Title: Fabrication of Tissue Engineered Osteochondral Allografts for Clinical Translation
Description: Damage to articular cartilage, whether through degeneration (i.e. osteoarthritis) or acute injury, is particularly debilitating due to the tissue's limited regenerative capacity. These impairments are common: nearly 27 million Americans suffer from osteoarthritis and 36% of athletes suffer from traumatic cartilage defects. Allografts are the preferred treatment for large cartilage defects, but demand for these tissues outweighs their supply. To generate additional replacement tissues, tissue engineering strategies have been studied as a cell-based alternative therapy. Our laboratory has had great success repeatedly achieving native or near-native mechanical and biochemical properties in grafts engineered from chondrocyte-seeded agarose hydrogels. The most common iteration of this technique yields a disk of ~4 mm diameter and ~2.3 mm thickness. However, much work is still needed to increase the potential for clinical translation of this product. Our laboratory operates under the premise that in vivo success is predicated on replicating native graft properties in vitro.

Compared to these engineered grafts, native grafts are larger in size. They consist of cartilage, which has properties varying in a depth-specific manner, anchored to a porous subchondral bone base. They are able to be stored between harvest and transplantation. This dissertation presents strategies to address a subset of the remaining challenges of reproducing these aspects in engineered grafts. First, graft macrostructure was addressed by incorporating a porous base to generate biomimetic osteochondral grafts. Previous studies have shown advantages to using porous metals as the bony base. Likewise, we confirmed that osteochondral constructs can be cultured to robust tissue properties using porous titanium bases. The titanium manufacturing method, selective laser melting, offers precise control, allowing for tailoring of base shape and pore geometry for optimal cartilage growth and osteointegration. In addition to viability studies, we investigated the influence of the porous base on the measured mechanical properties of the construct's gel region.

Through measurements and correlation analysis, we linked a decrease in measured mechanical properties to pore area. We promote characterization of such parameters as an important consideration for the generation of functional grafts using any porous base. Next, we investigated a high intensity focused ultrasound (HIFU) denaturation of gel-incorporated albumin as a strategy for inducing local tissue property changes in constructs during in vitro growth. HIFU is a low cost, non-contact, non-invasive ultrasound modality that is used clinically and in the laboratory for such applications as ablation of uterine fibroids and soft tissue tumors. Denaturing such proteins has been shown to increase local stiffness. We displayed the ability incorporate albumin into tissue engineering relevant hydrogels, alter transport properties, and visualize treatment with its denaturation. HIFU treatment is aided by the porous metal base, allowing for augmented heating. Though heating cartilage is used in the clinic, it is associated with cell death.

We investigated this effect, finding that the associated loss of viability remains localized to the treatment zone over time. This promotes the option of balancing desired changes in tissue properties against the concomitant cell viability loss. In order to match clinically utilized allografts, engineered constructs must be scaled up in size. This process is limited by diffusional transport of nutrients and other chemical factors, leading to preferential extracellular matrix deposition in the construct periphery. Many methods are being investigated for overcoming this limitation in fixed-size constructs. In this chapter, we investigated a novel strategy in which small constructs are cultured for future assembly. This modular assembly offers coverage of variable sized defects with more consistent growth with more uniform distribution of biochemical constituents than large constructs cultured on their own. Physiologic failure testing showed that integration of these tissues may be strengthened by increased subunit strength or assembled culture.

It is expected that bioadhesive caulking and/or the incorporation of osteochondral bases would further increase integration of the assembled large graft. Finally, we sought to provide a preservation/storage protocol for engineered cartilage constructs. Such a technique is critical for clinical translation, providing the engineered graft with a “shelf-life.” We adopted and evaluated the Missouri Osteochondral Allograft Preservation System (MOPS), which had been shown to maintain cell viability in native grafts for at least 63 days at room temperature without serum or growth factors. Within the current clinical of 28 days, MOPS maintained chondrocyte viability and 75% of the pre-preservation Young's modulus without significant decline in biochemical content, however it did not extend the clinical window as it had with native grafts. Refrigeration with MOPS did not show any benefit at day 28, but proved better with longer preservation times. These results are the first evaluating engineered cartilage storage.

Further optimization is necessary to extend storage tissue property maintenance in storage. Overall, this dissertation presents four strategies for increasing the translation potential of engineered articular cartilage grafts by better matching the clinically utilized native allograft system. Combining these techniques, one could ideally engineer small, interlocking ostechondral constructs with HIFU modified interface properties, which could be stored from maturity to implantation. Future optimization is required to better understand and utilize these methods to engineer fully functional, clinically relevant grafts.