389860 Biofabrication of Robust Tissue Engineered Vascular Media Employing Doxycycline Treatment

Monday, November 17, 2014: 2:36 PM
207 (Hilton Atlanta)
Vivek K. Bajpai, Chemical & Biological Engineering, University at Buffalo, The State University of New York, Amherst, NY, Panagiotis Mistriotis, Chemical and Biological Engineering, University at Buffalo, State University of New York, Amherst, NY, Zahra Chamanzar, Biomedical Engineering, University at Buffalo, Amherst, NY, Ryan Carpenter, Chemical & Biological Engineering, University at Buffalo, Amherst, NY and Stelios T. Andreadis, Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Amherst, NY

Introduction: Tissue engineered vessels (TEV) are attractive alternative to the shortage of autologus vascular grafts for coronary artery disease which is the prominent cause of mortality and morbidity in the United States (Lloyd-Jones et al. 2010). However, engineering of functionally vasoresponsive and mechanically strong blood vessel that can withstand physiological hydrodynamic environment remains a challenge.  Several strategies (including scaffold choice (natural and synthetic biomaterial) (Grassl ED et al. 2003, Yao L et al. 2005) and bioreactor based mechanical preconditioning (Syedain ZH et al. 2011) of the TEVs) to improve the function and mechanical properties of TEV have met with partial success. Here we report highly contractile and mechanically robust vascular media generation employing a small chemical molecule doxycycline.  

Materials & Methods: TEV were fabricated by mixing smooth muscle cells (SMC) progenitors (from three different type of SMC progenitors i.e. human hair follicle derived mesenchymal stem cells (hHF-MSC), bone marrow derived mesenchymal stem cells (BM-MSC) and dermal myofibroblasts) in fibrin hydrogels during polymerization and cultured in-vitro in vessel medium (10% MSC qualified serum plus 2ng/ml TGF-β1 in DMEM) supplemented with different concentrations of doxycyline (0 μg/ml, 0.5 μg/ml, 1 μg/ml, 10 μg/ml  and 50 μg/ml). After two weeks of culture, contractile forces (in response to various vasoagonists) and mechanical properties (linear modulus and ultimate tensile stress (UTS)) of TEV were measured. TEV were further analysed by quantitative real time PCR (qRT-PCR), immunoblotting, immunohistochemistry and hydroxyproline assay (total collagen content) to assess SMC specific differentiation and maturation within TEV.

Results and Discussion: Doxycycline significantly increased the contractile force (5-6 fold higher force in response to receptor mediated as well as non-receptor mediated agonists) and mechanical properties (3-7 fold increase in young's modulus and 2-3 fold increase in UTS) of TEVs, irrespective of SMC progenitors used for TEV fabrication. Interestingly, doxycycline exerted its effects only at low doses (0.5 μg/ml -10 μg/ml) while higher doses had no effect on improving TEV properties. SMC differentiation specific genes (ACTA2, MHC, TAGLN, SMTN, CNN1 and CALD1) and extracellular matrix (ECM) genes (COL1A1 and ELN) were significantly upregulated transcriptionally and translationally as evidenced by qRT-PCR, immunoblotting and immunohistochemical analysis of TEV supporting increased vasoreactivity and mechanical properties of TEVs in response to doxycycline treatment. In addition, total collagen content of TEV were ~6 fold higher in response to doxycycline treatment suggesting efficient secretion and remodeling of collagen, possibly leading to increased UTS of TEV.  Surprisingly, doxycycline's effect on SMC differentiation (of SMC progenitors) was unique to three dimensional (3D) environment as none of the SMC and ECM related genes changed (transcriptionally or translationally) in 2 dimensional cultures of SMC progenitors under the same culture conditions. To rule out that doxycycline's effect was not limited to fibrin matrix, we employed type I collagen instead of fibrin to fabricate TEV. Similar to fibrin based TEV, doxycycline increased the mechanical properties and SMC specific proteins (ACTA2 and CNN1) in collagen based TEV as well suggesting doxycycline's effect was not unique to fibrin.

mix1.tif

Figure 1. Schematic diagram of vascular media fabrication with different SMC progenitors, matrices (fibrin and collagen) and doxycycline doses (A); Contractile force in response to vasoagonists U46619 (TXA2 mimetic) and endothelin 1 (B); tensile strain vs. Stress curve of TEV (C) and representative immunoblot for SMC (ACTA2 and CNN1) and ECM (COL1A1 and ELN) specific proteins (D). 

Conclusions: Taken together, our results suggest doxycycline treatment as a novel strategy for robust and highly vasoreative vascular media fabrication for vascular tissue engineering applications.


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See more of this Session: Tissue Engineering Microenvironment
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