Longitudinal Monitoring of Angiogenesis in a Murine Window Chamber Model In Vivo

Abstract

Angiogenesis induced by growth factor administration, which can augment the blood supply in regenerative applications, has drawn wide attention in medical research. Longitudinal monitoring of vascular structure and development in vivo is important for understanding and evaluating the dynamics of involved biological processes. In this work, a dual-modality imaging system consisting of photoacoustic microscopy (PAM) and optical coherence tomography (OCT) was applied for noninvasive in vivo imaging of angiogenesis in a murine model. Fibrin scaffolds, with and without basic fibroblast growth factor (bFGF), were implanted in a flexible imaging window and longitudinally observed over 9 days. Imaging was conducted at 3, 5, 7, and 9 days after implantation to monitor vascularization in and around the scaffold. Several morphometric parameters were derived from the PAM images, including vessel area density (VAD), total vessel length (TVL), and vessel mean diameter (VMD). On days 7 and 9, mice receiving bFGF-laden fibrin gels exhibited significantly larger VAD and TVL compared to mice with fibrin-only gels. In addition, VMD significantly decreased in +bFGF mice versus fibrin-only mice on days 7 and 9. Blood vessel density, evaluated using immunohistochemical staining of explanted gels and underlying tissue on day 9, corroborated the findings from the PAM images. Overall, the experimental results highlight the utility of a dual-modality imaging system in longitudinally monitoring of vasculature in vivo with high resolution and sensitivity, thereby providing an effective tool to study angiogenesis.

Impact statement

In this study, we used a dual-modality imaging system with optical coherence tomography (OCT) and photoacoustic microscopy (PAM) capabilities to longitudinally monitor angiogenesis in a murine window chamber model with a flexible imaging window. OCT enabled visualization of the morphology of the fibrin hydrogel and surrounding tissue. Angiogenesis was detected using PAM and verified using histology. The experimental results highlight the utility of a dual-modality imaging system in longitudinally monitoring of vasculature in vivo with high resolution and sensitivity, thereby providing an effective tool to study angiogenesis.

Introduction

Rapid vascularization is required for most types of engineered tissue constructs following implantation, particularly for constructs of clinically relevant sizes. For example, with a prevascularized construct, which already contains a vessel network, inosculation of the preformed network with the host vasculature is required to main construct viability. Blood vessel growth can occur via distinct mechanisms such as vasculogenesis, arteriogenesis, and angiogenesis, with the last mechanism being largely responsible for vascular in-growth into engineered constructs. Various strategies have been implemented to facilitate vascularization, including the administration of exogenous growth factors/cytokines,¹ genetic vectors,² and/or cells³ within biocompatible hydrogels. Alternatively, hydrogels can be designed with mechanical cues that stimulate vessel growth.⁴

Noninvasive imaging techniques enable longitudinal assessment of vascular structure and function that cannot be easily obtained using conventional histological methods. With imaging, spatial resolution is inversely correlated with field of view (FOV) and imaging depth. Angiogenesis, which is the growth of new vessels from existing vessels, results in increased microvessel (e.g., capillary) density. Clinically utilized techniques such as magnetic resonance and ultrasound imaging modalities enable acquisition of clinically relevant FOVs at applicable depths. However, even with the use of contrast agents, conventional protocols of these techniques yield spatial resolutions that are too coarse to permit visualization of individual microvessels. Contrastingly, the utilization of optical methods such as microscopy (e.g., confocal, multiphoton) and laser speckle contrast imaging enables spatial resolution of microvessels; however, the imaging depths of these techniques are relatively superficial.

