Non-Destructive Analysis of Extracellular Matrix Development in Cardiovascular Tissue-Engineered Constructs

In the field of tissue engineering, there is an increasing demand for non-destructive methods to quantify the synthesis of extracellular matrix (ECM) components such as collagens, elastin or sulphated glycosaminoglycans (sGAGs) in vitro as a quality control before clinical use. In this study, procollagen I carboxyterminal peptide (PICP), procollagen III aminoterminal peptide (PIIINP), tropoelastin and sGAGs are investigated for their potential use as non-destructive markers in culture medium of statically cultivated cell-seeded fibrin gels. Measurement of PICP as marker for type I collagen synthesis, and PIIINP as marker of type III collagen turnover, correlated well with the hydroxyproline content of the fibrin gels, with a Pearson correlation coefficient of 0.98 and 0.97, respectively. The measurement of tropoelastin as marker of elastin synthesis correlated with the amount of elastin retained in fibrin gels with a Pearson correlation coefficient of 0.99. sGAGs were retained in fibrin gels, but were not detectable in culture medium at any time of measurement. In conclusion, this study demonstrates the potential of PICP and tropoelastin as non-destructive culture medium markers for collagen and elastin synthesis. To our knowledge, this is the first study in cardiovascular tissue engineering investigating the whole of here proposed biomarkers of ECM synthesis to monitor the maturation process of developing tissue non-invasively, but for comprehensive assessment of ECM development, these biomarkers need to be investigated in further studies, employing dynamic cultivation conditions and more complex tissue constructs.

[1]  D. Weiss,et al.  Value of fibrosis markers for staging liver fibrosis in patients with precirrhotic alcoholic liver disease. , 2008, Alcoholism, clinical and experimental research.

[2]  K J Halbhuber,et al.  Comparative study of cellular and extracellular matrix composition of native and tissue engineered heart valves. , 2004, Matrix biology : journal of the International Society for Matrix Biology.

[3]  M. Seibel,et al.  Molecular Markers of Bone Turnover: Biochemical, Technical and Analytical Aspects , 2000, Osteoporosis International.

[4]  Justin S. Weinbaum,et al.  Monitoring collagen transcription by vascular smooth muscle cells in fibrin-based tissue constructs. , 2010, Tissue engineering. Part C, Methods.

[5]  Michael S Sacks,et al.  The independent role of cyclic flexure in the early in vitro development of an engineered heart valve tissue. , 2005, Biomaterials.

[6]  T. Kita,et al.  Current understanding of biochemical markers in heart failure. , 2006, Medical science monitor : international medical journal of experimental and clinical research.

[7]  T. Halme,et al.  Lysyl oxidase activity and synthesis of desmosines in cultured human aortic cells and skin fibroblasts: comparison of cell lines from control subjects and patients with the Marfan syndrome or other annulo-aortic ectasia. , 1986, Scandinavian journal of clinical and laboratory investigation.

[8]  Thomas Schmitz-Rode,et al.  Nondestructive method to evaluate the collagen content of fibrin-based tissue engineered structures via ultrasound. , 2011, Tissue engineering. Part C, Methods.

[9]  F J Schoen,et al.  Tissue-engineered valved conduits in the pulmonary circulation. , 2000, The Journal of thoracic and cardiovascular surgery.

[10]  J. F. Woessner,et al.  The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. , 1961, Archives of biochemistry and biophysics.

[11]  Diane Hoffman-Kim,et al.  Comparison of three myofibroblast cell sources for the tissue engineering of cardiac valves. , 2005, Tissue engineering.

[12]  Y. Nishizawa,et al.  Dexamethasone enhances In vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells. , 1999, Arteriosclerosis, thrombosis, and vascular biology.

[13]  R. Burgeson,et al.  Collagen types. Molecular structure and tissue distribution. , 1992, Clinical orthopaedics and related research.

[14]  Thomas Schmitz-Rode,et al.  Tissue-engineered small-caliber vascular graft based on a novel biodegradable composite fibrin-polylactide scaffold. , 2009, Tissue engineering. Part A.

[15]  Justin S. Weinbaum,et al.  Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro. , 2010, Tissue engineering. Part A.

[16]  Laura Marcu,et al.  Noninvasive multimodal evaluation of bioengineered cartilage constructs combining time-resolved fluorescence and ultrasound imaging. , 2011, Tissue engineering. Part C, Methods.

[17]  Robert T Tranquillo,et al.  Elastic fiber production in cardiovascular tissue-equivalents. , 2003, Matrix biology : journal of the International Society for Matrix Biology.

[18]  L. T. Jensen,et al.  Collagen: scaffold for repair or execution. , 1997, Cardiovascular research.

[19]  Thomas Schmitz-Rode,et al.  The in vitro development of autologous fibrin-based tissue-engineered heart valves through optimised dynamic conditioning. , 2007, Biomaterials.

[20]  A. Weiss,et al.  Glycosaminoglycans Mediate the Coacervation of Human Tropoelastin through Dominant Charge Interactions Involving Lysine Side Chains* , 1999, The Journal of Biological Chemistry.

[21]  Joe S Mendez,et al.  Influence of strain on proteoglycan synthesis by valvular interstitial cells in three-dimensional culture. , 2008, Acta biomaterialia.

[22]  Peter J. Neame,et al.  Cartilage aggrecan. Biosynthesis, degradation and osteoarthritis. , 1994, The Journal of the Florida Medical Association.

[23]  K. König,et al.  Multiphoton autofluorescence imaging of intratissue elastic fibers. , 2005, Biomaterials.

[24]  Stefan Jockenhoevel,et al.  A collagen-glycosaminoglycan co-culture model for heart valve tissue engineering applications. , 2006, Biomaterials.

[25]  L. Krishnan,et al.  Glycosaminoglycans restrained in a fibrin matrix improve ECM remodelling by endothelial cells grown for vascular tissue engineering , 2009, Journal of tissue engineering and regenerative medicine.

[26]  Thomas Schmitz-Rode,et al.  Tranexamic acid--an alternative to aprotinin in fibrin-based cardiovascular tissue engineering. , 2009, Tissue engineering. Part A.

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