Bovine Cartilage Digestion and Characterization of Cellular Surface Proteins Using Atomic Force Microscopy

Cartilage is the avascular tissue surrounding moving joints. Because of its nature, cartilage tissues do not heal upon injury.  A possible solution for people suffering from cartilage damage is to implant a cartilage tissue in vivo in the site of damage. Researchers have been attempting to culture cartilage in vitro by use of bioreactors and scaffolds. However, we hypothesize that using a scaffold-free centrifugal bioreactor with both mechanical and biochemical stimuli will optimize the properties of in vitro engineered tissue to mimic in vivo cartilage. In engineering this cartilage, chondrocytes are seeded in the reactor with appropriate growth factors and mechanical conditions applied. To do that, bovine knee joints are obtained fresh as donations from local butchers. During cartilage harvesting, the tissue must be in an aseptic environment to eliminate risks of contamination. After harvesting the cartilage, cells are isolated by incubation in collagenase type II for 16 hours to break down the peptide bonds in collagen and to separate the extracellular matrix from the cells. After this, cells are strained and washed to separate the cells from leftover matrix. Once this is complete, cells are prepared for freezing and future experiments. Chondrocyte isolation is done under sterile conditions and the cells are free of contamination as confirmed by plating on Tryptic soy agar. The chondrocytes are later inoculated into the centrifugal bioreactor and cultured for 21 days to form a cartilage construct. The construct is later digested to isolate the engineered chondrocytes and map the location and density of N-cadherins and β₁-integrins on the cell surface using atomic force microscopy (AFM). AFM also is used to quantify the tissue elastic module. The data from the experiment described above are currently being analyzed and will be presented in the meeting. Data will be compared to recent results obtained for cartilage tissues grown using adipose-derived stem cells (ASCs) oscillating hydrostatic pressure (OHP) mechanical stimulation in the presence of transforming growth factor (TGF)-β3growth factor. The mechanical properties of articular cartilage tissues grown using ASCs were comparable to the elastic module of human cartilage tissues. We expect to see similar results for chondrocytes.

In conclusion, our research is helping identify how mechanical and chemical stimuli can synergistically alter the expression of proteins involved in mechanotransduction as well as the mechanical properties of resulting tissues.

Acknowledgements: We would like to thank National Science Foundation (EAGER REU supplement CBET # 1517370), Regeneron, Frank’s Custom Slaughter.