Introduction: The inability of the articular cartilage to spontaneously heal focal defects remains a major problem for orthopedic surgeons. Current techniques have various shortcomings including poor integration and fibrocartilaginous repair tissue. This has led to the development of alternative tissue engineering approaches to enhance cartilage regeneration through combinations of cells and three-dimensional biomaterial scaffolds. To date, however, stem cell-based tissue engineering is still faced with challenges associated with control of cell differentiation in vitro prior to implantation. Furthermore, man-made materials, by themselves, are generally not conducive to chondrogenesis and require exogenous growth factors to control stem cell behavior. To address this challenge, we recently developed a novel porous scaffold derived from freeze-dried, devitalized cartilage allograft tissue and demonstrated that these scaffolds can regulate the chondrogenic differentiation of adipose stromal cells and chondrocytes in vitro [1]. In this study, we set out to evaluate in vivo the efficacy of implanting these porous cartilage-derived matrix scaffolds (CDM) to repair focal articular cartilage defects in a preclinical (rabbit) model. Methods: CDM Fabrication – Cartilage fragments were dissected from load bearing regions of the articular cartilage of the porcine knee. CDM scaffolds were fabricated from these cartilage fragments through a series of mechanical homogenization followed by lyophilization, as previously described [1]. The scaffolds were sterilized using supercritical CO2 (NovaSterilis, Inc. Lansing, NY). Animal Surgeries – All animal surgeries were approved by the University Committee on Animal Resources (UCAR). A total of 64 skeletally mature, male NZW rabbits were used in this study. A trephine and a mallet were used to create 3.0mm diameter defects in the medial femoral condyles of both knees to a depth of 2.5-3.0 mm. An 18 gauge needle was used to induce bone marrow bleeding through the subchondral bone in both defect sites to simulate the microfracture technique used in human patients. The treatment group received a CDM implant, which was press fit within the defect following microfracture (CDM+µFx), while the control site received only the microfracture (µFx). The treatments were randomized a priori. Animals were euthanized at multiple time points up to 112 days (16 weeks) post surgery (n=8 per time point; n=4 for histology and n=4 for RT-PCR). Histology – Paraffin embedded sagittal sections were stained with Alcian Blue/Orange G. Histology grading of the cartilage repair tissue was performed using established Pineda [2] and O'Driscoll [3] scales by 3 independent observers blinded to the identity of the slides. Gene Expression – RNA was extracted at harvest using Trizol and real time RT-PCR was performed with the RNeasy kit (Qiagen) using primer sequences specific to rabbit type I collagen (Col1a2), type II collagen (Col2a1), aggrecan (Acan), and β-actin (house-keeping gene). Statistics – Histology rank scores were analyzed by nonparametric repeated-measures ANOVA. Gene expression data were analyzed using a 2-way ANOVA followed by Bonferroni post hoc comparisons. Significant differences were determined if p<0.05.