(340e) A Study of  Using Synergistic Factors on the Mechanical Properties and Phenotype of Engineered Articular Cartilage Using Atomic Force Microscopy and Immunohistochemistry

Articular cartilage (AC) is an avascular tissue with diffusion limited nutrients and oxygen transfer, affecting its ability to self-heal and regenerate. Osteoarthritis is a degenerative disease that causes the degradation of articular cartilage in the joints evolving frequently into a disability. More than 50 million adults are diagnosed with arthritis in the US, and it is estimated that the number will increase to more than 78 million by the year 2040. Currently, there are no effective long-term solutions for osteoarthritis, and treatments rely heavily on the use of pain killers, hyaluronic acid injections, and invasive surgeries, all of which treat the symptoms rather than the issue itself. Thus, a need for an engineered tissue has emerged. In ongoing research efforts, we are hypothesizing that optimal chondrogenesis will happen when synergistically 1) using an optimum ratio of articular chondrocytes (Ach) and human derived adipose stem cells (hASCs) co-culture, 2) mimicking the in vivooscillating hydrostatic pressure by the use of a novel centrifugal bioreactor (CBR) with a hydraulic piston, 3) adding growth factor stimulation, specifically TGF-β1, 4) incorporating antioxidant nutraceutical laden, e.g., curcumin or α-tocopherol, hydrogels that reduce reactive oxygen species, and 5) printing of a 3D-layered tissue gradated with cells, growth factors, and nutraceuticals by direct-writing.

Assessment of the engineered AC will be made with atomic force microscopy (AFM) to scan for cell surface proteins such as β-integrins and N-Cadherin expression. AFM will also be used to study the mechanical properties of the tissue by evaluating the tissue’s elastic modulus. To probe for cell surface proteins such as the β-integrin, AFM cantilevers modified with anti-β1-integrin antibodies will be used. Qualitative biochemical characterization of the tissue will be done using histology, and immunohistochemistry techniques to stain for total collagen and total proteoglycans (GAGs) using toluidine blue and trichrome masson respectively. More specific phenotyping of collagen two and aggrecan (GAGs) will be done by using anti-collagen antibodies and anti-aggrecan antibodies and imaging with a fluorescent secondary antibody.

Our results indicated an inverse relationship between the density of β1-integrins distributed on cellular surfaces and the elastic modulus of the engineered articular cartilage tissues grown using adipose derived stem cells under oscillating hydrostatic pressure and in the presence of TGF-b3. A decrease in β1-integrin counts coincided with a higher young’s modulus on the cells grown in the presence of the oscillating pressure in the CBR; whereas a higher β1-integrin count showed a lower young’s modulus in the micromass culture. This indicates that better mechanical properties are obtained by using oscillating hydrostatic pressure in the CBR.

 Our research efforts are ongoing. Currently a co-culture of bone-marrow mesenchymal stem cells and chondrocytes are being investigated for their abilities to grow an articular cartilage tissue with mechanical and chemical properties close to those of native articular cartilage. The eventual outcome of this research supported by preliminary work is expected to greatly advance regenerative medicine approaches in creating personalized treatment for people with osteoarthritis that is biocompatible, is robust long-term, and with tissue mimicry of natural cartilage in terms of structure and mechanical properties.