(157h) Biofabrication of Robust Tissue Engineered Vascular Media Employing Doxycycline Treatment

normal">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.  

normal">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.

normal">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.

id="_x0000_t75" coordsize="21600,21600" o:spt="75" o:preferrelative="t"
path="m@4@5l@4@11@9@11@9@5xe" filled="f" stroked="f">

















alt="mix1.tif" style='width:471.75pt;height:150pt;visibility:visible;
mso-wrap-style:square'>

Figure
1. mso-bidi-font-weight:bold">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). 

normal">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.