As a hybrid technology, photoacoustic imaging (PAI) combines the advantages of optical contrast and acoustic penetration, offering high resolution at desired depth with high sensitivity. Specifically, tissue chromophores are irradiated by short laser pulses, generating broadband ultrasound waves due to optical absorption and thermal expansion. These waves are detected at the tissue surface by an ultrasound transducer and used to reconstruct three-dimensional (3D) images of the internal tissue structure.^5–7 Photoacoustic microscopy (PAM), as one major implementation of PAI, has achieved spatial resolutions ranging from submicron to submillimeter, at maximum imaging depths ranging from a few hundred microns to a few millimeters.^8,9 Since hemoglobin is a major absorber of laser, angiogenesis, which plays a significant role in tissue remodeling and wound healing,^10,11 can be detected and characterized using PAM with high sensitivity.^12,13

Optical coherence tomography (OCT) produces high-resolution cross-sectional images of the internal microstructure of living tissue and has been widely applied to medical and biological applications.^14,15 By evaluating scattering effects and low-coherence interferometry signals, OCT provides structural information, which is complementary with the optical absorption information provided by PAM. The spatial resolution of OCT can reach the micron level in both lateral and axial directions, with a depth penetration of a few millimeters.¹⁶ Thus, the combination of OCT and PAM can present a comprehensive picture of the in vivo microenvironment.

To realize longitudinal in vivo imaging, a proper intravital imaging window is necessary to provide a clear and consistent imaging FOV. The dorsal skinfold chamber, which is installed following surgical removal of a full-thickness region of skin, offers a stable window for PAI. This model involves the installation of two chamber frames along with a glass coverslip to cover the excised skin. However, the stretched dorsal skin causes a huge burden on the animal, which can decrease the duration of an experiment and cause potential infection.^17,18

An abdominal imaging window, which can be used to study solid organs, requires less stretching of the skin but can still lead to infection due to the sharp edges of the glass coverslip.¹⁹ By comparison, a polydimethylsiloxane (PDMS)-based window, also known as a silicone window, is more flexible than a glass coverslip and can be glued directly to the skin adjacent to the tissue/organ of interest, thereby decreasing the burden on the mouse.²⁰ By utilizing a flexible window, vascular development can be monitored at micron resolution for an extended time, facilitating investigation of angiogenic progression.

In this work, in vivo longitudinal multimodality imaging was conducted through a PDMS-based window to monitor angiogenesis in a murine model. Fibrin hydrogels, with and without basic fibroblast growth factor (bFGF), were implanted within the window and subsequently imaged using PAM and OCT. bFGF is a pleiotropic factor that induces angiogenesis. Morphometric parameters derived from the PAM images were compared with vessel density acquired from histology. As will be shown, the histological analysis corroborated the results from the dual-modality imaging system, thus highlighting the utility of the imaging methodology.

Materials and Methods

Imaging system setup

As shown in Figure 1(a), a custom-built dual-modality imaging system was upgraded from our previous multimodality imaging setup.²¹ The system, which has high spatial resolution and fast image acquisition for noninvasive in vivo imaging, has a lateral resolution of 5 μm for both modalities as well as an axial resolution of 37.0 and 5.5 μm for PAM and OCT imaging modalities, respectively. Green and red light paths indicate the PAM and OCT working paths, respectively. They were integrated together through a dichroic mirror and shared the same scanning path during imaging. For PAM, a fiber laser (GLPM-16-1-10-M, IPG Photonics, Marlborough, MA) working at 532 nm was used as a light source, with a pulse repetition rate set to 50 kHz.

FIG. 1.

(a) Schematic diagram of the PAM and OCT dual-modality imaging system, in which the green light path represents PAM and red light path represents OCT. (b) Animal model and experimental timeline. A fibrin hydrogel, with or without bFGF, was implanted within the intravital imaging window that was surgically created within each mouse. Imaging experiments were performed on days 3, 5, 7, and 9 following implantation to assess vascular growth. After imaging on day 9, the mice were euthanized, and the hydrogels and underlying tissue were harvested for histological analysis. bFGF, basic fibroblast growth factor; BS, beam splitter; DAQ, data acquisition; DM, dichroic mirror; GM, galvo mirror; OCT, optical coherence tomography; PAM, photoacoustic microscopy; PD, photo diode; PH, pin hole; Ref Arm, reference arm; SLD, super luminescent diode; SMF, single-mode fiber. Color images are available online.

