Development of novel tissue engineering strategies toward the translation of articular cartilage implants
Articular cartilage diseases in the knee, initiated by age-related degenerative processes or trauma, compromise the material properties of the tissue and the function of the joint. Osteoarthritis, the most common cartilage disease, affects over 30 million people in the United States alone. Depending on the level of damage to the cartilage and/or the underlying bone, the symptoms can range from stiffness, pain, and swelling, to loss of function and disability. Current treatments aimed at treating cartilage diseases do not provide satisfactory outcomes. Osteochondral allografts are scarce, and autografts, aside from the risk of donor site morbidity, often present a mismatch to the articular surface of the recipient location. Other alternatives, including microfracture and MACI, fail to regenerate the tissue, and induce the formation of a mechanically inferior fibrocartilage repair tissue. As a result, patients eventually require revision surgeries or alternative treatments, such as synthetic implants, in as little as 5-10 years. Tissue engineering, capable of generating neocartilage constructs with properties akin to the native tissue, has emerged as a promising alternative to treating cartilage diseases. However, tissue engineering methodologies must be optimized so as to become clinically viable options; current technologies used to generate biomimetic neocartilage constructs 1) fail to recapitulate the properties of the native tissue, 2) jeopardize the translational potential of neocartilage constructs due to high cost, low reproducibility, and high technical complexity, and 3) do not produce neotissues akin to native tissue thickness. Toward overcoming these limitations, the global objectives of this work were 1) to provide a thorough assessment of the properties of native articular cartilage to inform tissue-engineering efforts, 2) to enhance the mechanical properties of neocartilage toward native tissue levels utilizing a repeatable and cost-effective ion modulation strategy, and 3) to develop a methodology to increase the thickness of neocartilage constructs.Toward assessing the properties of articular cartilage and providing an accurate set of data, this work first characterized the effects of storage time and temperature on the mechanical properties of juvenile bovine cartilage, a facsimile of a cartilage implant. In addition, an extensive assessment of the surface properties of cartilage was performed using conventional and novel methodologies, including non-contact vibrometry. Non-contact vibrometry was further developed as a new non-destructive measurement of the dynamic and viscoelastic mechanical properties of cartilage samples. Finally, the human medial femoral condyle, one of the knee compartments with the highest incidence of chondral degeneration, from young, healthy donors was topographically characterized using biochemical and biomechanical assays. It was determined that the articular cartilage of the human medial femoral condyle displays regional variations in the tensile and mechanical properties which may have repercussions on the injury patterns observed in the clinic; the Young’s modulus, ultimate tensile strength, aggregate modulus, and shear modulus in the posterior region were 1.0-fold, 2.8-fold, 1.1-fold, and 1.0-fold less than the values in the anterior region, respectively. As opposed to other tissues and species, including bovine cartilage, the coefficient of friction in human samples does not present regional variations, ranging 0.22-0.26 throughout the condyle, and is isotropic. It was also determined that particular storage conditions can affect the properties of cartilage tissues, and that vibrometry-based viscoelastic properties not only correlate to biochemical content, but also significantly correlate with moduli from stress relaxation and creep tests, with correlation strengths reaching up to 0.78. These findings are significant, as they not only establish a working framework for stored cartilage tissues and new non-contact testing methodologies, but also provide much needed design criteria. Moreover, the findings suggest that lower mechanical properties may predispose the tissue to chondral injuries, and propose new structure-function relationships, highlighting the importance of collagen crosslinks for the mechanical properties of cartilage. Taking into consideration the values obtained for human articular cartilage tissues, new methods to improve the mechanical properties of neocartilage constructs were developed. Mechanotransduction and hypoxia, two well-known stimuli that promote chondrogenesis, have been used to produce neocartilage constructs. However, both stimuli require the use of bioreactors that confound production of neocartilage, increasing the costs and complexity of the process. Modulation of ions, a cost-effective pharmacological methodology of mimicking mechanotransduction and hypoxia, was examined toward improving the material properties of neocartilage. It was determined that ionomycin, a calcium ionophore, caused a 61% and 115% increase in glycosaminoglycan and pyridinoline crosslink content, which translated to a 45% increase in the aggregate modulus and a 63% increase in the tensile Young’s modulus, respectively. Similarly, deferoxamine, an iron chelator, produced an 87% increase in pyridinoline crosslinks, and a 57% and 112% increases in the Young’s modulus and the ultimate tensile strength (UTS) of neocartilage constructs, respectively. Importantly, the combined use of both ion modulators resulted in significant increases of 150% and 176% in the Young’s modulus and UTS of neocartilage constructs, respectively. Ionomycin and deferoxamine can be used as analogs of mechanical and hypoxic stimuli, bypassing the need for bioreactors. The use of both analogs resulted in neocartilage constructs with a Young’s modulus of 11.76±3.29 MPa and UTS of 4.20±1.24 MPa, the highest reported by our group thus far. While the self-assembling process has been optimized to produce neocartilage constructs with biochemical and biomechanical properties on par to native tissue, it has been limited in its capability to grow tissues with biomimetic thickness. The existing neocartilage constructs are several times thinner compared to the average condylar cartilage, and thus, limit its therapeutic potential. Superficial irregularities between the native tissue and the repair tissue compromise the treatment outcome; disparities induce stress concentration, delamination of the native tissue, and dislodging of the implant. Because of this, a new self-assembling methodology, using sequential seeding and deferoxamine stimulation, was developed in this work to generate constructs with increased thickness. The new methodology utilizes sequential seeding, and expanded and rejuvenated chondrocytes previously stimulated with deferoxamine. An appropriate time between separate seeding events and deferoxamine stimulation prior to the self-assembling process was determined. Constructs derived from human articular chondrocytes, the current cell type used by cell-based treatment strategies, resulted in neocartilage with a 90% increase in thickness, an 8% increase in diameter, a 33% and a 41% increase in aggregate and shear moduli, and the maintenance of the tensile properties, compared to the control group. This is the first successful attempt to produce thicker biomimetic self-assembled tissues without a tradeoff in the mechanical properties. This work demonstrates the potential of tissue-engineered neocartilage implants for the surgical treatment of cartilage diseases, and altogether, the findings of this work provide new scalable tissue-engineering strategies toward developing clinically relevant neocartilage constructs.