PROJECT SUMMARY This proposal aims to tissue engineer an anisotropic neo-meniscus that also captures the regional variations present in the native tissue. Subsequently, allogeneic neo-meniscal constructs will be implanted in a leporine model to achieve both meniscus repair and replacement. It is hypothesized that: 1) regionally variant, anisotropic, meniscus-shaped constructs can be engineered by optimizing cell culture and scaffold-free culture conditions; 2) the strategic temporal application of multi-level stimuli (at cellular-, molecular-, and construct- levels) will allow for synergisms across the different levels of action to enhance the functional properties of the maturing neo-menisci; and 3) allogeneic constructs can be successfully implanted in a leporine model. These hypotheses will be tested via the following three specific aims: 1) To create an anisotropic neo-meniscus with regional variations mimicking native tissue; 2) to enhance functional and organizational properties of the neo- meniscus via multi-level exogenous stimulation synergized by temporal coordination; and 3) to develop surgical fixation techniques and implant the neo-menisci in the rabbit. Previously, the native meniscus was found to be highly anisotropic and regionally variant both morphologically and biomechanically, motivating our current tissue engineering approach to mimic these characteristics. Allogeneic leporine cells will be used to form organizationally and regionally mimetic neo-meniscal constructs in Aim 1; this goal will be accomplished via the use of novel spatial and temporally variant seeding techniques. The anisotropic and organizational properties of the engineered neo-meniscus will then be enhanced by manipulating molecular-, cellular-, and construct-level targets in Aim 2. Specifically, TGF-β1 and hydrostatic pressure will act on the cellular level to increase matrix production; lysophosphatidic acid and chondroitinase-ABC will be used to align and compact the matrix at the molecular level; and meniscus-specific mechanical stimulation will direct anisotropy at the construct level. In this proposal, to avoid the use of primary cells, we will investigate the use of passaged allogeneic cells toward in vivo repair and replacement of the meniscus (Aim 3). Upon successful demonstration of repair/replacement in the leporine model, we will determine methods to likewise expand sheep and human cells for future studies. This approach seeks to address the issue of tissue scarcity and aims to provide a solution to the complex problem of meniscus repair and replacement.