A Tissue Engineered Model of Aging: Interdependence and Cooperative Effects in Failing Tissues.

Aging remains a fundamental open problem in modern biology. Although there exist a number of theories on aging on the cellular scale, nearly nothing is known about how microscopic failures cascade to macroscopic failures of tissues, organs and ultimately the organism. The goal of this work is to bridge microscopic cell failure to macroscopic manifestations of aging. We use tissue engineered constructs to control the cellular-level damage and cell-cell distance in individual tissues to establish the role of complex interdependence and interactions between cells in aging tissues. We found that while microscopic mechanisms drive aging, the interdependency between cells plays a major role in tissue death, providing evidence on how cellular aging is connected to its higher systemic consequences.

Both sides nanopatterned tubular collagen scaffolds as tissue-engineered vascular grafts.

Two major requirements for a tissue-engineered vessel are the establishment of a continuous endothelium and adequate mechanical properties. In this study, a novel tubular collagen scaffold possessing nanopatterns in the form of channels (with a 650 nm periodicity) on both sides was designed and examined after seeding and co-culturing with vascular cells. Initially, the exterior of the tube was seeded with human vascular smooth muscle cells (VSMCs), cultured for 14 days, and then human internal thoracic artery endothelial cells (HITAECs) were seeded on the inside of the tube and cultured for a further week. Microscopy revealed that nano-scale patterns could be reproduced on collagen with high fidelity and preserved during incubation in vitro. The VSMCs were circumferentially orientated with the help of these nanopatterns and formed multilayers on the exterior, while HITAECs formed a continuous layer on the interior, as is the case in natural vessels. Both cell types were observed to proliferate and retain their phenotypes in the co-culture.

Influence of nanopatterns on endothelial cell adhesion: Enhanced cell retention under shear stress.

In this study, nanopatterned crosslinked films of collagen Type I were seeded with human microvascular endothelial cells and tested for their suitability for vascular tissue engineering. Since the films will be rolled into tubes with concentric layers of collagen, nutrient transfer through the collagen films is quite crucial. Molecular diffusivity through the collagen films, cell viability, cell proliferation and cell retention following shear stress were studied. Cells were seeded onto linearly nanogrooved films (groove widths of 332.5, 500 and 650nm), with the grooves aligned in the direction of flow. The nanopatterns did not affect cell proliferation or initial cell alignment; however, they significantly affected cell retention under fluid flow. While cell retention on unpatterned films was 35+/-10%, it was 75+/-4% on 332.5nm patterned films and even higher, 91+/-5%, on 650nm patterned films. The films were found to have diffusion coefficients of ca. 10(-6)cm(2)s(-1) for O(2) and 4-acetaminophenol, which is comparable to that observed in natural tissues. This constitutes another positive asset of these films for consideration as a scaffold material for vascular tissue engineering.

Nanopatterning of collagen scaffolds improve the mechanical properties of tissue engineered vascular grafts.

Tissue engineered constructs with cells growing in an organized manner have been shown to have improved mechanical properties. This can be especially important when constructing tissues that need to perform under load, such as cardiac and vascular tissue. Enhancement of mechanical properties of tissue engineered vascular grafts via orientation of smooth muscle cells by the help of topographical cues have not been reported yet. In the present study, collagen scaffolds with 650, 500, and 332.5 nm wide nanochannels and ridges were designed and seeded with smooth muscle cells isolated from the human saphenous vein. Cell alignment on the construct was shown by SEM and fluorescence microscopy. The ultimate tensile strength (UTS) and Young's modulus of the scaffolds were determined after 45 and 75 days. Alamar Blue assay was used to determine the number of viable cells on surfaces with different dimensioned patterns. Presence of nanopatterns increased the UTS from 0.55 +/- 0.11 to as much as 1.63 +/- 0.46 MPa, a value within the range of natural arteries and veins. Similarly, Young's modulus values were found to be around 4 MPa, again in the range of natural vessels. The study thus showed that nanopatterns as small as 332.5 nm could align the smooth muscle cells and that alignment significantly improved mechanical properties, indicating that nanopatterned collagen scaffolds have the potential for use in the tissue engineering of small diameter blood vessels.

