- Email: [email protected]
Collagen gel contraction assay for evaluation of porcine aortic valve interstitial cell glycolytic metabolism
e36
Abstracts / Cardiovascular Pathology 22 (2013) e29–e52
Collagen gel contraction assay for evaluation of porcine aortic valve interstitial cell glycolytic metabolism Peter Kamela, Paul Qua, Deepak Nagratha, Romain Harmanceyb, Heinrich Taegtmeyerb, K. Jane Grande-Allena a Rice University b University of Texas at Houston Medical School Purpose: Despite a high incidence of calcific aortic valve disease in metabolic syndrome, there is little information about the fundamental metabolism of heart valves. Furthermore, it is appreciated for other cell types that cell metabolism is a first responder to chemical and mechanical stimuli. It is unknown, however, how these strategies – which are often employed in the context of valve tissue engineering – may impact valvular interstitial cell (VIC) biology. Methods: To analyze basal metabolism, aortic VICs were harvested from porcine hearts, seeded into fibrin and collagen gels, and analyzed for gel contraction, lactate production, and glucose consumption in response to manipulation of concentrations of various metabolic substrates including glucose, galactose, pyruvate, and glutamine. Results: Gel contraction was a sensitive means of assessing VIC responses to metabolic manipulation, particularly in nutrientdepleted culture medium. VIC contraction of gels was optimal at a glucose concentration of 2 g/L, slower at 1 g/L and 4.5 g/L, and extremely delayed at 0–0.5 g/L. Substitution of galactose significantly delayed contraction and reduced lactate production. In low sugar concentrations, pyruvate depletion reduced contraction. Glutamine depletion reduced cell metabolism and viability. Conclusions: Nutrient depletion and manipulation of metabolic substrates impacts the viability, metabolism, and contractile behavior of VICs. Nonetheless, VICs readily utilize alternative substrates including pyruvate and glutamine to compensate for low sugar. Interestingly, high concentrations of specific nutrients reduced gel contraction, even without a substantial effect on cell viability. These results begin to link VIC metabolism and macroscopic behavior such as cell-matrix interaction and contraction.
http://dx.doi.org/10.1016/j.carpath.2013.01.030
Design valvular interstitial cell seeded scaffolds to mimic heart valve leaflet's structure and mechanical properties Nafiseh Masoumia,b, Benjamin L. Larsonc, Jesper Hjortnaesc,d, Ali Khademhosseinib,c,e a The Pennsylvania State University, Bioengineering Department, University Park, PA, USA b Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge MA, USA c Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA d Department of Cardiothoracic Surgery, University Medical Center Utrecht, Utrecht, The Netherlands e Wyss Institute of Biologically, Inspired Engineering, Harvard Medical School, Boston, MA, USA Purpose: We developed novel tri-layered scaffolds comprised of aligned elctrospun poly glycerol sebacate (PGS)/poly caprolactone (PCL) fibers and microfabricated PGS scaffolds and designed to emulate the anisotropic mechanical properties of native valvular tissues. Methods: PGS micromolding was used to create a scaffold with diamond-shaped pores. Two layers of aligned PGS/PCL electrospun scaffolds were formed and attached to the surface of a single layer PGS scaffolds to improve the mechanical stiffness of the single layer PGS microfabricated scaffold and enhance the tissue formation on the scaffold's 3D structure. Uniaxial mechanical testing was performed on
native porcine tissue and one layer and composite scaffolds for comparisons. The PGS/PCL concentration and ratio were set to match the composite scaffolds mechanical property and anisotropy in accordance with native tissue mechanical characteristics. Following the aortic porcine VICs isolation, the scaffolds were seeded for 5 weeks. Scaffolds and VIC-seeded constructs were characterized for their mechanical properties, and further characterized by confocal microscopy for cell orientation and phenotypic immunofluorescence, scanning electron microscopy for observation of general tissue formation, and by DNA and collagen assays. Results: The stiffness of aligned electrospun fibers with (20% w/v) PGS/ PCL in a (1:1) ratio, most closely matched the native tissue's anisotropic stiffness. Microfabricated PGS scaffolds offered elasticity and appropriate porosity to the composite scaffold. Cell orientation analysis showed that cell alignment was found in the direction of the long axis of the pores. DNA and collagen assays showed that there was a considerable amount of cellular material within all of the scaffolds. Conclusions: Collectively, the results of these studies will help better define scaffolds properties and cell seeding / cultivation requirements necessary for forming clinically relevant biomimetic tissue engineered heart valve leaflets. http://dx.doi.org/10.1016/j.carpath.2013.01.031