The laser was delivered to the imaging region after beam shaping followed by passing through a scan lens and the imaging window. The induced photoacoustic (PA) waves were detected by a custom-built, needle-shaped ultrasonic transducer (Optosonic Inc., Arcadia, CA) with a central frequency of 30 MHz and 60% bandwidth, which was coupled with the imaging window through gel drops (Systane, Alcon, TX). The PA signal was amplified by applying a homemade low-noise preamplifier and collected by a 250 MHz digitizer (RazorMax PCIe CSE161G4, Dynamic Signal Inc., San Bruno, CA).

For OCT, a spectral domain OCT platform (TEL321, Thorlabs, Newton, NJ) was integrated with the PAM to ensure coaxial illumination of both imaging modalities. For each imaging session, PAM was conducted first to assess vascular structure, followed by OCT to obtain scaffold structure. In PAM acquisition, the scanning time to capture a C-scan map image with a scanning area of 5 × 5 mm² was 2 s. To acquire an image of the same size using OCT, the acquisition time was 10 s.

Fibrin hydrogel fabrication

Hydrogels were prepared by combining bovine fibrinogen (Sigma-Aldrich, St. Louis, MO), bovine lung aprotinin (Sigma-Aldrich), and Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Waltham, MA). The mixture was vortexed and then placed in a vacuum chamber to ensure complete dissolution of the fibrinogen. Fibrin hydrogels (0.16 mL volume. 9.5 mm diameter, 2.3 mm height) were polymerized in custom plates by adding recombinant human thrombin (Recothrom, Baxter, Deerfield, IL). The final concentrations of fibrinogen, aprotinin, and thrombin were 10 mg/mL clottable protein, 0.05 U/mL, and 2 U/mL, respectively. In a subset of hydrogels, bFGF (GF003, Millipore, Temecula, CA) was added to the fibrinogen solution before polymerization at 10 μg per gel.

PDMS-based intravital imaging window

A PDMS silicone elastomer encapsulant kit (QSIL 216, PP&S, Tübingen, Germany), consisting of part A base and part B curing agent, was used to fabricate the imaging window. The base and curing agent were thoroughly mixed at a 10:1 (w/w) ratio in a Petri dish, and the mixture was placed under vacuum to remove bubbles produced during the mixing procedure. The mixture was aliquoted into a 24-well culture plate (well diameter: 16 mm) to yield a window with a nominal height of 100 μm. Subsequently, the plate was placed in a 77°C oven for 20 min to facilitate curing of the mixture. Following this, the membrane was removed from the mold and baked at 82°C for another 20 min to generate the imaging window. The windows were washed with 75% (v/v) ethanol and sterilized by autoclaving for eventual use in surgery.

Implantation of imaging window and hydrogel

This research was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan. Female CF-1 mice (n = 5 for fibrin-only group, n = 6 for fibrin + bFGF group, 8–12 weeks old, 26 ± 1 g bodyweight, Charles River Laboratories, Wilmington, MA) were anesthetized using isoflurane (2% for maintenance) and placed in a supine position on a heating pad to maintain body temperature. Hair was removed from the right side of the dorsal skin by shaving and applying depilatory cream. The skin was then disinfected using povidone iodine. Subsequently, a circular region of skin (diameter: 10 mm) was surgically excised and the fibrin hydrogel was placed atop the exposed hypodermis. Then the PDMS-based window was inserted such that the skin at the perimeter of the surgical site was overlying the window perimeter.

Normal saline was aliquoted into the window area to eliminate bubbles and ensure acoustic coupling. Once the window was fully inserted, tissue adhesive (Vetbond, 3M, St. Paul, MN) was applied to adhere and seal it to the skin. Due to skin overlapping the perimeter of the imaging window, the hydrogel occupied the entire window area that was visible (i.e., not covered by skin). The height of the hydrogel was such that it was held down against the underlying tissue by the window, thus preventing it from moving out of the window area. These hydrogel dimensions ensured that for each imaging time point, the FOV of the PAM captured an area of the implanted hydrogel within the imaging window.

In vivo longitudinal OCT and PAM imaging procedure

Mice were anesthetized using isoflurane (2% for maintenance) and placed in a supine position on a holder with the imaging window facing upwards. PAM and OCT were carried out in accordance with the descriptions in Section “Imaging system setup.” Figure 1(b) shows the experimental timeline. Imaging experiments started from 3 days after window implantation, ensuring that the mice had fully recovered from the surgery. The initial imaging conducted on day 3 was used as a baseline for further analysis. Subsequent longitudinal observations were performed on day 5, 7, and 9 to assess vascularization within the window region.

