Completely biological tissue replacements can be fabricated by entrapping cells in a molded fibrin gel. Over time, the fibrin is degraded and replaced with cell-produced extracellular matrix. However, the relationship between fibrin degradation and matrix deposition has not been elucidated. We developed techniques to quantify fibrin degradation products (FDP) and examine plasmin activity in the conditioned medium from fibrin-based constructs. Fibrin-based tissue constructs fabricated with vascular smooth muscle cells (vSMC) were cultured for 5 weeks in the presence of varied concentrations of the fibrinolysis inhibitor -aminocaproic acid and cellularity, and deposited collagen and elastin were measured weekly. These data revealed that increasing concentrations of -aminocaproic acid led to delayed and diminished FDP production, lower vSMC proliferation, and decreased collagen and elastin deposition. FDP were shown to have a direct biological effect on vSMC cultures and vSMC within the fibrin-based constructs. Supplementing construct cultures with 250 or 500μg/mL FDP led to 30% higher collagen deposition than the untreated controls. FDP concentrations as high as 250μg/mL were estimated to exist within the constructs, indicating that FDP generation during remodeling of the fibrin-based constructs exerted direct biological activity. These results help explain many of the positive outcomes reported with fibrin-based tissue constructs in the literature, as well as demonstrate the importance of regulating plasmin activity during their fabrication.

Human blood outgrowth endothelial cells (HBOECs) are expanded from circulating endothelial progenitor cells in peripheral blood and thus could provide a source of autologous endothelial cells for tissue-engineered vascular grafts. To examine the suitability of adult HBOECs for use in vascular tissue engineering, the shear stress responsiveness of these cells was examined on bioartificial tissue formed from dermal fibroblasts entrapped in tubular fibrin gels. HBOECs adhered to this surface, deposited collagen IV and laminin, and remained adherent when exposed to 15 dyn/cm(2) shear stress for 24 h. The shear stress responses of HBOECs were compared to human umbilical vein endothelial cells (HUVECs). As with HUVECs, HBOECs upregulated vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 when exposed to tumor necrosis factor (TNF)-α and shear stress decreased the expression of these adhesion molecules on TNF-α-activated monolayers. Nitric oxide production was elevated by shear stress, but did not vary between cell types. Both cell types decreased platelet adhesion to the bioartificial tissue, whereas pre-exposing the cells to flow decreased platelet adhesion further. These results illustrate the potential utility for HBOECs in vascular tissue engineering, as not only do the cells adhere to bioartificial tissue and remain adherent under physiological shear stress, they are also responsive to shear stress signaling.

Blood outgrowth endothelial cells (BOECs) are isolated by outgrowth of circulating progenitor cells from a patient blood sample and thus could provide a source of autologous endothelial cells for tissue‐engineered vascular grafts. To examine the suitability of these cells for use in vascular tissue engineering, late outgrowth endothelial cells isolated from human peripheral blood were seeded on tissue constructs formed from fibrin remodeled by entrapped neonatal human dermal fibroblasts (NHDFs). BOECs adhered to this surface and remained adherent when exposed to laminar shear flow of cell culture medium, at shear stresses of 15 dynes/cm2.We have examined shear stress effects on human BOEC activation state, by comparing VCAM‐1, ICAM‐1, and Thrombomodulin (TM) expression after activation with TNF‐α. These markers were chosen due to their reported response to changes in shear stress, as well as their implication in endothelial cell activation. BOECs seeded on collagen‐coated slides and tissue constructs were statically cultured or exposed to 5–15 dynes/cm2 shear stress for 24 hours. Expression of the activation markers was altered compared to static controls, while the cell densities did not vary significantly. TM expression was elevated by shear stress, while VCAM‐1 and ICAM‐1 showed decreased expression. These results illustrate the potential utility for BOECs in vascular tissue engineering, as not only do the cells adhere to fibrin‐based tissue constructs and remain adherent under physiological shear stress, they are also responsive to shear stress signaling. Funding from NIH R01 HL083880 (to RTT).

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