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Multimodality imaging reveals angiogenic evolution in vivo during calvarial bone defect healing

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Abstract

The healing of calvarial bone defects is a pressing clinical problem that involves the dynamic interplay between angiogenesis and osteogenesis within the osteogenic niche. Although structural and functional vascular remodeling (i.e., angiogenic evolution) in the osteogenic niche is a crucial modulator of oxygenation, inflammatory and bone precursor cells, most clinical and pre-clinical investigations have been limited to characterizing structural changes in the vasculature and bone. Therefore, we developed a new multimodality imaging approach that for the first time enabled the longitudinal (i.e., over four weeks) and dynamic characterization of multiple in vivo functional parameters in the remodeled vasculature and its effects on de novo osteogenesis, in a preclinical calvarial defect model. We employed multi-wavelength intrinsic optical signal (IOS) imaging to assess microvascular remodeling, intravascular oxygenation (SO2), and osteogenesis; laser speckle contrast (LSC) imaging to assess concomitant changes in blood flow and vascular maturity; and micro-computed tomography (μCT) to validate volumetric changes in calvarial bone. We found that angiogenic evolution was tightly coupled with calvarial bone regeneration and corresponded to distinct phases of bone healing, such as injury, hematoma formation, revascularization, and remodeling. The first three phases occurred during the initial two weeks of bone healing and were characterized by significant in vivo changes in vascular morphology, blood flow, oxygenation, and maturity. Overall, angiogenic evolution preceded osteogenesis, which only plateaued toward the end of bone healing (i.e., four weeks). Collectively, these data indicate the crucial role of angiogenic evolution in osteogenesis. We believe that such multimodality imaging approaches have the potential to inform the design of more efficacious tissue-engineering calvarial defect treatments.

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Acknowledgments

This work was supported by NIH/NCI grant nos. 2R01CA196701-06A1, 5R01CA237597-04 and 5R01DE027957-05.

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Author notes

  1. Arvind P. Pathak

    Present address: Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, 720 Rutland Ave, 217 Traylor Bldg, Baltimore, MD, 21205, USA

Authors and Affiliations

  1. Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Yunke Ren, Xinying Chu, Warren L. Grayson & Arvind P. Pathak

  2. Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, 720 Rutland Ave, 217 Traylor Bldg, Baltimore, MD, 21205, USA

    Janaka Senarathna & Akanksha Bhargava

  3. Kavli Neuroscience Discovery Institute, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Janaka Senarathna

  4. The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Arvind P. Pathak

  5. Department of Materials Science and Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Warren L. Grayson

  6. Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Warren L. Grayson

  7. Electrical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Arvind P. Pathak

  8. Translational Tissue Engineering Center, Johns Hopkins University, Baltimore, MD, USA

    Warren L. Grayson

  9. Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD, USA

    Warren L. Grayson & Arvind P. Pathak

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Contributions

"YR, WG and APP designed the experiments. YR and APP wrote the main manuscript text and prepared figures. XC, JS, AB, WG assisted with imaging, data/image analyses and sample processing. All authors reviewed the manuscript."

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Correspondence to Arvind P. Pathak.

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10456_2023_9899_MOESM1_ESM.tiff

Supplementary file1 (TIFF 24677 KB) Supplementary Fig. 1: Developing and mature vessel phenotypes exhibited distinct blood flow and morphology. (a) Data from a representative animal in which developing vessels (blue) exhibited lower blood flow compared to mature vessels (yellow) through all the stages of calvarial bone healing. (b) Compared to developing vessels, mature vessels exhibited a widerrange of radii. (c) For the entire cohort, the mean blood flow of developing vessels was significantly lower (p<0.05) than that of mature vessels and (d) the mean radii of developing vessels was significantly smaller (p<0.05) than that of mature vessels, across the phases of angiogenic evolution

Supplementary file2 (TIFF 24677 KB)

Supplementary file3 (MOV 9905 KB) Supplementary Video 1: 3D visualization of the bone and blood vessels using the VascuViz protocol

Supplementary file4 (MOV 5727 KB) Supplementary Video 2: Time-lapse movie illustrating structural and functional changes in the vasculature (i.e. angiogenic evolution) in a representative mouse over the four week calvarial defect healing cycle

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Ren, Y., Chu, X., Senarathna, J. et al. Multimodality imaging reveals angiogenic evolution in vivo during calvarial bone defect healing. Angiogenesis 27, 105–119 (2024). https://doi.org/10.1007/s10456-023-09899-0

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