3D image fusion

PAM and OCT imaging results were coregistered to provide a comprehensive depiction within the scaffold and surrounding tissue through 3D image fusion. Due to coaxially aligned illumination lights for both imaging modalities, the XY planes for these results were coregistered by simply scaling the image size. Z-axial positions were also calculated based on the imaging parameter settings. The PAM image provided vascular structure under the gel, and the OCT image provided tomographic details of the fibrin scaffold.

Vasculature quantification

To simplify the analysis model, two-dimensional (2D) PAM maximum intensity projection (MIP) images were utilized for vasculature quantification. An open-source software, OCTAVA,²² was applied. By importing PAM images into the software, several quantitative metrics for vasculature were extracted, including vessel area density (VAD), total vessel length (TVL), and vessel mean diameter (VMD). VAD is the total area of perfused blood vessels divided by total image area, TVL is the total length of all observable vessels measured along the vessel centerline, and VMD is the mean vessel diameter derived from a local thickness algorithm applied to single vessels.²³ In addition, all vessel diameters were extracted, and a diameter distribution was derived. Compared with VMD, the distribution provided a comprehensive evaluation of the vessel diameter in the FOV.

Histology

After imaging on day 9, the mice were euthanized. The implants were harvested with the surrounding tissue and fixed overnight in 10% buffered formalin phosphate (Fisher Scientific). Tissue samples were transferred into 70% (v/v) ethanol until processing and embedding in paraffin by the Tissue and Molecular Pathology Core at the University of Michigan. Serial sections (thickness: 5 μm) were cut from the embedded tissue and transferred onto precleaned glass slides (Fisherbrand Superfrost Plus, Thermo Fisher Scientific) for histological analyses. Tissue sections were stained with hematoxylin and eosin (H&E) to visualize the overall tissue morphology. Tissue sections were immunohistochemically stained using a rabbit anti-mouse CD31 primary antibody (ab182981, Abcam). A goat anti-rabbit secondary labeled polymer-horseradish peroxidase conjugate (Envision + System-HRP [DAB], Dako North America, Inc., Carpinteria, CA) was used with the primary antibody. Staining specificity was confirmed by staining with only the secondary conjugate.

Stained tissue sections were imaged with an inverted microscope (Eclipse Ti-E, Nikon, Melville, NY) with 4 × and 20 × objectives, color digital camera (DS-Fi3, Nikon), and associated software (NIS-Elements, Nikon). Vessel density was derived from CD31-stained images acquired at 20 × magnification, with five different fields of view per tissue section. Using ImageJ (National Institutes of Health, Bethesda, MD), vessel density was determined by dividing the total number of vessels in each image by the tissue area.

Statistics

All statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). All bar graphs are plotted as the mean ± standard error of the mean of measured quantities for n = 6 (fibrin + bFGF) and n = 5 (fibrin). Statistically significant differences between groups were determined using an unpaired t-test, with differences deemed as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Experiment

Longitudinal PAM imaging of angiogenesis

PAM imaging was conducted on days 3, 5, 7, and 9 after hydrogel implantation to monitor vascular structure within the imaging window. Figure 2a, b shows representative 2D MIP images from fibrin + bFGF and fibrin-only groups, respectively. Qualitatively, greater vascular density was observed in the fibrin + bFGF group compared to the fibrin-only group, with more small vessels in the + bFGF group. A zoomed-in panel is presented in Figure 2c and shows the branch-like neovascular structures. Furthermore, the profile across a single vessel (marked by a white line) is displayed. Using a Gaussian fit, the full width at half maximum of the signal intensity was ∼20 μm.

FIG. 2.

Longitudinal PAM imaging results for (a) fibrin + bFGF and (b) fibrin-only groups on days 3, 5, 7, and 9. (c) A zoomed-in area from the fibrin + bFGF (day 9) is shown (top). The intensity profile along the white line, which bisects a single microvessel, is shown (bottom). Scale bar: 1 mm. Color images are available online.

