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

Abstract

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 (H(2)O(2)) 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 H(2)O(2), 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.