Mechanically challenged tissue-engineered organs, such as blood vessels, traditionally relied on synthetic or modified biological materials for structural support. In this report, we present a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) cultured with ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This TEBV featured a well-defined, three-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, SMC reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated LDL, produced PGI2, and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength over 2000 mmHg. This is the first completely biological TEBV to display a burst strength comparable to that of human vessels. Short-term grafting experiment in a canine model demonstrated good handling and suturability characteristics. Taken together, these results suggest that this novel technique can produce completely biological vessels fulfilling the fundamental requirements for grafting: high burst strength, positive surgical handling, and a functional endothelium.

A completely biological tissue-engineered human blood vessel

The immune and the detoxication systems of animals are characterized by allelic polymorphisms, which underlie individual differences in ability to combat assaults from pathogens and toxic compounds. Previous studies have shown that females may improve offspring survival by selecting mates on the basis of sexual ornaments and signals that honestly reveal health. In many cases the expression of these ornaments appears to be particularly sensitive to oxidative stress. Activated immune and detoxication systems often generate oxidative stress by an extensive production of reactive metabolites and free radicals. Given that tolerance or resistance to toxic compounds and pathogens can be inherited, female choice should promote the evolution of male ornaments that reliably reveal the status of the bearers' level of oxidative stress. Hence, oxidative stress may be one important agent linking the expression of sexual ornaments to genetic variation in fitness-related traits, thus promoting the evolution of female mate choice and male sexual ornamentation, a controversial issue in evolutionary biology ever since Darwin.

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Good genes, oxidative stress and condition-dependent sexual signals

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.

L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissuelike substance by skin fibroblasts

Vitamin C (or ascorbic acid) is regarded as the most important water-soluble antioxidant in human plasma and mammalian cells which have mechanisms to recycle and accumulate it against a concentration gradient, suggesting that the vitamin might also have important intracellular functions. In this review we summarize evidence from human trials that have attempted an association between vitamin C supplementation and an effect on biomarkers of oxidative DNA damage. Most studies reviewed herein showed either a vitamin C-mediated reduction in oxidative DNA damage or a null effect, whereas only a few studies showed an increase in specific base lesions. We also address the possible beneficial effects of vitamin C supplementation for the prevention of cancer and cardiovascular disease. Finally, we discuss the contribution of cell culture studies to our understanding of the mode of action of vitamin C and we review recent evidence that vitamin C is able to modulate gene expression and cellular function, with a part...

Review: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C.

The mitogenic, chemotactic, and synthetic responses of rat periodontal ligament (PDL) fibroblastic cells to epidermal growth factor (EGF), transforming growth factorβ (TGF-β), recombinant human platelet-derived growth factor (rhPDGF)-AB, rhPDGFBB, natural (n) PDGF-AB, and insulin-like growth factor-I (IGF-I) were examined in vitro using PDL cells obtained from the coagulum of healing tooth sockets. PDGFs and IGF-I have potent and comparable mitogenic effects on PDL fibroblastic cells. The maximum mitogenic effect of PDGFs was observed at the concentration of 10 ng/ml, whereas that of IGF-I was seen at concentrations higher than 100 ng/ml. In contrast, EGF induced moderate, and TGF-β inhibitory mitogenic responses. The combination of rhPDGF-AB with either EGF or TGF-β demonstrated comparable mitogenic potency, equivalent to the level of PDGF alone regardless of the mitogenic effect of other growth factors. The combination of rhPDGF-AB and IGF-I, however, showed a synergistic effect revealing the highest mi...

Mitogenic, chemotactic, and synthetic responses of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro.

Epidermal growth factor (2-50 ng/ml), prepared from mouse submaxillary glands, stimulated growth and the synthesis of non-collagenous proteins and hyaluronic acid, but inhibited collagen synthesis in cultured human skin fibroblasts, both stimulation and inhibition being dose-dependent. All these effects may be intrinsic functions of the epidermal growth factor molecule, because these effects were cancelled by the co-presence of antiserum specific for epidermal growth factor and because they were also observed following the addition of human epidermal growth factor produced urogastrone cDNA. On the other hand, L-ascorbate (vitamin C) stimulated growth and collagen synthesis, as well as synthesis of non-collagenous proteins, with no significant effect on hyaluronic acid synthesis. Co-presence of epidermal growth factor and ascorbate gave additive effects on growth and protein synthesis of the cells. These results suggest that the two growth-promoting factors, epidermal growth factor and L-ascorbate, modulate metabolism of extracellular matrix components as well as cell growth in a quite different manner in human skin fibroblasts.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb13993.x

Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts

Glycosaminoglycan synthesis by liver parenchymal cell clones in culture and its change with transformation

Active synthesis of glycosaminoglycans by liver parenchymal cells in primary culture

Two lines of albumin-producing rat liver parenchymal cell clones (BB and BC) have been shown to synthesize collagen at a level of 1-2% of total proteins produced which was two to nine times higher than those by the fibroblastic cells (L-929 and RLG-1). Type analysis of the collagen by carboxymethylcellulose chromatography has shown that the former two cell lines synthesized type I collagen, [α1(I)]2α2, type X, composed of α1 chains and type Y similar to type III collagen, while a transformed liver cell line (M) did the former two collagen types.

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Active Synthesis of Collagen by Albumin-Producing Liver Parenchymal Cell Clones in Culture

Rat liver parenchymal and non-parenchymal cells were isolated by the collagenase perfusion method followed by Percoll density gradient centrifugation. The isolated parenchymal cells, when cultured for a few days, formed cords of polygonal cells which are typical for hepatocytes and produced a low (about 0.2% of total protein synthesized) but significant amount of collagen. This is the first demonstration of collagen production by hepatic parenchyma) cells in primary culture.

Yoshifumi Ninomiya

Papers

Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts

Glycosaminoglycan synthesis by liver parenchymal cell clones in culture and its change with transformation

Active synthesis of glycosaminoglycans by liver parenchymal cells in primary culture

Active Synthesis of Collagen by Albumin-Producing Liver Parenchymal Cell Clones in Culture

Collagen Production by Rat Liver Parenchymal Cells in Primary Culture