3D fusion images of morphology and vascularization

3D fusion images from different observation perspectives are shown in Figure 3. OCT signal, which is shown in grayscale, highlights the morphology of the implanted hydrogel and surrounding tissue. PA signal, which is shown in a hot colormap, indicates the vascular structures. The PDMS window, implanted hydrogel, and underlying tissue layer are marked in Figure 3b, d.

FIG. 3.

Fusion images show the overlaid OCT and PAM data. 3D rendering of the fibrin + bFGF group (a) and fibrin-only group (c). 2D renderings, which include a PAM B-scan, for fibrin + bFGF group (b) and fibrin-only group (d). Scale bar: 2 mm. 2D, two-dimensional; 3D, three-dimensional. Color images are available online.

Quantitative analysis of vascularization based on PAM imaging

Figure 4 displays the quantified morphometric parameters derived from the PAM images. Figures 4a–c show the unnormalized values of VAD, TVL, and VMD, whereas Figures 4d–f display the corresponding values normalized by the day 3 data. For the fibrin-only group, there were no significant changes in VAD, TVL, or VMD. Contrastingly, with the fibrin + bFGF group, there were significant increases in VAD and TVL relative to day 3 beginning on day 5 and 7, respectively. The VMD decreased significantly on day 7 and 9 when compared to day 3. The distributions of vessel diameters are plotted in Figure 4g, h. Qualitatively, the number of small vessels in the fibrin + bFGF group increased longitudinally, whereas in the fibrin-only group, the number of small vessels decreased.

FIG. 4.

The following morphometric parameters, which characterized vascular structure, were derived from the PAM images: (a) VAD, (b) TVL, and (c) VMD. Panels (d–f) show the morphometric parameters normalized by day 3 values. The distribution of blood vessel diameters is shown for fibrin + bFGF group (g) and fibrin-only group (h). The bin width was 10 μm. Statistically significant differences in panels (a–f) are denoted as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). VAD, vessel area density; VMD, vessel mean diameter; TVL, total vessel length. Color images are available online.

H&E and CD31 stained images

Representative H&E and CD31 stained images are shown in Figure 5. Qualitatively, more host cells migrated into the hydrogel in the fibrin + bFGF group versus the fibrin-only group, which remained largely acellular. Blood vessels were seen in the granulation tissue underlying the hydrogels, with qualitatively more vessels in the fibrin + bFGF group.

FIG. 5.

Hydrogels and surrounding tissues were harvested on day 9 and stained with H&E as well as immunohistochemically stained for CD31. CD31-stained slides were also counterstained with hematoxylin. H&E images of the fibrin + bFGF group (a, c) and fibrin-only group (e, g) are shown. Zoomed-in areas are displayed in (c, g). CD31 images of the fibrin + bFGF group (b, d) and fibrin-only group (f, h) are shown. Zoomed-in areas are displayed in (d, h). Black triangles denote the edges of the fibrin hydrogel. Scale bar: 1 mm (a, b, e, f), 100 μm (c, d, g, h). H&E, hematoxylin and eosin. Color images are available online.

Quantitative analysis of vascularization based on histology

Blood vessel density was quantified from the CD31-stained images. As displayed in Figure 6a, there was a significantly higher density of vessels in the fibrin + bFGF group compared to the fibrin-only group. This finding is corroborated by the VAD derived from the PA images (Fig. 6b). A histogram profile of the cross-sectional area of the vessels, computed from the CD31 images, is shown in Figure 6c. Vessel area, rather than diameter, is reported since many of the vessels were noncircular in shape. Dashed line in the figure indicated the peak of the distribution. The fibrin + bFGF group exhibited a distribution of vessels that was smaller than the fibrin-only group. This histological finding was consistent with the distribution of vessel diameter derived from the PA images (Fig. 6d).

FIG. 6.

Blood vessels were characterized using CD31 immunohistochemistry and PA-derived metric. Vessel density (a) and distribution of individual vessel area (c) on day 9 were obtained from CD31-stained images. As a comparison, VAD (b) and distribution of vessel diameter (d) derived from PA images on day 9 are also shown. Statistically significant differences in panels (a,b) are denoted as follows: p < 0.05 (*) and p < 0.0001 (****). PA, photoacoustic. Color images are available online.