Nanopatterned collagen tubes for vascular tissue engineering.

Nanopatterned (330 nm wide channels) type I collagen films were prepared by solvent casting on poly(dimethyl siloxane) (PDMS) templates. These films were rolled into tubular constructs and crosslinked. Tubular constructs were incubated under cell culture conditions for 28 days and examined by stereomicroscopy and scanning electron microscopy (SEM) for the integrity of the structure. The nanopatterned films were also seeded with human vascular smooth muscle cells (VSMCs) and examined after immunostaining with fluorescence microscopy and SEM to assess the cell phenotype and alignment on the nanopatterns on the films.

A novel construct as a cell carrier for tissue engineering.

A 3D scaffold, in the form of a foam, with the top surface carrying a micropattern, was constructed from biodegradable polyesters poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and poly(L-lactide-co-D,L-lactide) (P(L/DL)LA) to serve as a substitute for the extracellular matrix (ECM) of tissues with more than one cell type. The construct was tested in vitro for engineering of such tissues using fibroblasts (3T3) and epithelial cells (retinal pigment epithelial cells, D407). The patterned surface was seeded with D407 cells and the foam was seeded with 3T3 cells to represent a tissue with two different cell types. To improve cell adhesion, the construct was treated with fibronectin. The cells were seeded on the construct in a sequence allowing each type time for adhesion. Cell proliferation, studied by MTS assay, was significantly higher than that of tissue culture polystyrene control by day 14. Scanning electron and fluorescence microscopy showed that the foam side of the construct was highly porous and the pores were interconnected and this allowed cell mobility and proliferation. Immunostaining showed collagen deposition, indicating the secretion of the new ECM by the cells. On the film side of the construct D407 cells formed piles in the grooves and covered the surface completely. It was concluded that the 3D P(L/DL)LA-PHBV construct with one micropatterned surface has a serious potential for use as a tissue engineering carrier in the reconstruction of complex tissues with layered organization and different types of cells in each region.

Nanobiomaterials: a review of the existing science and technology, and new approaches.

Nanotechnology has made great strides forward in the creation of new surfaces, new materials and new forms which also find application in the biomedical field. Traditional biomedical applications started benefiting from the use nanotechnology in an array of areas, such as biosensors, tissue engineering, controlled release systems, intelligent systems and nanocomposites used in implant design. In this manuscript a review of developments in these areas will be provided along with some applications from our laboratories.

Cornea engineering on polyester carriers.

In this study, biodegradable polyester based carriers were designed for tissue engineering of the epithelial and the stromal layers of the cornea, and the final construct was tested in vitro. In the construction of the epithelial layer, micropatterned films were prepared from blends of biodegradable and biocompatible polyesters of natural (PHBV) and synthetic (P(L/DL)LA) origin, and these films were seeded with D407 (retinal pigment epithelial) cells. To improve cell adhesion and growth, the films were coated with fibronectin. To serve as the stromal layer of the cornea, highly porous foams of P(L/DL)LA-PHBV blends were seeded with 3T3 fibroblasts. Cell numbers on the polyester carriers were significantly higher than those on the tissue culture polystyrene control. The cells and the carriers were characterized scanning electron micrographs showed that the foam was highly porous and the pores were interconnected. 3T3 Fibroblasts were distributed quite homogeneously at the seeding site, but probably because of the high thickness of the carrier ( approximately 6 mm); they could not sufficiently populate the core (central parts of the foam) during the test duration. The D407 cells formed multilayers on the micropatterned polyester film. Immunohistochemical studies showed that the cells retained their phenotype during culturing; D407 cells formed tight junctions characteristic of epithelial cells, and 3T3 cells deposited collagen type I into the foams. On the basis of these results, we concluded that the micropatterned films and the foams made of P(L/DL)LA-PHBV blends have a serious potential as tissue engineering carriers for the reconstruction of the epithelial and stromal layers of the cornea.