Modulation, mechanism and angiogenic potential of macrophage polarization (M1/M2) on electrospun bioresorbable vascular grafts Koyal Garg, Nicholas A. Pullen, Carole A. Oskeritzian, John J. Ryan, Gary L. Bowlin Virginia Commonwealth University, VA Purpose: (a) Modulate the macrophage phenotype (M1/M2) by varying the pore-size of electrospun polydioxanone (PDO). (b) Assess the angiogenic potential of macrophages on PDO in vitro and in vivo (Directed in vivo angiogenesis assay (DIVAA)). (c) Study the involvement of MyD88, TLR-4 and NFκB in macrophage polarization. Methods: Bone marrow derived murine macrophages (BMMΦs, 106 cells) were seeded on TCP (24 well plate) and PDO scaffolds (15 mm discs) of varying pore-sizes (1 μm, 10 μm and 14 μm). After 24 hours, the cell lysates were analyzed for arginase (Arg1), and iNOS expression (Western blot) and cell culture supernatants were analyzed for nitric oxide (NO2), TNF-α, IL-6, VEGF, TGF-β1 (ELISA). A 3D bead assay was used to quantify the endothelial cell sprouting induced by conditioned media from BMMΦ:PDO interaction. BMMΦs were also prepared from MyD88−/− and TLR-4−/− knockout mice and seeded. Results: Arg1, TGF-β1, VEGF (M2 markers) expression was statistically higher on the 14μm pore-size. The expression of iNOS, NO2-, TNF-α, IL6 (M1 markers) was higher on the 1-μm pore-size. The 3D bead assay showed larger average length of sprouts and higher percentage density of sprouts from the conditioned media of BMMΦ:PDO (14 μm) interaction. BMMΦ from both MyD88−/− and TLR-4−/− mice showed impaired iNOS and Arg1 expression on both PDO and TCP indicating that they are both involved in BMMΦ:PDO interaction. The results from the ongoing studies on NFκB inhibition and the DIVAA assay should provide greater insight into the mechanism of phenotype modulation and the angiogenic response of PDO in vivo, respectively. http://dx.doi.org/10.1016/j.carpath.2013.01.032
Targeting extracellular DNA to deliver IGF-1 to the injured heart Raffay S. Khana, Jay C. Sya, Milton E. Browna, Mario D. Martineza, Niren Murthyb, Michael E. Davisa a Emory University, Atlanta, GA b Georgia Tech, Atlanta, GA
Abstracts / Cardiovascular Pathology 22 (2013) e29–e52
Collagen gel contraction assay for evaluation of porcine aortic valve interstitial cell glycolytic metabolism Peter Kamela, Paul Qua, Deepak Nagratha, Romain Harmanceyb, Heinrich Taegtmeyerb, K. Jane Grande-Allena a Rice University b University of Texas at Houston Medical School Purpose: Despite a high incidence of calcific aortic valve disease in metabolic syndrome, there is little information about the fundamental metabolism of heart valves. Furthermore, it is appreciated for other cell types that cell metabolism is a first responder to chemical and mechanical stimuli. It is unknown, however, how these strategies – which are often employed in the context of valve tissue engineering – may impact valvular interstitial cell (VIC) biology. Methods: To analyze basal metabolism, aortic VICs were harvested from porcine hearts, seeded into fibrin and collagen gels, and analyzed for gel contraction, lactate production, and glucose consumption in response to manipulation of concentrations of various metabolic substrates including glucose, galactose, pyruvate, and glutamine. Results: Gel contraction was a sensitive means of assessing VIC responses to metabolic manipulation, particularly in nutrientdepleted culture medium. VIC contraction of gels was optimal at a glucose concentration of 2 g/L, slower at 1 g/L and 4.5 g/L, and extremely delayed at 0–0.5 g/L. Substitution of galactose significantly delayed contraction and reduced lactate production. In low sugar concentrations, pyruvate depletion reduced contraction. Glutamine depletion reduced cell metabolism and viability. Conclusions: Nutrient depletion and manipulation of metabolic substrates impacts the viability, metabolism, and contractile behavior of VICs. Nonetheless, VICs readily utilize alternative substrates including pyruvate and glutamine to compensate for low sugar. Interestingly, high concentrations of specific nutrients reduced gel contraction, even without a substantial effect on cell viability. These results begin to link VIC metabolism and macroscopic behavior such as cell-matrix interaction and contraction.
