# Evaluation of the Osteogenic Potential of a NOTCH1 Agonist and Poloxamer 407 Hydrogel Regarding Osteoblasts

## TL;DR

This study shows that a NOTCH1 activator and a hydrogel can promote bone cell growth and mineralization, with potential for bone healing therapies.

## Contribution

The novel contribution is demonstrating the combined osteogenic potential of a NOTCH1 agonist and Poloxamer 407 hydrogel in vitro.

## Key findings

- Yhhu3792 increased NOTCH1 signaling and mineralization in osteoblasts with no toxicity.
- Combining Yhhu3792 with P407 significantly enhanced bone marrow stromal cell proliferation.
- Both agents showed increased expression of bone formation and NOTCH1 target genes.

## Abstract

To facilitate healing of bone injuries, there is a need for small-molecule therapeutics and bone substitutes that are osteogenic, non-toxic, and capable of releasing therapeutics. In this study, we assessed the importance of the NOTCH1 agonist Yhhu3792 and the Poloxamer 407 (P407) hydrogel on bone-forming osteoblasts and bone marrow stromal cells. Our findings indicate that Yhhu3792 has an anabolic effect on osteoblast proliferation and differentiation, promoting mineralization with little or no toxicity. P407 exhibits osteogenic effects at low doses, and the combination of Yhhu3792 with P407 significantly enhances bone marrow stroma cell functions compared to treatment with either agent alone. Therefore, our in vitro findings demonstrating the osteogenic effects of Yhhu3792 and P407 hydrogel warrant confirmation in vivo in animal fracture healing models.

In this study, the osteoinductive activity of a small-molecule NOTCH1 activator, Yhhu3792, and Poloxamer 407, an FDA-approved hydrogel, was evaluated independently regarding osteoblast functions in vitro using primary cultures of osteoblasts derived from C57BL/6J mice. We found that treatment with Yhhu3792 increased the number of NOTCH1-positive osteoblasts (36%) compared to the vehicle control (19%) after antibody staining, suggesting increased NOTCH1 signaling after Yhhu3792 treatment. Osteoblasts treated with varying doses (5, 10, and 20 μM) of Yhhu3792 and P407 (1–25%) stimulated both osteoblast proliferation and differentiation by 25–45% (p < 0.05) compared to the vehicle control. Accordingly, 10 µM Yhhu3792 treatment for 9 days increased the alizarin red-stained mineralized nodule area (8.69 ± 0.97 vs. 4.05 ± 1.51 arbitrary units; p < 0.05) compared to the vehicle treatment. Similarly, osteoblasts treated with 10% P407 also significantly increased mineralized nodule formation. The Cell Tox Green dye assay revealed that the dosage of Yhhu3792 used was not cytotoxic. Gene expression studies measured by real-time PCR revealed that a 24 h treatment with 10 µM Yhhu3792 significantly increased expression levels of bone formation markers (Vegf, Osteocalcin) and NOTCH1 targets (c-myc, Cox2, and Hes1) in osteoblasts. A low dose of P407 in combination with 10 µM Yhhu3792 stimulated a significant increase (>40%) in the proliferation of bone marrow stromal cells. In conclusion, our in vitro findings showing osteogenic effects of the small molecule Yhhu3792 and P407 hydrogel should be confirmed in vivo in animal fracture healing models.