Spatially-directed angiogenesis using ultrasound-controlled release of basic fibroblast growth factor from acoustically-responsive scaffolds

Abstract

Vascularization is a critical step following implantation of an engineered tissue construct in order to maintain its viability. The ability to spatially pattern or direct vascularization could be therapeutically beneficial for anastomosis and vessel in-growth. However, acellular and cell-based strategies to stimulate vascularization typically do not afford this control. We have developed an ultrasound-based method of spatially- controlling regenerative processes using acellular, composite hydrogels termed acoustically-responsive scaffolds (ARSs). An ARS consists of a fibrin matrix doped with a phase-shift double emulsion (PSDE). A therapeutic payload, which is initially contained within the PSDE, is released by an ultrasound-mediated process called acoustic droplet vaporization (ADV). During ADV, the perfluorocarbon (PFC) phase within the PSDE is vaporized into a gas bubble. In this study, we generated ex situ four different spatial patterns of ADV within ARSs containing basic fibroblast growth factor (bFGF), which were subcutaneously implanted in mice. The PFC species within the PSDE significantly affected the morphology of the ARS, based on the stability of the gas bubble generated by ADV, which impacted host cell migration. Irrespective of PFC, significantly greater cell proliferation (i.e., up to 2.9-fold) and angiogenesis (i.e., up to 3.7-fold) were observed adjacent to +ADV regions of the ARSs compared to -ADV regions. The morphology of the PSDE, macrophage infiltration, and perfusion in the implant region were also quantified. These results demonstrate that spatially-defined patterns of ADV within an ARS can elicit spatially-defined patterns of angiogenesis. Overall, these finding can be applied to improve strategies for spatially-controlling vascularization. STATEMENT OF SIGNIFICANCE: Vascularization is a critical step following implantation of an engineered tissue. The ability to spatially pattern or direct vascularization could be therapeutically beneficial for inosculation and vessel in-growth. However, acellular and cell-based strategies to stimulate vascularization typically do not afford this control. We have developed an ultrasound-based method of spatially-controlling angiogenesis using acellular, composite hydrogels termed acoustically-responsive scaffolds (ARSs). An ARS consists of a fibrin matrix doped with a phase-shift double emulsion (PSDE). An ultrasound-mediated process called acoustic droplet vaporization (ADV) was used to release basic fibroblast growth factor (bFGF), which was initially contained within the PSDE. We demonstrate that spatially-defined patterns of ADV within an ARS can elicit spatially-defined patterns of angiogenesis in vivo. Overall, these finding can improve strategies for spatially-controlling vascularization.

Keywords: Acoustic droplet vaporization; Angiogenesis; Basic fibroblast growth factor; Drug delivery; Fibrin; Ultrasound.

Figure 1.

An ultrasound-based mechanism termed acoustic droplet vaporization (ADV) was used to release basic fibroblast growth factor (bFGF) from an acoustically-responsive scaffold (ARS). A) bFGF was encapsulated within a phase-shift double emulsion (PDSE). At subthreshold acoustic pressures (P < P_ADV), the perfluorocarbon (PFC) phase within the PSDE remains a liquid. At suprathreshold acoustic pressures (P > P_ADV), the PFC phase is vaporized into a gas bubble, which causes release of bFGF. Depending on the molecular weight of the PFC, the generated bubble either is stable and continues to grow in size due to in-gassing or is transient and recondenses back into liquid PFC. B) ARSs were generated by incorporating the PSDE into a fibrin hydrogel. The ARSs were exposed ex situ to four different spatial patterns of ADV and then implanted subcutaneously in mice. As demonstrated with immunohistochemical staining, angiogenesis correlated with the pattern of ADV. The figure was created with BioRender.com

Figure 2.

The morphology of the ARS after ADV and extent of host cell migration into the ARS were dependent on the PFC species within the PSDE and pattern of ADV. A) H&E images of ARSs explanted after 14 days reveal stable bubbles in C6-ARSs whereas no stable bubbles were evident in C8-ARSs. Host cell migration was more evident in C8-ARSs. The white and black arrows denote the upper and lower interfaces of the ARSs proximal to the overlying skin and underlying muscle, respectively. Low magnification images are shown in Supplemental Figure 2. B) The migration distance of host cells into the upper and lower regions of the implanted scaffolds was quantified based on the H&E images. Data are represented as mean ± standard error of the mean (N=5 per group). Significant differences (p < 0.05) are denoted by brackets and Greek letters, which are defined in section 2.7.

Figure 3.

ADV impacted the size and density of the PSDE within the ARSs. A) Tissue sections were immunohistochemically stained for bFGF and counterstained with hematoxylin. The white and black arrows denote the upper and lower interfaces of the ARSs proximal to the overlying skin and underlying muscle, respectively. Low magnification images are shown in Supplemental Figure 3. The droplet diameter (B), droplet density (C), and bFGF+ area percent (D) were quantified in upper and lower regions of the ARSs. Data are represented as mean ± standard error of the mean (N=5 per group). Significant differences (p < 0.05) are denoted by brackets and Greek letters, which are defined in section 2.7.

Figure 4.

Cell proliferation around the ARSs correlated with the pattern of ADV. A) Tissue sections were immunohistochemically stained for Ki-67 and counterstained with hematoxylin. The white and black arrows denote the upper and lower interfaces of the ARSs proximal to the overlying skin and underlying muscle, respectively. Low magnification images are shown in Supplemental Figure 4. B) The density of Ki-67+ cells was quantified in adjacent tissues in regions above (i.e., upper) and below (i.e., lower) the implants. Data are represented as mean ± standard error of the mean (N=5 per group). Significant differences (p < 0.05) are denoted by brackets and Greek letters, which are defined in section 2.7.

Figure 5

Angiogenesis around the ARSs correlated with the pattern of ADV. A) Tissue sections were immunohistochemically stained for CD31 and counterstained with hematoxylin. The white and black arrows denote the upper and lower interfaces of the ARSs proximal to the overlying skin and underlying muscle, respectively. Low magnification images are shown in Supplemental Figure 5. B) The density of CD31+ blood vessels was quantified in adjacent tissues in regions above (i.e., upper) and below (i.e., lower) the implants. Data are represented as mean ± standard error of the mean (N=5 per group). Significant differences (p < 0.05) are denoted by brackets and Greek letters, which are defined in section 2.7.

Figure 6.

Laser speckle contrast analysis (LASCA) imaging was used to monitor perfusion in the implant region. A) Longitudinal images display an implanted C6-ARS (+ADV whole) within a mouse, which was imaged in a lateral recumbent position. The white circle denotes the region of interest (ROI) that was used for quantitative analysis. B) The change in perfusion was quantified by calculating the average perfusion within an ROI at a given day and then comparing it to the average perfusion on day 0. Data are represented as mean ± standard error of the mean (N=5 per group). Significant differences (p < 0.05) are denoted by Greek letters, which are defined in section 2.7.