Discussion

In this study, we used a dual-modality imaging system with OCT and PAM capabilities to longitudinally monitor angiogenesis in a murine window chamber model with a flexible imaging window. OCT enabled visualization of the morphology of the fibrin hydrogel and surrounding tissue. Angiogenesis was detected using PAM and verified using histology.

bFGF stimulates angiogenesis by inducing migration and proliferation of endothelial cells as well as recruiting inflammatory cells. In this study, inclusion of bFGF within the fibrin hydrogel at 62.5 μg/mL significantly impacted vascular metrics as early as day 5 relative to fibrin-only controls for normalized VAD and day 7 for normalized TVL and VMD (Fig. 4). In a window chamber study conducted in rats, implantation of alginate beads with vascular endothelial growth factor increased the total number and density of vessels with diameters less than 25 μm 7 days after implantation.²⁴ Following incorporation within a type I collagen hydrogel, bFGF induced a dose-dependent angiogenic response within the window chamber model over the concentration range of 30 ng/mL to 3 μg/mL, above which the response became saturated.²⁵

In this previous study, angiogenesis was first observed on day 10 in a severe combined immunodeficient mouse model. The earlier onset of angiogenesis could be attributed to our use of an immunocompetent mouse model. A prior study, which utilized an abdominal imaging window, demonstrated that following implantation of prevascularized constructs, neovascularization was enhanced in immunocompetent versus immunocompromised mice.²⁶ As seen in Figure 5, blood vessels did not penetrate into either the fibrin + bFGF or fibrin-only hydrogels but rather were observed within the underlying tissue. This is consistent with previous acellular studies where delivered bFGF elicited an angiogenic response in the granulation tissue surrounding the fibrin implant.^27,28

Prior studies have assessed changes in vascular metrics within the window chamber model using PAI. Dual wavelength PAM microscopy enabled mapping of vascular structure and blood oxygenation following implantation of acellular dermal matrices.²⁹ Unlike the current study, angiogenesis was observed within the periphery of the implanted matrix. An advantage of using OCT is the visualization of the 3D morphology of the implant and surrounding tissue, unlike in the aforementioned study where only a 2D view was achievable. A commercially available PAI system revealed that the seeding density of adipose-derived microvascular fragments within collagen-glycosaminoglycan matrices correlated with blood oxygenation in the implant region.³⁰ In contrast to our PAM system, the PAT system adopted not only a linear-array ultrasound transducer, thus achieving deeper penetration, but also sacrificing spatial resolution. PAM showed that vessel diameter, vascular density, and tortuosity increased longitudinally following implantation of a 4T1 mammary tumor within the window chamber.³¹ This study presented exquisite PAM-based images of tumor vasculature, but the lack of a secondary imaging modality prevented study of the overall tumor morphology. An injection of gold nanorods, a PA contrast agent, improved the visualization of a prostate tumor implanted within the window chamber.³² Compared with our work, ultrasound imaging offered similar morphology results as OCT, but the use of a 14 MHz linear array was unable to achieve micron resolution in the lateral direction.

One limitation of this study was the use of a single-laser wavelength at 532 nm for generation of PA signal, which did not allow the measurement of blood oxygen saturation. As another limitation, this study assessed the angiogenesis induced by an acellular fibrin hydrogel containing bFGF. When directly incorporated into a 10 mg/mL fibrin hydrogel, bFGF exhibits a burst release pattern.³³ Future studies will investigate how this dual-modality system can assess the angiogenic response following controlled release of multiple growth factors as well as in prevascularized constructs.

Authors' Contributions

Z.X.: Methodology, Experiment, Data analysis, and Writing—Original draft preparation. W.Z.: Methodology and Experiment. C.Q.: Experiment. X.W.: Supervision. M.F.: Supervision and Writing—Reviewing and Editing.

Data Availability Statement

No large datasets were generated or analyzed during the current study. Minimal datasets necessary to interpret and replicate data in this article are available upon request to the corresponding author.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by NIH grant R01HL139656.