(622s) ß1-Integrin Surface Expression on Adipose Stem Cells Cultured in a Centrifugal Bioreactor with TGF-ß3 and Cyclic Hydrostatic Pressure

Introduction: Articular cartilage (AC) is a smooth white tissue covering the articulating surfaces of bones to act as a lubricant and load distributer.  Chondrocytes, the primary cell type in cartilage, remodel the tissue’s extracellular matrix in response to mechanochemical cues from their environment. When injured or damaged, cartilage tissues have limited regenerative capabilities because of lack of vascularization. Unfortunately, damage to AC is common with millions of Americans suffering from the joint disease, osteoarthritis, every year. Current treatments for osteoarthritis are inadequate in restoring the structure or function of native cartilage, making AC a good candidate for tissue engineering. This project aims to culture human adipose-derived stem cells (hASCs) in our novel centrifugal bioreactor (CBR) with oscillating hydrostatic pressure and transforming growth factor-β3 (TGF-β3). These cues lead to differentiation of the hASCs into chondrocytes to form a cartilage tissue with desirable properties. In our work, we use atomic force microscopy (AFM) to measure the mechanical properties of the cell and cartilage tissue as well as to map the location and density of β1-integrins on the cell surface as a measure of mechanotransduction. We hypothesize that biomechanical and chemical stimulation will be sensed by the cell via mechanotransduction through β1-integrins and this will prompt cells to secrete extracellular matrix. We further hypothesize that this stimulation will improve the Young’s modulus of cartilage tissues by increasing it to near native cartilage properties.

Materials and Methods: hASCs were cultured for 21 days in micromass, pellet and in the CBR with and without TGF-β3. Cells in the CBR were subjected to oscillating hydrostatic pressure. To determine tissue mechanics, a 10 µm² scan area was indented via AFM using gold-coated colloidal cantilevers in phosphate buffered saline. At least 256 points on each of 3 scan areas were indented per treatment group. Force-indentation profiles were fit to a Hertz model of contact mechanics to extract the elastic modulus of the engineered tissue. These values were then statistically analyzed to represent the mean and median Young’s moduli for each treatment group. To determine the cell mechanical properties as well as the surface expression of β1-integrins on the cell, the tissue from each treatment group was digested with type II collagenase to isolate the cells. These cells were seeded onto poly-L-lysine coated glass slides for further AFM studies. A gold-coated sharp cantilever modified with a monoclonal antibody against β1-integrins was used to map the β1-integrin localization on the cell surface. As the cantilever probes the cell surface, specific adhesion interactions can be identified as single-molecule signatures that represent antibody-antigen binding. The location and sum of these binding events was recorded and compared across treatment groups.

Results and Discussion: CBR tissues grown with only oscillating hydrostatic pressure had Young’s moduli 44-fold higher than the micromass or pellet static controls. With the addition of TGF-β3 to the CBR tissues, a 1.8-fold increase was observed, bringing the Young’s modulus nearer to that of native cartilage. It was found that the average adhesion force for a single antibody-β1-integrin interaction was 80 pN, independent of treatment group. This fits the typical protein-protein interaction magnitudes found in the literature which are typically under 100 pN. Higher force magnitudes followed patterns indicating that multiple antibody-antigen interactions were present. The micromass and pellet cultures had two to five-fold larger β1-integrin counts compared to the cultures engineered in the CBR. We expect that this is a result of the cell responding to a lack of extracellular matrix (ECM). Because β1-integrins facilitate cell-ECM attachment, a deficient ECM may require more β1-integrins to attach to the cell. This would explain why static cultures with lower Young’s moduli have more β1-integrins.

Conclusions:  Cartilage tissues grown from hASCs had higher Young’s moduli, nearing that of native cartilage, when cultured with oscillating hydrostatic pressure. The improvement in the Young’s moduli was furthered by the supplementation with TGF-β3. The surface expression of β1-integrin is dependent on the tissue culture method and is lower in the CBR samples with oscillating pressure compared to static cultures. These data support the hypothesis that a larger number of β1-integrins are necessary when sub-optimal ECM is present.

Acknowledgements: This work was supported by an NSF EAGER grant 1212573, the NIH Protein Biotechnology Training Program 24280305, a NASA Space Grant, a WSU DRADS fellowship, and a Harold P. Curtis Scholarship.