Fibrillin-1 microfibril deposition is dependent on fibronectin assembly.

Newly deposited microfibrils strongly colocalise with fibronectin in primary fibroblasts. Microfibril formation is grossly inhibited by fibronectin depletion, but rescued by supplementation with exogenous cellular fibronectin. As integrin receptors are key determinants of fibronectin assembly, we investigated whether they also influenced microfibril deposition. Analysis of beta1-integrin-receptor-null fibroblasts, blockage of cell surface integrin receptors that regulate fibronectin assembly and disruption of Rho kinase all result in suppressed deposition of both fibronectin and microfibrils. Antibody activation of beta1 integrins in fibronectin-depleted cultures is insufficient to rescue microfibril assembly. In fibronectin(RGE/RGE) mutant mouse fibroblast cultures, which do not engage alpha5beta1 integrin, extracellular assembly of both fibronectin and microfibrils is markedly reduced. Thus, pericellular microfibril assembly is regulated by fibronectin fibrillogenesis.

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment.

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

Gravity spun polycaprolactone fibers for applications in vascular tissue engineering: proliferation and function of human vascular endothelial cells.

Poly(epsilon-caprolactone) (PCL) fibers produced by wet spinning from solutions in acetone under lowshear (gravity-flow) conditions resulted in fiber strength of 8 MPa and stiffness of 0.08 Gpa. Cold drawing to an extension of 500% resulted in an increase in fiber strength to 43 MPa and stiffness to 0.3 GPa. The growth rate of human umbilical vein endothelial cells (HUVECs) (seeded at a density of 5 x 10(4) cells/mL) on as-spun fibers was consistently lower than that measured on tissue culture plastic (TCP) beyond day 2. Cell proliferation was similar on gelatin-coated fibers and TCP over 7 days and higher by a factor of 1.9 on 500% cold-drawn PCL fibers relative to TCP up to 4 days. Cell growth on PCL fibers exceeded that on Dacron monofilament by at least a factor of 3.7 at 9 days. Scanning electron microscopy revealed formation of a cell layer on samples of cold-drawn and gelatin-coated fibers after 24 hours in culture. Similar levels of ICAM-1 expression by HUVECs attached to PCL fibers and TCP were measured using RT-PCR and flow cytometry, indicative of low levels of immune activation. Retention of a specific function of HUVECs attached to PCL fibers was demonstrated by measuring their immune response to lipopolysaccharide. Levels of ICAM-1 expression increased by approximately 11% in cells attached to PCL fibers and TCP. The high fiber compliance, favorable endothelial cell proliferation rates, and retention of an important immune response of attached HUVECS support the use of gravity spun PCL fibers for three-dimensional scaffold production in vascular tissue engineering.

Gravity spun polycaprolactone fibres: controlling release of a hydrophilic macromolecule (ovalbumin) and a lipophilic drug (progesterone).

A hydrophilic macromolecule (ovalbumin (OVA)) and a lipophilic drug (progesterone) were incorporated in polycaprolactone (PCL) fibres by gravity spinning using particulate dispersions and co-solutions of PCL and steroid, respectively. PCL fibres loaded with 1% (w/w) OVA powder displayed a pronounced burst release phase (60% of the protein load) over 2 days in PBS at 37 degrees C. The release profile then tended to plateau. In contrast, OVA nanoparticle-loaded fibres exhibited delayed protein release initially and then a major increase at day 14. This behaviour may be useful for sequential release of polypeptide growth factors which are influential at specific time points in the wound healing process. SDS-PAGE analysis revealed that the protein molecular weight was conserved during fibre spinning. The amount of progesterone release from PCL fibres in PBS increased with drug loading but the cumulative release profiles (% w/w) were little affected by the initial drug loading of the fibres (1.5 and 3.5% w/w) or the concentration of the PCL spinning solution (12.5 and 20% w/v). Steroid delivery was rapid due to the high fibre surface area and high permeability of PCL resulting in complete drug loss over 24h. Released progesterone inhibited the growth of MCF-7 breast epithelial cells in culture, demonstrating retention of bioactivity. Gravity spinning shows potential for producing PCL fibre-based platforms for programmed delivery of bioactive molecules of utility for tissue engineering and drug delivery.

Gravity spinning of polycaprolactone fibres for applications in tissue engineering.

Poly(epsilon-caprolactone) (PCL) fibres have been produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. The tensile strength and stiffness of as-spun fibres were highly dependent on the concentration of the spinning solution. Use of a 6% w/v solution resulted in fibres having strength and stiffness of 1.8 MPa and 0.01 GPa, respectively, whereas these values increased to 9.9 MPa and 0.1 GPa when fibres were produced from 20% w/v solutions. Cold drawing to an extension of 500% resulted in further increases in fibre strength (up to 50 MPa) and stiffness (0.3 GPa). The surface morphology of as-spun fibres was modified, to yield a directional grooved pattern by drying in contact with a mandrel having a machined topography characterised by a peak-peak separation of 91 microm and a peak height of 30 microm. Limited in vitro studies of cell behaviour in contact with the fibres were performed using cell culture. The number of attached fibroblasts and myoblasts on as-spun PCL fibres after 5 days in cell culture was lower than on tissue culture plastic by a factor 2 and 1.5, respectively, but higher than on Dacron monofilament by a factor of 4 and 11, respectively. The high fibre compliance and the potential for controlling the fibre surface architecture to promote contact guidance effects together with the maintained proliferation of fibroblasts and myoblasts on as-spun PCL fibres in vitro recommends their use for 3-D scaffold production in soft tissue engineering.