Quantitative three-dimensional analysis of vascular microstructure in tissue engineering

[摘要] Organ and tissue shortages have driven a need for alternatives to traditional reconstructive and transplantation techniques. Tissue engineering has arisen amidst the promise of producing patient-specific, biocompatible substitutes that could revolutionize plastic and transplantation surgery. Although successful in a few clinical situations, an increase in the number of tissue engineered products is limited, in part, by the formation of a patent blood supply within the tissue. The mass transfer limitations of engineered tissue are well-documented, but little progress has been made in addressing this need.In this thesis, a systematic approach was used to study angiogenesis. First, a comprehensive method was developed using 6-mum serial sections of tissue, immunostaining for CD31, brightfield microscopy, automated alignment of two-dimensional sections, and volume rendering to produce high-resolution, three-dimensional, quantifiable images of microvascular structure. Comparing measurements from automated and manually aligned MRI and fibrin samples verified quantitation. Automation removes concerns of observer bias or variation and increases the speed of analysis. Data obtained from these automatically aligned images agreed with those obtained using manual analysis, and the results were consistent with data from traditional methods.Next, three-dimensional analysis and double immunohistochemical stains were used to show that desmin and smooth muscle alpha actin (SMA) positive cells are present during vessel sprout formation in an in vivo fibrin gel model. Although the level of staining with desmin and SMA was the same, their distribution during angiogenesis was quite different. In addition, mural cell coated vessels were present in the gels at 14 days, but absent at 21 days following loss of the fibrin. These results indicate that the presence of desmin or SMA positive cells surrounding a vessel surface is not conclusive of vessel stability.The fibrin gel results suggest that growth of both endothelial cells and mural cells are important to normal angiogenic processes. Therefore, we hypothesized that a mutant, cysteine-free version of fibroblast growth factor one (FGF-1cys-) would increase angiogenesis over wild type FGF-1 (wtFGF-1) in an in vivo collagen gel model. FGF-1 cys- increased angiogenesis over wtFGF-1 at 12 days but did not alter the mural cell presence.

[发布机构] Rice University

[效力级别] engineering