Enzymatically cross-linked gelatin-phenol hydrogels with a broader stiffness range for osteogenic differentiation of human mesenchymal stem cells

An injectable hydrogel system, composed of gelatin–hydroxyphenylpropionic acid (Gtn–HPA) conjugates chemically cross-linked by an enzyme-mediated oxidation reaction, has been designed as a biodegradable scaffold for tissue engineering. In light of the role of substrate stiffness on cell differentiation, we herein report a newly improved Gtn hydrogel system with a broader range of stiffness control that uses Gtn–HPA–tyramine (Gtn–HPA–Tyr) conjugates to stimulate the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The Gtn–HPA–Tyr conjugate was successfully synthesized through a further conjugation of Tyr to Gtn–HPA conjugate by means of a general carbodiimide/active ester-mediated coupling reaction. Proton nuclear magnetic resonance and UV–visible measurements showed a higher total phenol content in the Gtn–HPA–Tyr conjugate than that content in the Gtn–HPA conjugate. The Gtn–HPA–Tyr hydrogels were formed by the oxidative coupling of phenol moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Rheological studies revealed that a broader range of storage modulus (G′) of Gtn–HPA–Tyr hydrogel (600–26,800 Pa) was achieved using different concentrations of H2O2, while the G′ of the predecessor Gtn–HPA hydrogels was limited to the range of 1000 to 13,500 Pa. The hMSCs on Gtn–HPA–Tyr hydrogel with G′ greater than 20,000 showed significantly up-regulated expressions of osteocalcin and runt-related transcription factor 2 (RUNX2) on both the gene and protein level, with the presence of alkaline phosphatase, and the evidence of calcium accumulation. These studies with the Gtn–HPA–Tyr hydrogel with G′ greater than 20,000 collectively suggest the stimulation of the hMSCs into osteogenic differentiation, while these same observations were not found with the Gtn–HPA hydrogel with a G′ of 13,500.