Engineering of articular cartilage: Challenges and prospects
Nehal Abu-Lail, Chrystal R. Quisenberry, Arshan Nazempour, and Bernard Van Wie
Voiland School of Chemical Engineering & Bioengineering, Washington State University, Pullman, WA 99164-6515.
Introduction: Articular cartilage (AC) is the smooth white connective tissue of diarthrodial joints. It acts as a lubricant to the moving joins as well as a distributor of loads applied on them. However, AC tissues are subjected to extreme mechanical loads, harsh chemical conditions and worse of all, they are devoid of blood and nerve vessels. Mainly due to the latter, AC tissues have limited regenerative capabilities when injured or damaged. AC failure results in osteoarthritis (OA) or can cause musculoskeletal morbidity. Currently, no successful long-term treatments of OA are present. Patients with OA usually have to replace joints. In the US alone, a joint is replaced ~ every 90 seconds and it is even worse in Europe. With no treatments, AC presents an ideal candidate for tissue engineering. Tissue engineering is an interdisciplinary field in which biological concepts are combined with engineering tools and quantitative methods to model environments capable of producing functional tissues that can be used to replace native diseased tissues. Here, we are utilizing a unique centrifugal bioreactor, an array of mechanical and chemical cues and a variety of cell types to engineer AC in vitro. We hypothesize that biomechanical and chemical stimulation will be sensed by the cell via mechanotransduction through β1-integrins and N-cadherins 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
Chondrocytes, the primary cell type in AC, remodel the tissue’s extracellular matrix (ECM) in response to mechanochemical cues from their environment. Unfortunately, during growth, they dedifferentiate. With that limit, human adipose-derived stem cells (hASCs) or bone marrow stem cells can be used as a cell source. Cells were seeded in our novel centrifugal bioreactor (CBR) in a variety of ways. To mimic native AC environment, oscillating hydrostatic pressure and transforming growth factor-β3 (TGF-β3) were also applied. These cues lead to differentiation of stem cells into chondrocytes to form a cartilage tissue with desirable properties. Cells were generally grown for 21 days in the reactor as well as in static controls of micromass and pellet. The functionality of the engineered AC tissues was tested via an array of techniques. First, atomic force microscopy (AFM) was used 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. mRNA sequencing was used to probe for 10 markers of chondrogenesis. Finally, biochemical analyses were carried to quantify DNA and glycosaminoglycans (GAG) contents of the engineered cartilage.
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. The micromass and pellet cultures had two to five-fold larger β1-integrin counts compared to the cultures engineered in the CBR. Because β1-integrins facilitate cell-ECM attachment, a deficient ECM may require more β1-integrins to attach to the cell. Analysis of N-cadherins on the surfaces of cells in response to stresses is ongoing.
Conclusions: Cartilage tissues grown from hASCs had higher Young’s moduli, nearing that of native cartilage, when oscillating hydrostatic pressure and TGF-β3 were applied synergistically. The surface expression of β1-integrin was dependent on the tissue culture method. In the future, adding a scaffold with gradients in its properties to the CBR may bring us a step close to engineering AC with properties close to those of native cartilage.
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.
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