Phenotyping the Microvasculature in Critical-Sized Calvarial Defects via Multimodal Optical Imaging

Abstract

Tissue-engineered scaffolds are a powerful means of healing craniofacial bone defects arising from trauma or disease. Murine models of critical-sized bone defects are especially useful in understanding the role of microenvironmental factors such as vascularization on bone regeneration. Here, we demonstrate the capability of a novel multimodality imaging platform capable of acquiring in vivo images of microvascular architecture, microvascular blood flow, and tracer/cell tracking via intrinsic optical signaling (IOS), laser speckle contrast (LSC), and fluorescence (FL) imaging, respectively, in a critical-sized calvarial defect model. Defects that were 4 mm in diameter were made in the calvarial regions of mice followed by the implantation of osteoconductive scaffolds loaded with human adipose-derived stem cells embedded in fibrin gel. Using IOS imaging, we were able to visualize microvascular angiogenesis at the graft site and extracted morphological information such as vessel radius, length, and tortuosity two weeks after scaffold implantation. FL imaging allowed us to assess functional characteristics of the angiogenic vessel bed, such as time-to-peak of a fluorescent tracer, and also allowed us to track the distribution of fluorescently tagged human umbilical vein endothelial cells. Finally, we used LSC to characterize the in vivo hemodynamic response and maturity of the remodeled microvessels in the scaffold microenvironment. In this study, we provide a methodical framework for imaging tissue-engineered scaffolds, processing the images to extract key microenvironmental parameters, and visualizing these data in a manner that enables the characterization of the vascular phenotype and its effect on bone regeneration. Such multimodality imaging platforms can inform optimization and design of tissue-engineered scaffolds and elucidate the factors that promote enhanced vascularization and bone formation.

VascuViz: a multimodality and multiscale imaging and visualization pipeline for vascular systems biology

“…Next, a thinned-skull cranial window was surgically prepared according to 17 after which the animal’s head was secured in a custom-designed stereotaxic frame. Following this, in vivo optical imaging was conducted using a benchtop imaging system 18 at high spatial (7.7 μm) and temporal (5 s) resolution. This included imaging of total hemoglobin (HbT) absorption or cerebral blood volume (CBV) via intrinsic optical signal (IOS) contrast imaging under 570 ± 5 nm illumination and cerebral blood flow (CBF) via laser speckle contrast (LSC) imaging under 632.8 nm illumination, respectively.…”

Heparin‐Conjugated Decellularized Bone Particles Promote Enhanced Osteogenic Signaling of PDGF‐BB to Adipose‐Derived Stem Cells in Tissue Engineered Bone Grafts

“…ASC Culture and Scaffold Seeding : ASCs from two donors were isolated according to a previously described protocol with approval from Johns Hopkins Medical Institutions institutional review board . Following isolation, ASCs were expanded using growth medium containing DMEM with 4.5 g L −1 glucose (Gibco) + 10% fetal bovine serum (Atlanta Biologicals) + 1% penicillin‐streptomycin (Corning) + 1 ng mL −1 fibroblast growth factor‐2 (Peprotech).…”

Tracking Strain-Specific Morphogenesis and Angiogenesis of Murine Calvaria with Large-Scale Optoacoustic and Ultrasound Microscopy

“…Moreover, multimodal imaging would be a powerful tool to understand the dynamics of BM cells, vascularization, and bone formation. (6,43) Other widefield optical microscopy techniques could be integrated into the dual-modal U-LSOM to provide complementary information on, e.g., blood flow. (44) Prolonged isoflurane anesthesia is associated with a significant increase in brain water content, leading to edema formation.…”