# Evaluating blood vessel phenotype in tissue-engineered craniofacial bone grafts using quantitative 3D light-sheet microscopy

> **NIH NIH F31** · JOHNS HOPKINS UNIVERSITY · 2020 · $46,320

## Abstract

Over 2 million patients worldwide are treated annually with bone grafts to fill critical-sized craniofacial defects.
Since autografts, the current gold standard treatment, introduce a high risk of donor site morbidity and have
significant geometric constraints, tissue-engineered bone grafts (TEBGs) present a promising alternative with
the potential to effectively regenerate geometrically complex, vascularized craniofacial tissues. However,
translational application of TEBGs has been limited by significant knowledge gaps regarding the relationship
between vascular structure and bone regeneration. The overall objective of this study is to develop and
implement novel quantitative 3D imaging techniques to gain a fundamental understanding of how neo-
vasculature impacts bone formation in TEBGs. While strategies have been developed to promote angiogenesis
in regenerating bone, published reports demonstrate that the amount of vasculature formed within TEBGs has
no correlation with the quantity or quality of regenerated bone. In native bone, microenvironmental interactions
between vessels and bone cells are essential to bone growth and maintenance. In particular, scientists have
recently identified a vessel phenotype high in CD31 and endomucin expression, termed “Type H”, that is
intimately associated with osteoprogenitors, and necessary for bone homeostasis. To determine whether Type
H vessels are related to regenerating bone in TEBGs, this proposed work will integrate whole-mount
immunostaining with a novel optical clearing method and light-sheet microscopy to image entire TEBGs (>mm3
volume) in 3D at single-cell resolution. Combining these technologies will enable unprecedented 3D quantitative
characterization of vessel phenotypes and vessel-bone cell relationships. Vessel and bone formation will be
evaluated with previously investigated TEBGs used to treat 4-mm murine critical-sized defects. In Aim 1,
protocols for whole-mount immunostaining, clearing, and light-sheet imaging native murine calvaria and
implanted calvarial TEBGs will be developed to enable 3D quantitative characterization of vessel phenotypes
and spatial relationships between vessels and bone cells. In Aim 2, this 3D quantitative imaging technique will
be applied to determine whether specific vessel phenotypes are correlated with enhanced bone formation in
TEBGs. First, vessel and bone formation will be compared in TEBGs known to yield two distinct levels of bone
regeneration in order to determine whether Type H vessel development contributes to bone healing. Second,
the effects of angiogenic and osteogenic growth factors on vascularized bone formation in TEBGs will be
evaluated to further elucidate the relationship of vessel phenotypes to bone regeneration. These findings will
enable the development of targeted strategies to promote vascularized bone regeneration. This research will
have a substantial positive impact on developing improved treatments for patients with debilitating cr...

## Key facts

- **NIH application ID:** 10064965
- **Project number:** 5F31DE029109-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Alexandra Rindone
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $46,320
- **Award type:** 5
- **Project period:** 2019-09-01 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10064965

## Citation

> US National Institutes of Health, RePORTER application 10064965, Evaluating blood vessel phenotype in tissue-engineered craniofacial bone grafts using quantitative 3D light-sheet microscopy (5F31DE029109-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10064965. Licensed CC0.

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