Culture and differentiation of mesenchymal stem cells into chondrocyte- and osteoblast-like cells using alginate-based hydrogel enriched with type I collagen and calcium phosphate compounds

Abstract

Horseradish peroxidase (HRP)-catalyzed hydrogels are considered to be suitable platforms for engineered scaffolds both in physiological and pathological conditions. In this study, the phenol conjugated alginate hydrogel was synthesized by the addition of hydrogen peroxide to an aqueous alginate solution (1.2 wt. %) in the presence of HRP. The oxidation of phenol moieties resulted in the formation of alginate-phenol hydrogel. To induce osteogenic capacity, collagen (0.5 wt. %) and nano-hydroxyapatite (1 wt. %) were added to the alginate backbone via using a similar method. Finally four groups of hydrogels including alginate-phenol, alginate phenol/nano-haydrohyapatite, alginate-phenol/collagen, alginate phenol/collagen/nanohydroxyapatite were synthesized and their physio-mechanical properties were examined and compared. Hydrogels with optimized characterizations were selected for encapsulation of amniotic membrane mesenchymal stem cell line and in vitro investigations. The cytocompatibility of the collagen-included hydrogels was investigated via using MTT assay, DAPI , and H&E staining. Alizarin Red S and Alkaline Phosphatase were used to measure osteogenic capacity. The protein and expression levels of factors related to chondrogenic and osteogenic potential were evaluated using western blotting and Real-Time PCR assays. PCR array analysis of Wnt signaling pathway was also performed to evaluate the osteogenic potential of the alginate/collagen/nano-hydroxyapatite hydrogel. Data showed that mechanical properties such as biodegradation, swelling rate, stiffness and porosity were efficiently changed by the addition of collagen and apatite compared to the alginate-phenol groups. Gene and protein expression assay showed that the combination of collagen led to enhanced expression of COL2A1, ACAN , TGF-β1, SOX9 compared to the alginate-phenol group (p<0.05). The addition of hydroxyapatite promoted the expression of osteogenic gene and protein markers such as osteocalcin, RUNX2, COL1A1 and also many diffrenet genes incorporated in Wnt-ꞵcatenin signaling pathway. in apatite-included hydrogels. This study has established that alginate-phenol/collagen and alginate-phenol/collagen/nano-hydroxyapatite hydrogels can be used as chondrogenic and osteogenic inducer matrixes for stem cells. Keywords: Amniotic membrane mesenchymal stem cells; Alginate; Collagen; nano-hydroxyapatite; Phenolation; Chondrogenesis, Osteogenesis.