# A Hydrogel Scaffold Incorporating Fennel Seed Extract Induces Osteogenic Differentiation in Mesenchymal Stem Cells

## TL;DR

A hydrogel made with fennel seed extract helps mesenchymal stem cells turn into bone cells, offering a new approach for bone tissue repair.

## Contribution

The study introduces a novel chitosan-based hydrogel incorporating fennel seed extract to promote osteogenic differentiation of MSCs.

## Key findings

- MSCs cultured on chitosan hydrogels with 1% fennel extract showed increased hydroxyapatite deposits and ALP activity.
- The extract upregulated osteogenic genes (ALP, Runx2, COL1A1) and enhanced calcium deposition.
- The treatment was non-toxic and significantly improved MSC proliferation at 0.5% extract concentration.

## Abstract

Tissue engineering utilizing hydrogels and scaffolds composed of organic and synthetic materials has emerged as a promising approach for bone repair. Chitosan, a biocompatible natural polymer, supports bone healing and possesses antibacterial and antioxidant properties. Fennel (Foeniculum vulgare), known for its traditional use in treating osteoporosis in females, exhibits phytoestrogenic properties.

This study investigated the osteogenic differentiation of mesenchymal stem cells (MSCs) cultured within chitosan hydrogels incorporating fennel seed extract.

Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM) were employed to characterize the chitosan films containing fennel extract. MSCs were isolated from ovine foetal bone marrow and subjected to various concentrations of fennel hydroalcoholic extract and hydrogels containing these extracts. Cell viability was assessed using the MTT assay. Following osteoblast differentiation, gene expression of osteogenic markers (ALP, Runx2 and COL1A1) was evaluated using real‐time PCR, alongside assessments of calcium deposition, alkaline phosphatase activity and ALP expression. Alizarin red staining was performed to quantify mineralized matrix deposition.

The MTT assay revealed that the 0.5% extract treatment group significantly enhanced MSC proliferation. Notably, MSCs cultured on the chitosan film containing 1% fennel extract demonstrated increased numbers of hydroxyapatite deposits, elevated ALP activity and calcium content and upregulated expression of osteogenic genes (ALP, Runx2 and COL1A1). Acridine orange/ethidium bromide staining confirmed the non‐toxicity of the extract in all treatment groups.

These findings demonstrate that chitosan hydrogels incorporating fennel seed extract can effectively induce osteogenic differentiation of MSCs, highlighting their potential as a promising strategy for bone tissue regeneration.

Graphical description of the osteogenic differentiation of ovine foetal bone marrow mesenchymal stem cells cultured within chitosan hydrogels incorporating fennel seed extract. After 21 days, their differentiation was evaluated using morphological, biochemical and molecular assays. The results demonstrated their effective osteogenic differentiation.