BCSRC COMPETITION 2015

Determinants of Cell
Spectral Markers for
Assessment of
Engineered Cartilage:
Are Fibroblasts
Present?
Amanda Craine
Owner
[Pick the date]

Table of Contents

Introduction
Tissue engineering is being applied to create new cartilage tissue for articular cartilage
repair in the knee. However, engineered tissue is also used for other reasons. Research is
currently being conducted in medical labs to determine the viability of engineered cartilage
transplants for rheumatic disease treatment (Sittinger and Burmenster). Also, engineered tissue
can be used for facial reconstruction in the event of a nasal or auricular injury. Cartilage tissue is
composed of collagen, proteoglycan, and water (Hollister). The primary purpose of articular
cartilage is to protect the ends of bones from damage due to mechanical loading, and to help
enable joint articulation (Boskey and Pleshko). There are different types of cells that go into the
formation of cartilage (change that wording). Fibroblasts produce proteins including collagen
type 1 and fibronectin. However, collagen type 1 is not present in articular cartilage. Fibroblasts
also create the extracellular matrix of (cells in general or only cartilage??) and are a precursor
cell for chondrocytes. Chondrocytes are the only cells found in hyaline cartilage and produce
collagen type 2 and aggrecan. Collagen type 2 is the only type of collagen found in hyaline
cartilage, while aggrecan is the major type of proteoglycan in hyaline cartilage (too repetitive?).
When analyzing the properties of tissue engineered cartilage, there are several different factors to
observe, including mechanical properties, composition of the matrix, and cell type. This research
project involved the assessment of the cells in tissue engineered cartilage samples and fibroblast
cells to evaluate the cell population present in the cartilage. Fourier Transform Infrared Imaging
Spectroscopy (FTIR-IS) was used to quantitatively and spatially analyze protein, proteoglycan,
and collagen contents of articular cartilage.

Goal
The goal of this project was to identify the unknown cells in engineered cartilage samples
using frequencies in the infrared spectral range pertaining to different cells.
Materials and Methods
The tissue engineered cartilage was grown in the Imaging and Spectroscopy Lab on
February 11th 2013. First, the researchers harvested the bovine chondrocytes. Then, the
chondrocytes were seeded on the proteoglycolic acid mesh scaffolds. Finally, the tissue was
cultured in the cell culture for three to six weeks.

Figure 1: Growth of Cartilage

The fibroblast cells were cultured in the Tissue Imaging and Spectroscopy Lab (TISL). Fourier
Transform Infrared Imaging Spectroscopy (FTIR-IS) was used to quantitatively and spatially
analyze protein, proteoglycan, and collagen contents of articular cartilage. The spectrometer was
coupled to a light microscope, which examined the chemical composition of the cells by
evaluation of specific molecular vibrations as each cell was exposed to light. Data was collected
using the Perkin Elmer Spotlight 400 imaging spectrometer with a spatial resolution of 8cm-1, a
spatial resolution of 6.25m, and 128 co-added scans. These parameters were necessary to
provide the most accurate image for cells of this specific size (too broad?).
Data
The spectroscopic data was collected and analyzed using the computer software iSYS,
which created infrared maps of the raw spectra and the second derivative of the spectra. The
second derivative was taken in order to observe shoulders that are difficult to see with the raw
spectra, this making the peak more accurate. All images were taken at a wavelength of 1396cm-1,
a previously determined cell marker for fibroblasts.

Figure 2: FTIS-IS Fibroblast Images

Figure 3: Second Derivative Fibroblast Images

Figure 4: Engineered Cartilage Comparison

Analysis
The histology samples were stained with Hematoxylin and Eosin (H&E Staining), which
resulted in the appearance of abnormal tissue present (pink tissue). Previous research concluded
that the blue areas of the histology were composed of proteoglycan, a major type of protein
present in hyaline cartilage. However, the pink areas of histology were unknown because the
pink area had a different cell marker than the blue. This suggested that the two areas were
composed of different cell types. Using the gold standard H&E staining technique, this years
data suggests that the pink area of the cell is composed of fibroblasts. Hematoxylin is basic and
binds with acidic structures to stain them blue (Peckham et al). Eosin is acidic and stains basic
structures pink (Peckham et al). Proteoglycan is an acidic protein, which explains its blue color.
Sugars, carbohydrates, and ECM are stained pink. Since fibroblasts create a basic ECM, the area
in which they reside should be stained pink.
Using a cell marker of 1396cm-1, the areas of highest absorbance in the infrared image
correspond to the unknown pink areas in the histology stain.
In order for cartilage to form, cells must differentiate. Fibroblasts differentiate into
fibrochondrocytes and fibrochondrocytes differentiate into chondrocytes. The chondrocytes then
proliferate and secrete ECM, forming articular cartilage. The presence of fibroblasts in
engineered cartilage deduces that the chondrocytes de-differentiated back into fibroblasts.
Conclusion

The de-differentiation of cells causes the cartilage matrix to disband and form other types
of cartilage due to the cells secreting other proteins not specific to articular cartilage. This causes
the aticular cartilage to lose its mechanical properties and it is no longer able to function
optimally. Understanding that this cartilage cannot be used on patients allows scientists to
improve upon the engineering process in the future.