Ultrasound-guided photoacoustic imaging-directed re-endothelialization of acellular vasculature leads to improved vascular performance

Abstract

As increasing effort is dedicated to investigating the regenerative capacity of decellularized tissues, research has progressed to recellularizing these tissues prior to implantation. The delivery and support of cells seeded throughout acellular scaffolds are typically conducted through the vascular axis of the tissues. However, it is unclear how cell concentration and injection frequency can affect the distribution of cells throughout the scaffold. Furthermore, what effects re-endothelialization have on vascular patency and function are not well understood. We investigated the use of ultrasound-guided photoacoustic (US/PA) imaging as a technique to visualize the distribution of microvascular endothelial cells within an optimized acellular construct upon re-endothelialization and perfusion conditioning. We also evaluated the vascular performance of the re-endothelialized scaffold using quantitative vascular corrosion casting (qVCC) and whole-blood perfusion. We found US/PA imaging was an effective technique to visualize the distribution of cells. Cellular retention following perfusion conditioning was also detected with US/PA imaging. Finally, we demonstrated that a partial recovery of vascular performance is possible following re-endothelialization-confirmed by fewer extravasations in qVCC and improved blood clearance following whole-blood perfusion.

Statement of significance: Re-endothelialization is a method that enables decellularized tissue to become useful as a tissue engineering construct by creating a nutrient delivery and waste removal system for the entire construct. Our approach utilizes a decellularization method that retains the basement ECM of a highly vascularized tissue upon which endothelial cells can be injected to form an endothelium. The US/PA method allows for rapid visualization of cells within a construct several cm thick. This approach can be experimentally used to observe changes in cellular distribution over large intervals of time, to help optimize cell seeding parameters, and to verify cell retention within re-endothelialized constructs. This approach has temporal and depth advantages compared to section reconstruction and imaged fluorophores respectively.

Keywords: Acellular biological matrices; Angiogenesis and vasculogenesis; Bioartificial organ; Biomimetic materials; Extracellular matrix.

Figure 1

Effects of nanoparticle incubation on HDMEC morphology. HDMECs incubated with gold nanoparticles overnight, then allowed proliferate near confluency (A–C) displayed similar morphology as HDMECs plated without nanoparticles (D–F). The same field of view was captured in phase-contrast, dark-field and bright-field images. Phase-contrast imaging reveals cobblestone morphology in nanoparticle and control groups (A, D) and a departure from spindle-like morphology. Dark-field and bright-field imaging demonstrate the presence of nanoparticles (light in dark-field imaging, black in bright-field imaging) (B, C) compared to control (E, F). Scale bar = 50μm. HDMECs injected into OA decellularized scaffolds with staining for CD-31 (G), VE-Cadherin (I). Tissue was not extensively cellularized, intimating at the need for more optimization. CD-31 positive staining matched up with VE-Cadherin with an association with nanoparticles (H). Scale bar = 200 μm. MTS assay for HDMECs incubated with gold nanotracers (J). Following incubation with gold nanotracers for 24 hours, HDMECs were maintained for 24 and 72 hours then incubated with MTS for four hours. The absorbance of the culture media and MTS dye were then measured on a 96-well plate in triplicate at 490 nm, n = 3. HDMECs incubated with NPs did not exhibit abnormal metabolic activity when compared to HDMECs without NPs.

Figure 2

Ultrasound-guided photoacoustic imaging of an optimized acellular scaffold devoid of cells (A–C, G–I) and re-endothelialized with HDMECs (D–F; J–R). Images A–F represent a top down view of the overlay of photoactoustic signal and ultrasound; whereas G-R represent individual cross-sections of the lung. Three injections of 3.3×10⁵ HDMECs, with gold nanoparticles in 3.3 ml media over two hours were delivered to an OA scaffold. Cells were allowed to adhere overnight before gradually increasing perfusion flow was administered (3.2 dyn/cm² for 24 hours, 8.7 dyn/ cm² for 12 hours, 19.6 dyn/ cm² for 24 hours). Photoacoustic signal from cells at 750 nm indicates the retention of cells distributed within the entirety of an OA scaffold at the conclusion of the culture, whereas barren OA scaffolds presented no signal. Media was rinsed out then tissue was fixed prior to US/PA imaging at 750 nm. A void in the scaffold representing the bronchus can be seen in cross-sections of the ultrasound images (arrow).

Figure 3

Vascular corrosion casts of fresh (A, D), decellularized (B, E) and re-endothelialized scaffolds following perfusion culture for 60 hours (C, F). Media was rinsed from scaffolds then fixed before vascular corrosion casting performed. Capillary networks from re-endothelialized scaffolds (C) closely resembled those of fresh tissue (A), compared to decellularized scaffolds alone (B). In fresh (D) and re-endothelialized (F) scaffolds, the larger vessels appear to be irregularly formed (arrowheads), denoting cellularity, whereas decellularized tissues have smooth vessels (asterisk) (E). Scale bar = 20 μm. Total extravasation volume was calculated per mm² by cubic micron for VCCs of fresh, decellularized, and re-endothelialized lung (G), n = 2. Re-endothelialization with HDMECs clearly rescues some of the vascular patency of the OA tissue.

Figure 4

Photographs of whole blood perfusion into re-endothelialized OA scaffolds (A–E, L–M), OA decellularized scaffolds (F–H), and fresh lung (I–K). 50 ml of blood was perfused into the pulmonary artery (B, G, J) of the tissue then cleared with 100 ml PBS (C–E, H, K). Although some blood aggregation occurred in fresh tissue (K), this was drastically less than in re-endothelialized (C–E), and OA decellularized (H). Whereas blood pooled within OA decellularized tissue with no clearance (H), in re-endothelialized tissue, clearing of blood occurred (C–E). Images (D) and (M) show increased clearing of image (C) and (L) when PBS perfusion is continued (arrows). Image E shows the underside of the re-endothelialized scaffold has also been remodeled. The OA scaffold alone had no regions where blood could be readily perfused out and cleared.

Figure 5

OA scaffolds re-endothelialized with HDMECs help decrease leakage of whole blood. Following re-endothelialization and whole-blood perfusion, scaffolds were perfused with PBS to clear blood. Sections were stained for CD-31 (A, C), staining positively for endothelial cells and platelets (red), and DAPI (blue). The majority of positive staining for nuclei was likely from white blood cells from blood. Extensive leakage of blood unable to be cleared was seen (A, B). Blood leakage can be visualized in (A) and (B) around the top of the vessel (demarked with a dashed line). Although occlusion failed to occur, the resultant leakage can be traced. An overlay with brightfield imaging (B) shows the presence of red blood cells (arrow). (C) and (D) demonstrate a non-occluded vascular pair. Some damage in the form of cell aggregation can be seen (asterisk) which failed to occur in some of the regions containing nanoparticles (arrowhead). In contrast, occlusions were present in other vessels (dagger). Scale bar = 200 μm