(546.1) Characterizing the Correlation Between Angiogenesis and Osteogenesis In Vivo Using Multicontrast Functional Imaging in a Calvarial Defect Model

Poster Board Number: E1

Yunke Ren (Johns Hopkins University School of Medicine), Akanksha Bhargava (Johns Hopkins University School of Medicine), Janaka Senarathna (Johns Hopkins University School of Medicine), Xinying Chu (Johns Hopkins University), Warren Grayson (Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, Johns Hopkins University, Johns Hopkins University, Johns Hopkins University), Arvind Pathak (Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine)

Angiogenesis is a key factor in bone healing that allows the delivery of oxygen, nutrients, inflammatory and bone precursor cells to the defect site. It induces dramatic structural and functional remodeling of the vasculature during the first 4 weeks of bone healing. Although recent advances in optical imaging have elucidated the in vivo relationship between angiogenesis and osteogenesis in a calvarial defect model, these efforts were mostly limited to structural imaging of the vasculature and bone. Therefore, to better characterize changes in vascular function (i.e. blood flow, oxygenation, etc.) during the bone healing cascade we developed a multicontrast optical imaging framework to assess in vivo changes in microvascular architecture using intrinsic optical signal (IOS) imaging; changes in blood flow with laser speckle contrast (LSC) imaging; and bone formation with bright-field imaging at high spatial (5 µm) and temporal (200 ms) resolutions. With this system we acquired multicontrast images from 10 animals with calvarial defects every 2 days, over 4 weeks (Fig. 1a, d). The bone and vasculature were then digitally segmented from the bright-field and IOS images for quantitative analysis (Fig. 1b, c). We also euthanized an animal each week for assessing 3D changes in bone volume and vascular architecture using an ex vivo, high-resolution (10 µm) CT imaging workflow we recently developed called VascuViz (Fig. 2a, b, d). Using this framework, we demonstrated that angiogenic remodeling within the bone defect microenvironment was most robust from post injury day (D) 4 to D10, during which blood vessel length, volume and density all showed significant increase (Fig. 2c). The blood flow exhibited a sudden increase at D6 when angiogenesis started, and the angiogenic vessels were most perfused at D12 (Fig. 1d). These vascular changes peaked by the end of week 2 and plateaued during the next 2 weeks which were correlated with a large increase in bone volume during the first 2 weeks and smaller increase during the last 2 weeks (Fig. 2e). These results indicate that both structural and functional changes of the microvascular system strongly correlate with bone growth during the early stages of bone healing.

Next, we plan to include additional indices of vascular function such as vessel maturity and intravascular oxygenation, as well as image-based hemodynamic models to further characterize the role of the vascular microenvironment during osteogenesis. We believe that this novel imaging framework for characterizing the defect microenvironment can be utilized to inform the design of novel tissue engineering (TE) constructs for craniofacial bone regeneration. Collectively, these advances herald a new era of “image-based TE”.

This work was supported by NIH grants 51R01CA196701-05, 1R01CA237597-01A1 and 5R01DE027957-02.





Fig. 1: Multicontrast In vivo imaging in a calvarial defect model over 4 weeks (a) Intrinsic Optical Signal (IOS) images showing bone and vessel morphology. Grey and red dashed lines represent the 3mm diameter cranial window and the 2mm diameter calvarial defect. (b) Segmentation of bone illustrates robust osteogenesis. (c) Segmentation of blood vessels illustrates angiogenesis within the defect. (d) Laser Speckle Contrast (LSC) images shows blood flow changes during bone healing.; Fig. 2: Correlation between osteogenesis and angiogenesis in a calvarial defect model over 4 weeks:(a) CT images of bone and vascular changes. Red dashed lines indicate the defects. (b) Segmented 3D bone volume within the defects shows osteogenesis. (c) Quantitative analysis shows vascular remodeling. Each data point represents mean ± SEM (n=3). *Significant difference (p < 0.05). (d) 3D visualization of vascular and bone remodeling. (e) Correlation between microvascular density and bone growth.