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Scaffolds Covalently Immobilized with VEGF and Angiopoietin-1 to Promote Angiogenesis in Engineered Cardiac Tissues

Loraine L. Y. Chiu, Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada and Milica Radisic, IBBME/Chemical Engineering and Applied Chemistry, University of Toronto, 164 College Street, Room 407, Toronto, ON M5S 3G9, Canada.

Introduction:  Cardiovascular diseases have been the main cause of death in Canada for the past 25 years, and accounted for more than 30% of deaths in 20041. Myocardium infarction causes irreversible damage to the heart, and the heart becomes incapable of regeneration due to the non-proliferative nature of terminally differentiated adult cardiomyocytes. As a result, there is motivation to engineer cardiac tissue patches in vitro by culturing cardiac cells in a scaffold matrix, after which these patches can be grafted into the diseased heart to regenerate the injured myocardium.

One of the recent challenges in cardiac tissue engineering is that functional blood supply is needed for constructs larger than 100-200 microns in thickness in order to provide sufficient oxygen and nutrients. Oxygen transport only occurs up to a distance of 100μm under normal medium diffusion2. Cell density and viability is compromised under limited oxygen supply3. One solution is to design a physiologically interactive replacement consisting functional blood vessels for the injured vascular tissue. By inducing vascularization within the engineered tissue in vitro, limited transport capacity of oxygen and nutrients into the tissue can be overcome, thus improving its survival both in vitro and in vivo4.

Neovascularization requires coordination of multiple endothelial growth factors, receptors and intracellular signaling pathways. Amongst these angiogenic growth factors, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) have been shown to be the most important regulators of blood vessel formation. Past studies have shown that VEGF promotes the formation of new capillary vessels, while Ang1 induces the maturation and stabilization of new vessel networks, suggesting a complementary relationship between these two angiogenic factors5.

In this study, we immobilized VEGF and Ang1 in collagen scaffolds to protect growth factors against cellular inactivation and digestion, and to allow highly sustained and localized activity. We hypothesize that VEGF and Ang1, when covalently immobilized in collagen scaffolds, work together to first form new vessels, and then to stabilize these vessels.

Materials and Methods:  A metal borer was used to cut circular collagen scaffolds with diameter of 7mm and thickness of 2mm from a sheet of porous collagen sponge (Ultrafoam collagen sponge, Davol, 1050050). Both mouse recombinant VEGF-165 (Cell Sciences, CRV014B) and human recombinant Ang1 (R&D Systems, 923-AN-025) were immobilized into the scaffolds using EDC chemistry. Briefly, EDC chemistry was performed by immersing the collagen scaffolds into 150L sterile filtered solution of EDC (N-(3-dimenthylaminopropyl)-N'-ethylcarbodiimide hydrochloride, Sigma, E7750) and Sulfo-NHS (N-hydroxysulfosuccinimide, Pierce Chemicals, 24510) in a 96-well plate (EDC/sulfo-NHS concentrations of 24mg/60mg per 1mL 1M PBS). The activation of the collagen scaffolds was allowed to proceed for 20 minutes at room temperature. The scaffolds were then removed from the solution, and immersed into 100L solution of a) 1g/mL VEGF (VEGF+PBS), b) 1g/mL Ang1 (Ang1+PBS), c) 0.5g/mL VEGF and 0.5g/mL Ang1 (1/2VEGF+1/2Ang1), or d) 1g/mL VEGF and 1g/mL Ang1 (VEGF+Ang1) in PBS. For control samples (PBS control), collagen sponges were immersed in PBS at room temperature. The reaction was allowed to proceed for 1 hour at room temperature. The scaffolds were immersed in fresh PBS for 8 times to wash away the uncrosslinked VEGF, EDC and sulfo-NHS. After washing in PBS, the scaffolds were incubated in culture medium for 1 hour and dried on autoclaved Kim-wipes prior to cell seeding.

For in vitro studies, the collagen scaffolds were transferred to a clean 12-well plate after scaffold preparation. 50000 H5V endothelial cells were seeded onto the scaffolds in 10L culture medium. The scaffolds were incubated for 40 minutes at 37oC for cells to attach. After incubation, 1mL fresh culture medium was added to each well. The samples were cultured for 3 or 7 days with a 100% change and collection of culture medium on Day 3, Day 5 and Day 7. Collected culture medium was stored at -20oC for further lactate and glucose assays. The samples were analyzed for final cell number by XTT assay, lactate production rate by lactate assay, glucose consumption rate by glucose assay, and cell density at various heights of the scaffold by cryosectioning and DAPI staining. The effective VEGF and Ang1 concentrations were quantified by ELISA.

