summarized below.
 

MULTI-MODAL IMAGING


We have constructed an instrument that can gather information on a TEMP using multiple imaging modalities. This means that each channel of imaging data provides different but complimentary information. Optical coherence microscopy (OCM) was chosen as the technique to image scaffold, cell, and tissue structure because of its unique combination of high resolution ("1 mm) and high sensitivity (> 100 dB). OCM is an interferometric technique that uses both confocal and coherence gating mechanisms for stray light rejection, rendering it comparable in resolution to laser scanning confocal microscopy but far superior in imaging depth. We have added confocal fluorescence microscopy (CFM) to the OCM to collect information on cell function using traditional cell staining techniques. In our collinear instrument, we collect volumetric images of cell and scaffold structure using the OCM channel and cell function using the CFM channel. Each channel is then overlayed in the rendered image for maximal insight.
 
Figure 1 displays merged and registered OCM and CFM images 145 mm below the surface of a TEMP. The TEMP consists of a volume fraction of 50 % poly(e-caprolactone) (PCL) scaffold that was cultured with fetal chick osteoblasts for 10 weeks and stained with a nuclear
Figure 1 displays merged and registered OCM and CFM images 145 mm below the surface of a TEMP. The TEMP consists of a volume fraction of 50 % poly(ecaprolactone) (PCL) scaffold that was cultured with fetal chick osteoblasts for 10 weeks and stained with a nuclear
 
stain. In Figure 1, the regions of low OCM signal are red (pores), high OCM signal are black (scaffold), and regions of high CFM signal are yellow (cells). CFM complements OCM by allowing us to positively identify stained tissues at more shallow depths. Once identified, OCM allows us to discriminate these tissues from scaffold, and thus view them at a greater depth. This will form the basis of structure-property relationships for TEMPs based on microscopic characterization of scaffold properties and concomitant cellular responses.
 

QUANTITATIVE VISUALIZATION


Quantification of scaffold properties must be performed to establish scaffold structure and cell function relationships and to optimize scaffold design. One of our goals is to design an approach that is valid with any pore structure. Figure 2 displays a volumetric view of the PCL scaffold images collected using X-ray computed tomography. In this figure, the pores are colored in red and the scaffold in green. From viewing this image, one gets a qualitative sense of the need for such an approach. Heterogeneity in the microstructure is exhibited by the difference in pore size, shape and anisotropy as seen on the different faces of the volume. Pore volume, size distribution, tortuosity, and connectivity are metrics of interest for the scaffold microstructure.
 
An example of the type of information gleaned from the imaging data is shown below. Figure 3 displays the pore size as measured by the chord length distribution function (CLDF). The CLDF is the probability of finding a chord of length l between x and x + dx entirely in one phase. Chords are defined as the segments formed by the intersection of lines with the interface between two phases
 
Figure 2: X-ray computed tomographic reconstruction of a PCL based scaffold. Dimensions: 1.0 mm on each side.
Figure 2: X-ray computed tomographic reconstruction of a PCL based scaffold. Dimensions: 1.0 mm on each side.
 
Figure 3: Example of a CLDF from one image plane in Figure 2.
Figure 3: Example of a CLDF from one image plane in Figure 2.
 
Another important aspect of the effort to optimize scaffold design is the need to balance competing requirements. High porosity is required because of the need for cell migration, proliferation, and nutrient influx. However, the drive towards higher and higher pore volumes opposes the need for certain mechanical property requirements, especially in orthopedic applications. To this end, we are using 3D finite element analysis (FEA) to develop an analytical tool to predict the relationship between the effective properties and individual constituent properties of TEMPs based on real material images. This relationship, plus the analysis of the structural problem of interest, provides a means of optimizing the performance of TEMPs by varying individual constituent properties without conducting a variety of time-consuming experiments. Initially, the properties of interest are anisotropic elastic constants. Figure 4 displays a typical mesh of FEA based on a section of the PCL scaffold from Figure 2.
 

Figure 4: Finite element mesh of a subsection of scaffold shown in Figure 2. The dimension is 274 mm on each side.

Figure 4: Finite element mesh of a subsection of scaffold shown in Figure 2. The dimension is 274 mm on each side.
 
This work represents a systematic, integrated approach to the study of structure/function relationships and optimal design in TEMPs. We will be able to extract metrics for the anisotropic scaffold microstructure and properties and use that information to understand their influence on cell function, and on a larger scale, TEMP viability.
For More Information on this Topic
F. Landis, M. Chiang (Polymers Division, NIST)
M.T. Cicerone, J.P. Dunkers, and N.R. Washburn, "Towards in-situ Monitoring of Cell Growth in Tissue Engineering Scaffolds: High Resolution Optical Techniques," in Tissue Engineered Medical Products (TEMPs) ASTM STP, 1452, November 3, 2002, Miami Beach, FL.
For information involving chemical imaging of TEMPs, see the project page entitled: "Coherent Anti-Stokes Raman Microspectroscopy (m-CARS) for Understanding Tissue Growth in Scaffold Constructs" by M. Cicerone and T. Kee.
 
 
 
 
 
 
 
 
 
 
 
 
NIST Material Science & Engineering Laboratory - Polymers Division