UC Irvine

Hyaline articular cartilage, the smooth, white, tissue that is found at the ends of long bones, does not regenerate. Although it is a mechanically robust tissue that repeatedly supports up to six-times body weight, its innate lack of vasculature limits access to nutrients and progenitor cells that are necessary for tissue repair. Currently, clinicians attempt to limit further degeneration when focal defects are manifested on the articular cartilage surface. Unfortunately, for the 250,000 Americans that receive clinical treatment each year, the available options do not produce biomimetic repair tissue necessary to substantiate a long-term treatment option. Tissue-engineered articular cartilage can be designed and manipulated in vitro to fill this significant clinical need. In particular, mechanical stimulation during neocartilage culture can be used to enhance mechanical properties and drive extracellular matrix content toward biomimetic levels. However, before an effective tissue-engineering strategy for treating focal articular cartilage defects can be translated to the clinic, the FDA requires that both local and systemic safety be rigorously demonstrated in a large animal model. Thus, toward translating tissue engineering technologies to clinical applications, the global objectives of this research are: 1) to enhance the mechanical and extracellular matrix properties of neocartilage using mechanical stimulation and bioactive factors, and 2) to evaluate the local and systemic safety of neocartilage implanted in an orthotopic location in a large animal model.

To address these objectives, this research: 1) enhanced the mechanical and extracellular matrix properties of neocartilage using shear stress stimulation, 2) further improved neocartilage properties by exploring combinations of mechanical stimulation strategies with bioactive factors, and 3) evaluated the safety of mechanically stimulated neocartilage implanted in the femoral condyles of Yucatan minipigs.

Shear stress stimulation was shown to improve compressive moduli and extracellular matrix content in neocartilage created from bovine articular chondrocytes, minipig costochondrocytes, and human articular chondrocytes. In particular, it was shown that fluid-induced shear stress applied within a range of 0.05-0.21Pa improved the compressive moduli by 72% - 450% in all types of self-assembled neocartilage. Additionally, the modes of action for fluid-induced shear stress were investigated and it was determined that shear stress upregulated genes encoding a mechanically gated ion channel on the primary cilia of chondrocytes, which have previously been shown to be key sensors of mechanical stimulation. These results indicated that fluid-induced shear stress is an effective strategy for the improvement of mechanical properties in neocartilage constructs.

Shear stress was combined with other forms of mechanical stimulation (i.e. compression stress or tension stress). The tensile properties of neocartilage constructs that were stimulated with a combination of tension and shear stress were significantly improved over shear stress only constructs. Compressive properties were also significantly improved over non-stimulated controls, but not over neocartilage stimulated with only shear stress. However, when bioactive factors were included, the neocartilage constructs that received only shear stress and bioactive factors were the most mechanically robust in both compressive and tensile properties. This research indicates that neocartilage should be stimulated with bioactive factors and shear stress before implantation.

Finally, to assess the local and systemic safety of mechanically stimulated implants, an in vivo study was performed in the femoral condyles of Yucatan minipigs. It was found that the implants did not elicit local or systemic inflammatory responses in minipigs. In particular, there were no adverse effects in the gross morphology of the native tissue surrounding the implants. Furthermore, histological staining did not show evidence of fibrosis or infiltrating immune cells. Finally, the systemic evaluation of the minipigs via complete blood count and blood phenotyping chemistry panels did not show differences between animals that received implants and animals that did not receive implants. These large animal in vivo studies conclude that there are no adverse local or systemic safety effects of neocartilage implants that are treated with mechanical stimulation and bioactive factors.

Overall, this research is significant because it has elucidated strategies to improve the properties of neocartilage toward native articular cartilage, as well as demonstrated the safety of neocartilage implants in a clinically relevant location in a large animal model. This work is foundational for future preclinical studies because it has presented evidence of both the local and systemic safety of self-assembled, mechanically stimulated, neocartilage implants. With further research, the efficacy of tissue-engineered neocartilage implants can be investigated and have the potential to transform clinical treatment options for patients suffering from articular cartilage degeneration.