PROJECT SUMMARY / ABSTRACT The objective of this proposal is to study how a new human-derived biomaterial with granulated structure, NatruLage, stimulates adult stem cell differentiation for integrative cartilage repair. Recent advances in the treatment of articular cartilage and bone injuries have shown exciting potential to address matrix degeneration associated with osteoarthritis (OA), a joint disease afflicting millions of people in the United States. New candidate therapies utilize animal- or human-sourced materials to formulate unique microenvironments favorable to stem cell responses that promote healing and integration. In healthy osteochondral tissue, the extracellular matrix is organized into interstitial and pericellular domains with specialized cells and signaling molecules. Together, these components define tissue-specific and structure-function relationships which are lost during disease progression. Unfortunately, integrative cartilage repair still remains as a major medical need, and new products are required for enhanced and improved therapies. Successful adoption of osteochondral tissue therapies requires extensive technical and manufacturing de-risking to meet stringent regulatory approval requirements. New innovations must meet current good manufacturing practice (cGMP) specifications and demonstrate pre-clinical evidence of positive cellular and biological responses as well as lack of toxicity prior to submission of an investigational device exemption (IDE) package and, later, execution of a clinical trial. To improve integrative cartilage repair, we developed a novel granulated extracellular matrix with a proprietary crosslinking technology, termed NatruLage, that is injected into the defect in bone and cartilage layers to form osteochondral tissue that closely mimics the natural zonal tissue structure. Our device is ‘flowable’ to deliver densely-packed tissue particles, and enable forming and shaping after delivery through unique crosslinking via thiol-functionalized macromolecules. We have evaluated the same NatruLage technology, using porcine instead of human tissue, and found promising regeneration and integration in a 12- month large animal (goat) study of osteochondral repair. We are now focused on the development of NatruLage using human-derived allograft tissue for delivery of distinct layers of osteochondral tissue. We will define the appropriate chemistry, manufacturing, and quality controls to support the cGMP production of NatruLage for future clinical trials, and we will contribute to understanding the stem cell responses to NatruLage. We will pursue three related specific aims. In Aim 1, we will adapt established cGMP facilities for production of human NatruLage. In Aim 2, we will define specifications for NatruLage reproducibility and donor variability. In Aim 3, we will quantify NatruLage stimulation of adult stem cell proliferation and differentiation. If successful, we will create an ideal secondary osteoc...