Tissue engineering in cardiovascular surgery: new approach to develop completely human autologous tissue.

OBJECTIVE In cardiovascular tissue engineering, three-dimensional scaffolds serve as physical supports and templates for cell attachment and tissue development. Currently used scaffolds are still far from ideal, they are potentially immunogenic and they show toxic degradation and inflammatory reactions. The aim of this study is to develop a new method for a three-dimensional completely autologous human tissue without using any scaffold materials. METHODS Human aortic tissue is harvested from the ascending aorta in the operation room and worked up to pure human myofibroblasts cultures. These human aortic myofibroblasts cultures (1.5x10(6) cells, passage 3) were seeded into 15-cm culture dishes. Cells were cultured with Dulbecco' s modified Eagle's medium supplemented with 1 mM L-ascorbic acid 2-phosphate for 4 weeks to form myofibroblast sheets. The harvested cell sheets were folded to form four-layer sheets. The folded sheets were then framed up and cultured for another 4 weeks. Tissue development was evaluated by biochemical assay and light and electron microscopy. RESULTS After 4 weeks of culture in ascorbic acid supplemented medium, myofibroblasts formed thin cell sheets in culture dishes. The cell sheets presented in a multi-layered pattern surrounded by extracellular matrices. Cultured for additional 4 weeks on the frames, the folded sheets further developed into more solid and flexible tissues. Light microscopy documented a structure resembling to a native tissue with confluent extracellular matrix. Under transmission electron microscope, viable cells and confluent bundles of striated mature collagen fibers were observed. Hydroxyproline assays showed significant increase of collagen content after culturing on the frames and were 80.5% of that of natural human pericardium. CONCLUSIONS Improved cell culture technique may render human aortic myofibroblasts to a native tissue-like structure. A three-dimensional completely autologous human tissue may be further developed on the base of this structure with no show toxic degradation or inflammatory reactions.

[1]  Mark A. Randolph,et al.  Tissue Engineered Neocartilage Using Plasma Derived Polymer Substrates and Chondrocytes , 1998, Plastic and reconstructive surgery.

[2]  F. Grinnell,et al.  Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid. , 1989, Experimental cell research.

[3]  S. Hoerstrup,et al.  Tissue engineering: a new approach in cardiovascular surgery: Seeding of human fibroblasts followed by human endothelial cells on resorbable mesh. , 1998, European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery.

[4]  R Langer,et al.  Creation of viable pulmonary artery autografts through tissue engineering. , 1998, The Journal of thoracic and cardiovascular surgery.

[5]  D J Mooney,et al.  Development of biocompatible synthetic extracellular matrices for tissue engineering. , 1998, Trends in biotechnology.

[6]  G. Naughton,et al.  Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts. , 1997, Journal of biomedical materials research.

[7]  C K Breuer,et al.  The in vitro construction of a tissue engineered bioprosthetic heart valve. , 1997, European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery.

[8]  R. I. Schwarz,et al.  Ascorbate stimulation of PAT cells causes an increase in transcription rates and a decrease in degradation rates of procollagen mRNA. , 1984, Nucleic acids research.

[9]  I Vesely,et al.  Aortic valve cusp microstructure: the role of elastin. , 1995, The Annals of thoracic surgery.

[10]  S. Hoerstrup,et al.  Tissue engineering of a bioprosthetic heart valve: stimulation of extracellular matrix assessed by hydroxyproline assay. , 1999, ASAIO journal.

[11]  S. Pinnell,et al.  Regulation of collagen synthesis by ascorbic acid. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[12]  T. Matsuda,et al.  Mechanical stress induced cellular orientation and phenotypic modulation of 3-D cultured smooth muscle cells. , 1993, ASAIO journal.

[13]  Robert Langer,et al.  Biodegradable Polymer Scaffolds for Tissue Engineering , 1994, Bio/Technology.

[14]  W. Roberts,et al.  Histologic and ultrastructural features of normal human parietal pericardium. , 1980, The American journal of cardiology.

[15]  Y. Miyachi,et al.  Morphological and biochemical analyses on fibroblasts and self‐produced collagens in a novel three‐dimensional culture , 1997, The British journal of dermatology.

[16]  S. Pinnell,et al.  Regulation of collagen synthesis by ascorbic acid. Ascorbic acid increases type I procollagen mRNA. , 1982, Biochemical and biophysical research communications.

[17]  C. Enwemeka,et al.  A simplified method for the analysis of hydroxyproline in biological tissues. , 1996, Clinical biochemistry.

[18]  R. C. Chiu,et al.  Myocardial regeneration with satellite cell implantation. , 1994, Transplantation proceedings.

[19]  C K Breuer,et al.  Tissue-engineered heart valves. Autologous valve leaflet replacement study in a lamb model. , 1996, Circulation.

[20]  Y. Ninomiya,et al.  Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts. , 1988, European journal of biochemistry.

[21]  C K Breuer,et al.  Tissue engineering heart valves: valve leaflet replacement study in a lamb model. , 1995, The Annals of thoracic surgery.

[22]  H. Senoo,et al.  L‐ascorbic acid 2‐phosphate stimulates collagen accumulation, cell proliferation, and formation of a three‐dimensional tissuelike substance by skin fibroblasts , 1989, Journal of cellular physiology.