In Situ-Forming Collagen/poly-γ-glutamic Acid Hydrogel System with Mesenchymal Stem Cells and Bone Morphogenetic Protein-2 for Bone Tissue Regeneration in a Mouse Calvarial Bone Defect Model

Tissue Eng Regen Med. 2022 Oct;19(5):1099-1111. doi: 10.1007/s13770-022-00454-4. Epub 2022 Apr 23.


Background: Bone marrow-derived mesenchymal stem cells (BMSCs) and bone morphogenetic protein-2 (BMP-2) have been studied for bone repair because they have regenerative potential to differentiate into osteoblasts. The development of injectable and in situ three-dimensional (3D) scaffolds to proliferate and differentiate BMSCs and deliver BMP-2 is a crucial technology in BMSC-based tissue engineering.

Methods: The proliferation of mouse BMSCs (mBMSCs) in collagen/poly-γ-glutamic acid (Col/γ-PGA) hydrogel was evaluated using LIVE/DEAD and acridine orange and propidium iodide assays. In vitro osteogenic differentiation and the gene expression level of Col/γ-PGA(mBMSC/BMP-2) were assessed by alizarin red S staining and quantitative reverse-transcription polymerase chain reaction. The bone regeneration effect of Col/γ-PGA(mBMSC/BMP-2) was evaluated in a mouse calvarial bone defect model. The cranial bones of the mice were monitored by micro-computed tomography and histological analysis.

Results: The developed Col/γ-PGA hydrogel showed low viscosity below ambient temperature, while it provided a high elastic modulus and viscous modulus at body temperature. After gelation, the Col/γ-PGA hydrogel showed a 3D and interconnected porous structure, which helped the effective proliferation of BMSCs with BMP-2. The Col/γ-PGA (mBMSC/BMP-2) expressed more osteogenic genes and showed effective orthotopic bone formation in a mouse model with a critical-sized bone defect in only 3-4 weeks.

Conclusion: The Col/γ-PGA(mBMSC/BMP-2) hydrogel was suggested to be a promising platform by combining collagen as a major component of the extracellular matrix and γ-PGA as a viscosity reducer for easy handling at room temperature in BMSC-based bone tissue engineering scaffolds.

Keywords: Bone morphogenetic protein-2; Bone regeneration; Calvarial defect; Hydrogel scaffold; Mesenchymal stem cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acridine Orange / metabolism
  • Acridine Orange / pharmacology
  • Animals
  • Bone Regeneration
  • Collagen / metabolism
  • Glutamic Acid / metabolism
  • Glutamic Acid / pharmacology
  • Hydrogels* / chemistry
  • Hydrogels* / pharmacology
  • Mesenchymal Stem Cells* / metabolism
  • Mice
  • Osteogenesis
  • Polyglutamic Acid / analogs & derivatives
  • Propidium / metabolism
  • Propidium / pharmacology
  • X-Ray Microtomography


  • Hydrogels
  • poly(gamma-glutamic acid)
  • Polyglutamic Acid
  • Propidium
  • Glutamic Acid
  • Collagen
  • Acridine Orange