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Impact of electrospun conduit fiber diameter and enclosing pouch pore size on vascular constructs grown within rat peritoneal cavities.

ABSTRACT: The generation of vascular grafts by recruiting autologous cells within the peritoneal cavity has shown promise. However, the microenvironment affects cell differentiation and elastic matrix production. Therefore, this study determined the impact of systematic changes in the average fiber diameter of electrospun poly(?-caprolactone) conduits, and the pore size of pouches used to enclose the conduits, on recruited cells. After 2 weeks in the peritoneal cavity, fibrous capsules formed containing macrophages, ?-smooth muscle actin (?-SMA)(+) and SM22?(+) myofibroblastic or smooth muscle like-cells, and what appeared to be mesothelial cells on the outer surfaces. These cells infiltrated and deposited matrix (e.g., collagen, hyaluoronan, and limited elastin) within conduit walls. Constructs enclosed within the largest pore pouches exhibited significantly better tissue generation responses (e.g., better cell infiltration, elongation, and matrix deposition). Additionally, the healing response was impacted by the conduit average fiber diameter, and consequently, the effective pore diameter, with the largest diameter fibers promoting the most positive healing response (e.g., greater total cellularity, extracellular matrix deposition, and ?-SMA(+) cells). Six weeks post-intra-aortal grafting, constructs were occluded, but significant remodeling also occurred in the arterial microenvironment. Overall, these results demonstrate the importance of microenvironmental cues on recruited peritoneal cells and the necessity of developing strategies to further improve elastic matrix synthesis.

SUBMITTER: Bashur CA

PROVIDER: S-EPMC3589886 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Impact of electrospun conduit fiber diameter and enclosing pouch pore size on vascular constructs grown within rat peritoneal cavities.

Bashur Chris A CA Eagleton Matthew J MJ Ramamurthi Anand A

Tissue engineering. Part A 20121130 7-8

The generation of vascular grafts by recruiting autologous cells within the peritoneal cavity has shown promise. However, the microenvironment affects cell differentiation and elastic matrix production. Therefore, this study determined the impact of systematic changes in the average fiber diameter of electrospun poly(ɛ-caprolactone) conduits, and the pore size of pouches used to enclose the conduits, on recruited cells. After 2 weeks in the peritoneal cavity, fibrous capsules formed containing m ...[more]

PMID: 23075322

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Project description:Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2.

Project description:BackgroundWe tested the hypothesis that the mouse peritoneum can function like a bioreactor to generate directed bio-engineered tissues such as those used for bypass grafting. Additionally, we reasoned that the mouse animal model would allow us to elucidate the underlying cellular and molecular mechanisms that are responsible for the generation of tissue in peritoneal cavity.MethodsPlastic tubes (two tubes/mouse) were implanted into the peritoneal cavity of three strains of mice (C57BL/6, BALB/c, and MRL). The tubes were harvested, tissue capsule surrounding the tubes was removed, and analyzed by immunostaining (five capsules/five mice/strain) and microarray (three capsules/three mice/strain). In addition, the tissue capsules that were harvested from MRL mice (n = 21) were grafted into abdominal aorta of the same mice as autografts. The patency of all grafts was monitored by micro-ultrasound, and their functionality was assessed by laser Doppler imaging of blood flow in femoral arteries. Venous (n = 13) and arterial isografts (n = 11) were used as positive controls. In a negative control group (five mice/strain), the abdominal aorta was occluded by double ligation with 9-0 silk.ResultsThe implanted plastic tubes required at least 8 weeks of incubation in the peritoneum of the three strains of mice in order to generate useful grafts. No vascular cells were found in the tissue capsules. Microarray analysis of tissue capsules revealed that the capsular cells express a gene expression program that is vastly shared among the three strains of mice, and the cells exhibit a high degree of plasticity. The micro-ultrasound analysis of the grafts showed that 62% of autografts remained patent compared with 77% of venous isografts and 91% of arterial isografts. The laser Doppler imaging analysis showed that blood flow dropped by 40% and 35% in the autografts and vein isografts, respectively, 1 day after surgery. The flow, however, rebounded to the level of arterial isografts 1 month post-surgery and remained unchanged among all grafts for the next 4 months. Immunostaining of the autografts showed a thick vessel wall with endothelial cells that lined the lumen and smooth muscle cells that constituted the graft wall.ConclusionThe mouse peritoneal cavity of mice has the ability to function like a bioreactor to generate bio-engineered tissues. The tissue capsules harvested from peritoneal cavity of a mouse are composed of nonvascular cells that display phenotype of progenitor cells. After grafting, however, the capsule autografts become arterialized and remained patent for at least 4 months after surgery, similar to venous or arterial isografts.

Dataset Information

Impact of electrospun conduit fiber diameter and enclosing pouch pore size on vascular constructs grown within rat peritoneal cavities.

Publications

Impact of electrospun conduit fiber diameter and enclosing pouch pore size on vascular constructs grown within rat peritoneal cavities.

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