Stem cell lineage commitment in response to biomaterial cues offer attractive alternative means for complex tissue regeneration. The goal of this project is to design, develop, and evaluate a scaffold platform that can instruct stem cells in a 3D micro-environment through material stiffness and bio-physical cues. We propose to evaluate this scaffold technology to study osteochondral (OC) tissue development with an interface as a potential solution to complex tissue repair, an unment clinical need. The OC tissue regeneration continues to be a major clinical hurdle and despite many merits, current biological and tissue engineered grafts fail to provide successful long-term clinical outcomes. Incomplete tissue regeneration, quality of newly formed cartilage (fibrocartilage/hyaline), and lack of zonal structure formations lead to poor host tissue integration. The current knowledge in tissue engineering elucidates the role of biomaterials and their cues in the form of surface chemistry, topography, and matrix stiffness in regulating stem cell fate and lineage commitment in 2D cultures. However, limited efforts have been made to incorporate material and structural cues in 3D-scaffolds to induce stem cell lineage commitment. The primary objective of this proposal is to develop a scaffold platform with imbued structural and material cues to drive mesenchymal stem cell (MSC) lineage commitment, differentiation, and zonal structure formation to regenerate OC tissue. Our recent publications and unpublished work suggest layered OC tissue formation within the scaffold structure by the cultured MSCs under controlled in vitro and in vivo conditions. The current scaffold technology is innovative because it uses a single material to create pore gradients in zonal configurations to avoid material compatibility issues and delamination. Additionally, the literature lacks methodology to characterize a zonal tissue such as OC and scaffolds presented in this application. We propose to develop and validate a heat map methodology as a new analytical tool to measure material stiffness and validate quantitatively with histological findings of regenerated tissue from in vitro and in vivo samples. We hypothesize that scaffold architecture imbued with varied matrix stiffness and growth factors will promote implanted adult stem cell differentiation towards complete OC tissue regeneration with zonal structure. The specific aims of this project are: Aim 1: Optimization of a 3D-scaffold platform embedded with structural and physical cues for interface engineering. Aim 2: Elucidate biomaterial-cell interactions and the mechanistic role of local matrix stiffness and structure in influencing MSC lineage commitment in vitro. Aim 3: Assess the engineered scaffold system with bio-physical cues for OC interface formation in a rabbit model. The outcomes of this project may lead to (i) development of an enabling scaffold technology to engineer stem cells, and (ii) development of an OC tes...