For in vivo studies, the collagen scaffolds immobilized with solutions of a) 0g/mL VEGF (control), b) 0.5g/mL VEGF (low VEGF), and 2g/mL VEGF (high VEGF) in PBS were surgically inserted into the right ventricular outflow tract (RVOT) of adult rat hearts to repair transmural defects. At Week 1 and Week 4, the biomaterials were analyzed for patch thickness, cellular density and angiogenesis (i.e. vessel density and vessel diameter).

Results and Discussion:  At Day 3, collagen scaffolds with immobilized VEGF (VEGF+PBS, 1/2VEGF+1/2Ang1 and VEGF+Ang1 groups), showed increased final cell number (1.6-fold, 1.8-fold and 1.7-fold respectively) compared to PBS control in vitro (one-way ANOVA, P = 0.0213). This is consistent with the cell proliferative effect of VEGF. Although Ang1+PBS group had a 1.4-fold higher cell number than PBS control, it did not show statistically significant difference in final cell number compared to PBS control. This is due to the fact that Ang1 is responsible for endothelial cell survival rather than cell proliferation.

Scaffolds were also cryosectioned and stained with DAPI to count number of cell nuclei at various heights. At Day 3, groups with immobilized VEGF and/or Ang1 showed higher cell density than PBS control (two-way ANOVA, P < 0.0001). At 800m and 1000m from the top of the scaffold, VEGF+PBS group showed higher cell density than Ang1+PBS (Bonferroni post-tests, P < 0.05 for 800m, P < 0.01 for 1000m). At the top of the scaffold, 1/2VEGF+1/2Ang1 group showed higher cell density than VEGF+PBS group (Bonferroni post-tests, P < 0.05). At 400m, 1/2VEGF+1/2Ang1 group showed higher cell density than Ang1+PBS group (Bonferroni post-tests, P < 0.001). At 600m, VEGF+Ang1 group showed higher cell density than 1/2VEGF+1/2Ang1 (Bonferroni post-tests, P < 0.05).

Lactate production rate and glucose consumption rate both increased from Day 3 to Day 7 for all experimental groups in vitro. More importantly, VEGF+PBS, Ang1+PBS, 1/2VEGF+1/2Ang1 and VEGF+Ang1 groups all showed higher lactate production rates compared to PBS control at Day 7 (2.9-fold, 2.6-fold, 3.5-fold and 2.6-fold respectively). 1/2VEGF+1/2Ang1 showed significantly higher lactate production rate than PBS control at Day 7 (two-way ANOVA Bonferroni post-tests, P < 0.0001). VEGF+PBS, Ang1+PBS, 1/2VEGF+1/2Ang1 and VEGF+Ang1 groups all showed higher glucose consumption rates compared to PBS control at Day 7 (2.8-fold, 1.4-fold, 2.7-fold and 2.5-fold respectively). Specifically, VEGF+PBS and VEGF+Ang1 groups showed significantly higher glucose consumption rate than PBS control at Day 7 (two-way ANOVA Bonferroni post-tests, P < 0.05).

For in vivo studies, trichrome stained images and the corresponding image analysis showed significant increase in thickness of the biomaterial for high VEGF patches compared to control patches at 4 weeks (P < 0.001). Vessel density within the biomaterial was also significantly greater for high VEGF sponge compared to the control sponge at both 1 week and 4 weeks (P = 0.019 for 1 week; P = 0.008 for 4 weeks). This is consistent with the angiogenic effect of VEGF. However, the vessel diameters were not significantly different between high VEGF and control sponges, suggesting a need for an additional growth factor such as Ang1 for vessel stabilization.

Further in vivo studies showed that vessel density was significantly higher in high VEGF patches compared to low VEGF patches at Week 4 (two-way ANOVA Bonferroni post-tests, P < 0.05).

 

Conclusions:

The covalent immobilization of combined growth factors VEGF and Ang1 in collagen sponges leads to endothelial cell proliferation and survival, and increased cell metabolism in vitro. More importantly, angiogenic effect was evident when collagen scaffolds with immobilized VEGF was used in vivo to repair the right ventricular outflow tract of adult rat hearts. Further studies will be performed to investigate the effect of combined immobilized growth factors on angiogenesis in vivo.

References:

1. Statistics Canada. Mortality, Summary List of Causes (2004), 2007.

2. Morritt AN, Bortolotto SK, Dilley RJ, Han X, Kompa AR, McCombe D, Wright CE, Itescu S, Angus JA, Morrison WA, Circulation 115:353-360, 2007.

3. Radisic M, Malda J, Epping E, Geng W, Langer R, Vunjak-Novakovic G, Biotechnology and Bioengineering 93:332-343, 2006.

4. Shen YH, Shoichet MS, Radisic M, Acta Biomaterialia 4:477-489, 2008.

5. Kim SH, Kiick KL, Peptides 28:2125-2136, 2007.