This thesis addresses some approach for tissue engineering in cardiovascular regeneration. Cell source, biomaterials, ex vivo and in vivo assessment are all discussed step by step. This dissertation provides a good understanding on cardiovascular disease clinically and scientifically. It is essential to understand cardiovascular disease because there are more than 80 million people in USA suffered from cardiovascular disease in 2007. Aging, smoking, diet, lake of exercise, mental stress and genetic inheritance will result in vascular abnormality such as atherosclerosis, stenosis and aneurysm which might eventually lead to stroke or heart attack. Autologous and allogeneic grafts are the most promising solutions for vascular replacement like coronary artery or lower limp bypass but their availability is very limited.^One promising solution to vascular replacement is tissue-engineered vascular grafts for its great potential in customization with autologous cells and biocompatible materials. The major objective to a successful tissue engineered vascular graft is long term patency. Optimization of matrix, stem cell source, surface coating, and physiologically-mimic animal environment, surgically-friendly technique and real time monitoring are all elements of developing compatible, durable and functional vascular grafts. Regardless of significant progress in development of biomaterials and advanced technique to cultivate vascular constructs, cell source remains an important issue. Isolation of cells from patients' vessels not only created a second wound but also is limited by the health and age of the donors. Researchers widely studied different stem cell source including embryonic stem cells, adult stem cells and induced pluripotent stem cells.^We explored the potential of hair-follicle adult mesenchymal stem cells in differentiating toward smooth muscle cells which may provide an accessible autologous cell source for cardiovascular therapy. Hair-follicle smooth muscle cells (HF-SMCs) were isolated from a hair follicle based on tissue specific promoter. These cells expressed similar markers and exhibited similar functionality as vascular smooth muscle. Hair follicle stem cells also can be differentiated into mesenchymal lineages such as adipocytes, chondrocytes and osteoblasts by induction medium condition which need to optimize concentration of serum, growth factors, amino acid, minerals and chemicals to maintain stem cells properties or induce differentiation toward specific lineage. Fibrin hydrogel was widely applied for its biocompatible and biodegradable properties. The vascular constructs showed good compatibility to HF-SMCS and stimulate significant cellular remodeling. The disadvantage is its low mechanical strength which limits fibrin application for arterial reconstruction. Thus, a mechanically-strong and biologically-compatible biomaterial - small intestine submucosa (SIS) was chosen for tissue-engineered arterial substitute. 2 D model was first tested to analyze the compatibility and interaction between cells and matrix. Within two weeks in culture, cells migrated into SIS and secreted decent amount of extra cellular matrix - collagen and elastin, two major extracellular matrix components in the vessel wall. Vascular reactivity increased significantly while mechanical properties were similar to those of native carotid arteries. To make an implantable vascular graft, high concentration fibrin glue was applied to construct a tubular SIS conduit. The SIS grafts can hold pressure up to more than 1000mmHg which is 5 times than the normal physiological level. An implantable vascular graft not only needs to be mechanically-strong, but also need to be anti-thrombogenic. Endothelial cells seeding on biomaterial or modification of lumen on the synthetic grafts can prevent thrombosis in the circulation. Currently, in vitro model systems are inadequate to test elements of thrombogenicity and vascular dynamic functional properties while in vivo implantation is surgically-complicated, labor-intensive and cost-ineffective. To test our vascular grafts and validate the development protocol for cells seeding, we proposed an ex vivo ovine arteriovenous shunt model in which we can test the patency and physical properties of vascular grafts under physiologic conditions. Carotid arteries and jugular veins were collected freshly from the sheep to be compared with SIS grafts. Anti-coagulation properties of grafts were assessed via scanning electron microscopy imaging, en face immunostaining and histology. Seeding endothelial cells in the lumen greatly decreased attachment of thrombotic components. The most significant characteristic of this model is its convenience and accessibility in a suture free and relatively high throughput environment where multiple samples can be stably processed within one animal. After optimizing implantable SIS grafts, we moved further to preclinical animal implantation to test long-term patency of tissue engineered vascular grafts (TEVs). Surgical technique, medication of anticoagulation drug and immunosuppressor are all critical to contribute to a successful implantation. We developed a platform in which the grafts can be implanted easily without damaging risk of endothelium or compromising animal cardiovascular physiology. Animals were followed up and monitored up to 3 month. Pulsatile flow inside grafts was measured by Doppler ultrasound. In angiogram the TEV diameter remained consistent without evidence of dilation or aneurysm. When scarifying animals, the lumen of the grafts remain smooth and shining suggested there was no blood coagulation in the lumen or anastomosis site. No stenosis, graft failures, occlusions or anastomotic complications were observed. Significant cells infiltration was seen in histological analysis as wells as an intact endothelium layer on the lumen. This study suggested an implantable and biocompatible arterial substitute was successfully made by Hair follicle stem cells and clinically-available acellular matrix- small intestinal submucosa for cardiovascular regeneration.