Degradation of the extracellular matrix, facilitated by matrix metalloproteinases (MMPs), can lead to mechanical failure of vascular constructs, suggesting that MMP inhibition could improve survival of constructs. Therefore, we investigated the role of MMP-9 in collagen remodeling in vitro, focusing on the three major steps of production, degradation, and organization. Because an adequate blood supply is essential for survival of tissue-engineered constructs, we also evaluated the influence of MMP-9 deficiency on angiogenesis in vivo by implantation of thin biodegradable polymer scaffolds. Using aortic smooth muscle cells (SMCs) from wild-type and genetically deficient (9KO) mice, we examined the role of MMP-9 in collagen mRNA expression and protein accumulation, both with and without ascorbic acid treatment. We measured collagen assembly in a fibrillogenesis assay. We investigated in vivo angiogenesis and cell invasion, using fluorescence microangiography and histology. MMP-9 deficiency did not affect collagen mRNA production or polymer scaffold degradation, but collagen accumulation was greater in cultures of 9KO SMCs than in wild-type SMCs. Both MMP-9 deficiency and chemical inhibition impaired collagen degradation. Ascorbic acid treatment enhanced collagen production by 9KO SMCs compared with wild-type SMCs at 3 days, but by 7 days this effect was reversed. MMP-9 improved fibrillogenesis of collagen, significantly more on ascorbic acid treatment. MMP-9 deficiency dramatically decreased inflammatory cell invasion, but also capillary formation within biodegradable polymer scaffolds in vivo. Our data suggest that MMP inhibition, by impairing collagen organization and angiogenesis, might have detrimental effects on the survival of vascular constructs.