# Development of 3D Printed Synthetic Bone Graft Containing Small Molecules for Sequential Activation of Hedgehog and Hypoxia Signaling for Treatment of Nonunion Fractures

> **NIH VA I01** · VA LOMA LINDA HEALTHCARE SYSTEM · 2023 · —

## Abstract

ABSTRACT
 Bone injuries are a major health problem. There are 7.9 million bone fractures sustained annually in the U.S. Healing
is impaired in about 10% of these fractures with seriously delayed union or non-union, causing morbidity for patients and
enormous healthcare costs. While strategies such as bone grafting, synthetic polymers, low intensity pulsed ultrasound and
electromagnetic fields, growth factors and cell therapy are currently being used or investigated to promote bone healing,
each of these therapies have their own advantages and disadvantages in terms of cost, effectiveness and safety. Thus, there
is a compelling need to find novel effective therapies that promote fracture healing. Vitamin C and thyroid hormone (TH)
are known to play key roles in endochondral bone formation (EBF). Our recent studies on the molecular pathways for TH
and vitamin C actions revealed evidence that sequential activation of hedgehog and hypoxia signaling pathways contribute
to key steps involved in EBF. Our focus in this project is on the therapeutic utility and mechanisms of action of two small
molecules, SAG 21k and IOX2, that activate hedgehog and hypoxia signaling pathways to promote EBF at the fracture site.
In this proof of concept study, we propose to deliver SAG21K and IOX2 locally using 3D printed fibrin gel/β-tricalcium
phosphate (βTCP) scaffolds at the defect site to provide mechanical strength and minimize unwanted side effects on other
tissues. A clinically relevant segmental defect model in the femoral midshaft in which a 2.5-mm defect is stabilized by an
intramedullary threaded rod with attached plastic spacers that does not heal over a prolonged period will be used. Three
aims are proposed. In aim 1, we will 3D print fibrin gel/β-tricalcium phosphate (βTCP) scaffold preparations containing
SAG21k and IOX2 and evaluate the suitability of these preparations for delivery of effective concentrations of SAG21k
and IOX2 at the optimal therapeutic time window for activation of hedgehog and hypoxia signaling at the fracture site by
measurement of downstream signaling targets of these signaling pathways by immunohistochemistry (IHC) and real time
PCR in the fracture callus of mice at different times. In aim 2, we will test the hypothesis that sequential activation of
hedgehog followed by hypoxia signaling will be effective in promoting healing of femoral segmental defects. We will
compare the efficacy of bone healing with SAG21k and IOX2 with that of autografts, a gold standard used for healing of
nonunion defects. We will use validated microCT, bone strength and histological measurements to evaluate the fracture
healing phenotype. Therapeutic effectiveness of SAG21k/IOX2 combination therapy will be studied using aged and
diabetic mice with impaired fracture healing. In aim 3, we will test the hypothesis that sequential activation of sonic
hedgehog and hypoxia signaling induces bone healing by promoting direct conversion of chondrocytes-to-osteoblas...

## Key facts

- **NIH application ID:** 10664885
- **Project number:** 5I01BX005263-03
- **Recipient organization:** VA LOMA LINDA HEALTHCARE SYSTEM
- **Principal Investigator:** SUBBURAMAN MOHAN
- **Activity code:** I01 (R01, R21, SBIR, etc.)
- **Funding institute:** VA
- **Fiscal year:** 2023
- **Award amount:** —
- **Award type:** 5
- **Project period:** 2021-07-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10664885

## Citation

> US National Institutes of Health, RePORTER application 10664885, Development of 3D Printed Synthetic Bone Graft Containing Small Molecules for Sequential Activation of Hedgehog and Hypoxia Signaling for Treatment of Nonunion Fractures (5I01BX005263-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10664885. Licensed CC0.

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