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, 31 (14), 3840-7

Biomimetic Hydrogels With Pro-Angiogenic Properties

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Biomimetic Hydrogels With Pro-Angiogenic Properties

James J Moon et al. Biomaterials.

Abstract

To achieve the task of fabricating functional tissues, scaffold materials that can be sufficiently vascularized to mimic functionality and complexity of native tissues are yet to be developed. Here, we report development of synthetic, biomimetic hydrogels that allow the rapid formation of a stable and mature vascular network both in vitro and in vivo. Hydrogels were fabricated with integrin binding sites and protease-sensitive substrates to mimic the natural provisional extracellular matrices, and endothelial cells cultured in these hydrogels organized into stable, intricate networks of capillary-like structures. The resulting structures were further stabilized by recruitment of mesenchymal progenitor cells that differentiated into a smooth muscle cell lineage and deposited collagen IV and laminin in vitro. In addition, hydrogels transplanted into mouse corneas were infiltrated with host vasculature, resulting in extensive vascularization with functional blood vessels. These results indicate that these hydrogels may be useful for applications in basic biological research, tissue engineering, and regenerative medicine.

Figures

Figure 1
Figure 1. Proteolytically-degradable PEG hydrogels with intermediate rigidity support endothelial tubule formation and maintenance
A) The schematic illustration shows fabrication of MMP-sensitive PEG hydrogels by UV polymerization of PEG-RGDS and PEG chain with MMP-sensitive peptide in its backbone in the presence of cells. B) Hydrogels were degraded by collagenase (10 μg/ml), but not by plasmin (10 μg/ml) or buffer solutions. C) Hydrogels fabricated with different polymer weight percentages exhibited varying degradation profiles in collagenase (10 μg/ml) solution. D) Confocal and bright field images of HUVECs and 10T1/2 cells cultured for 3 and 6 days in hydrogels with varying polymer weight percentages. HUVECs and 10T1/2 cells were pre-stained with CytoTracker green and red, respectively, prior to encapsulation in hydrogels. E) The total tubule length formed and F) the number of branching point were measured during 6 day culture period in hydrogels formulated with varying polymer weight percentages. Only in hydrogels with intermediate polymer weight percentage (10%), HUVECs and 10T1/2 cells formed stable tubule-like structures. G) Compressive moduli ranging from 30 to 110 kPa were measured in hydrogels with and without encapsulated cells. Data represent mean ± SD. (n = 4 for B,C,G; n = 9 for E,F). * P < 0.05, analyzed by two-way ANOVA followed by Tukey's HSD test. Scale bars = 50 μm.
Figure 2
Figure 2. Time-lapse confocal videomicroscopy shows HUVECs and 10T1/2 undergoing tubule formation in hydrogels
Cellular interactions in hydrogels encapsulated with either A) HUVECs only or B) HUVECs and 10T1/2 cells were visualized over ∼70 hrs with time-lapse confocal videomicroscopy. A) In HUVECs mono-culture conditions, tubule-like structures initially formed by cells failed to maintain their networks and quickly regressed after ∼50 hrs of encapsulation in hydrogels. B) Tubule-like structures formed in co-culture conditions maintained their morphologies throughout the time-lapse experiments. C) The movies were analyzed quantitatively to plot tubule-length formed over time. Whereas the total tubule-length formed by HUVECs alone initially increased and sharply declined, there was a gradual increase and maintenance of the tubule-length in co-culture conditions. D) HUVECs with large vacuoles in their cell bodies and E) in the process of intercellular fusion of multiple vesicles to form large lumens were observed. 10T1/2 cells were recruited to the newly formed lumen or tubular structures. A,B,D,E) HUVECs and 10T1/2 cells were pre-stained with CellTracker Green and Red, respectively, prior to encapsulation into the hydrogels. F) HUVECs and 10T1/2 cells cultured for 28 days in hydrogels maintained the highly interconnected networks of tubules. F-actin and nuclei were stained by phalloidin-TRITC and DAPI, respectively. Data represent mean ± SD (n = 10-12). *P < 0.05, analyzed by two-way ANOVA followed by Tukey's HSD test compared to the HUVEC mono-culture groups. Scale bars = 50 μm.
Figure 3
Figure 3. The tubule structures are stabilized by smooth muscle-like cells differentiated from 10T1/2 cells and by new deposition of collagen IV and laminin in hydrogels
A) HUVECs and 10T1/2 cells each cultured in mono-culture condition had minimal expression of SM-α actin, calponin, and caldesmon on tissue culture wells and in 3D network of hydrogels as shown by Western blotting. In contrast, in co-culture conditions, the expression levels of these SMC protein markers were dramatically up-regulated both on tissue culture wells and in the hydrogels by day 6. B) In hydrogels with HUVECs and 10T1/2 cells, there was minimal expression of SM-α actin on day 1, but C) by day 6, expression of SM-α actin was localized adjacent to CD31 staining specific for ECs, indicating that 10T1/2 cells up-regulated expression of the SMC marker protein. In the PEG hydrogels cultured with HUVECs and 10T1/2 cells for 1 day, there was minimal expression of D) collagen type IV or F) laminin; however, by day 6, the tubule-like structures were highly decorated with E) collagen type IV and G) laminin, indicating that the encapsulated cells are actively producing their own set of ECM proteins, thereby remodeling the synthetic matrices. B,C) Anti-CD31 immunostainings and anti-SM-α actin are shown in green and red, respectively. D-G) Anti-CD31 is shown in red, while anti-collagen type IV (D,E) and anti-laminin (F,G) are shown in green. Scale bars = 50 μm.
Figure 4
Figure 4. Proteolytically-degradable PEG hydrogels promote neovascularization in murine cornea
Hydrogels incorporated with soluble and immobilized form of VEGF via PEG linkage were implanted into cornea in Flk1-myr∷mCherry transgenic mice. Newly formed blood vessels were visualized by confocal microscopy, and depth profiles of the vessels were generated to reveal their Z position with respect to the hydrogels. A-D) Non-degradable hydrogels releasing soluble VEGF supported angiogenesis adjacent to the hydrogels, but the depth decoding graph indicates lack of vessel penetration into the hydrogels. E-H) MMP-sensitive PEG hydrogels with 10% polymer weight promoted robust neovascularization adjacent to the hydrogels and extensive infiltration of host vasculature into the hydrogels. The arrow in E) indicates a portion hydrogel undergoing active degradation, and the arrow in H) points to regions within hydrogels with vessel infiltration. I-L) MMP-sensitive PEG hydrogels with 15% polymer weight had blood vessel growth on surface of hydrogels as pointed by the arrow in L), but the hydrogels remained mostly intact (I) without any significant vessel infiltration into the core. Scale bars = 100 μm in A-L).
Figure 5
Figure 5. Newly formed blood vessels in hydrogels are functional
A,B) Small incision was made in cornea and photopolymerized hydrogels were implanted into the micropocket. C). Blood vessels formed in the hydrogels were perfused with Dextran-Texas red (70KDa MW) injected intravenously into mice.

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