# Enhanced Bone Formation in Segmental Defect Healing Using 3D Printed Scaffolds Containing Bone Marrow Stromal Cells and Small Molecules Targeting Chondrogenesis and Osteogenesis

## TL;DR

This study shows that 3D printed scaffolds with bone marrow cells and targeted molecules can enhance bone healing in large bone injuries.

## Contribution

A novel 3D printed scaffold with sequential small molecule release and BMSCs is introduced for segmental bone defect healing.

## Key findings

- SAG21k/IOX2-treated mice showed increased PTCH1 and HIF1α expression in fracture callus tissue.
- Therapy-receiving mice had increased bone formation compared to controls in microCT and histology analyses.

## Abstract

Background/Objectives: Nonunion bone healing results from a critical size defect that fails to bridge a bone injury to produce bony union. Novel approaches are critical for refining therapy in clinically challenging bone injuries, but the complex and coordinated nature of fracture callus tissue development requires study outside of the simple closed murine fracture model. Methods: We have utilized a three-dimensional printing approach to develop a scaffold construct with layers designed to sequentially release small molecule therapy within the tissues of a murine endochondral segmental defect to augment different mechanisms of fracture repair during critical stages of nonunion bone healing. Initially, a sonic hedgehog (SHH) agonist is released from a fibrin layer to promote chondrogenesis. A prolyl-hydroxylase domain (PHD)2 inhibitor is subsequently released from a β-tricalcium phosphate (β-TCP) layer to promote hypoxia-inducible factor (HIF)-1α regulation of angiogenesis. This sequential approach to therapy delivery is assisted by the inclusion of bone marrow stromal cells (BMSCs) to increase the cell substrate available for the small molecule therapy. Results: Immunohistochemistry of fracture callus tissue revealed increased expression of PTCH1 and HIF1α, targets of hedgehog and hypoxia signaling pathways, respectively, in the SAG21k/IOX2-treated mice compared to vehicle control. MicroCT and histology analyses showed increased bone in the fracture callus of mice that received therapy compared to control vehicle scaffolds. Conclusions: While our findings establish feasibility for the use of BMSCs and small molecules in the fibrin gel/β-TCP scaffolds to promote new bone formation for segmental defect healing, further optimization of these approaches is required to develop a fracture callus capable of completing bony union in a large defect.