http://dx.doi.org/10.1016/j.carpath.2013.01.030
Design valvular interstitial cell seeded scaffolds to mimic heart valve leaflet's structure and mechanical properties Nafiseh Masoumia,b, Benjamin L. Larsonc, Jesper Hjortnaesc,d, Ali Khademhosseinib,c,e a The Pennsylvania State University, Bioengineering Department, University Park, PA, USA b Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge MA, USA c Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA d Department of Cardiothoracic Surgery, University Medical Center Utrecht, Utrecht, The Netherlands e Wyss Institute of Biologically, Inspired Engineering, Harvard Medical School, Boston, MA, USA Purpose: We developed novel tri-layered scaffolds comprised of aligned elctrospun poly glycerol sebacate (PGS)/poly caprolactone (PCL) fibers and microfabricated PGS scaffolds and designed to emulate the anisotropic mechanical properties of native valvular tissues. Methods: PGS micromolding was used to create a scaffold with diamond-shaped pores. Two layers of aligned PGS/PCL electrospun scaffolds were formed and attached to the surface of a single layer PGS scaffolds to improve the mechanical stiffness of the single layer PGS microfabricated scaffold and enhance the tissue formation on the scaffold's 3D structure. Uniaxial mechanical testing was performed on
native porcine tissue and one layer and composite scaffolds for comparisons. The PGS/PCL concentration and ratio were set to match the composite scaffolds mechanical property and anisotropy in accordance with native tissue mechanical characteristics. Following the aortic porcine VICs isolation, the scaffolds were seeded for 5 weeks. Scaffolds and VIC-seeded constructs were characterized for their mechanical properties, and further characterized by confocal microscopy for cell orientation and phenotypic immunofluorescence, scanning electron microscopy for observation of general tissue formation, and by DNA and collagen assays. Results: The stiffness of aligned electrospun fibers with (20% w/v) PGS/ PCL in a (1:1) ratio, most closely matched the native tissue's anisotropic stiffness. Microfabricated PGS scaffolds offered elasticity and appropriate porosity to the composite scaffold. Cell orientation analysis showed that cell alignment was found in the direction of the long axis of the pores. DNA and collagen assays showed that there was a considerable amount of cellular material within all of the scaffolds. Conclusions: Collectively, the results of these studies will help better define scaffolds properties and cell seeding / cultivation requirements necessary for forming clinically relevant biomimetic tissue engineered heart valve leaflets. http://dx.doi.org/10.1016/j.carpath.2013.01.031
Modulation, mechanism and angiogenic potential of macrophage polarization (M1/M2) on electrospun bioresorbable vascular grafts Koyal Garg, Nicholas A. Pullen, Carole A. Oskeritzian, John J. Ryan, Gary L. Bowlin Virginia Commonwealth University, VA Purpose: (a) Modulate the macrophage phenotype (M1/M2) by varying the pore-size of electrospun polydioxanone (PDO). (b) Assess the angiogenic potential of macrophages on PDO in vitro and in vivo (Directed in vivo angiogenesis assay (DIVAA)). (c) Study the involvement of MyD88, TLR-4 and NFκB in macrophage polarization. Methods: Bone marrow derived murine macrophages (BMMΦs, 106 cells) were seeded on TCP (24 well plate) and PDO scaffolds (15 mm discs) of varying pore-sizes (1 μm, 10 μm and 14 μm). After 24 hours, the cell lysates were analyzed for arginase (Arg1), and iNOS expression (Western blot) and cell culture supernatants were analyzed for nitric oxide (NO2), TNF-α, IL-6, VEGF, TGF-β1 (ELISA). A 3D bead assay was used to quantify the endothelial cell sprouting induced by conditioned media from BMMΦ:PDO interaction. BMMΦs were also prepared from MyD88−/− and TLR-4−/− knockout mice and seeded. Results: Arg1, TGF-β1, VEGF (M2 markers) expression was statistically higher on the 14μm pore-size. The expression of iNOS, NO2-, TNF-α, IL6 (M1 markers) was higher on the 1-μm pore-size. The 3D bead assay showed larger average length of sprouts and higher percentage density of sprouts from the conditioned media of BMMΦ:PDO (14 μm) interaction. BMMΦ from both MyD88−/− and TLR-4−/− mice showed impaired iNOS and Arg1 expression on both PDO and TCP indicating that they are both involved in BMMΦ:PDO interaction. The results from the ongoing studies on NFκB inhibition and the DIVAA assay should provide greater insight into the mechanism of phenotype modulation and the angiogenic response of PDO in vivo, respectively. http://dx.doi.org/10.1016/j.carpath.2013.01.032
Targeting extracellular DNA to deliver IGF-1 to the injured heart Raffay S. Khana, Jay C. Sya, Milton E. Browna, Mario D. Martineza, Niren Murthyb, Michael E. Davisa a Emory University, Atlanta, GA b Georgia Tech, Atlanta, GA