Cryoprotectant enables structural control of porous scaffolds for exploration of cellular mechano-responsiveness in 3D

Shumeng Jiang,Cheng Lyu,Yanan Du,Chenyu Huang,Wenjing Li,Guy M Genin,Peng Zhao,Wenyu Kong

doi:10.1038/s41467-019-11397-1

Abstract

Despite the wide applications, systematic mechanobiological investigation of 3D porous scaffolds has yet to be performed due to the lack of methodologies for decoupling the complex interplay between structural and mechanical properties. Here, we discover the regulatory effect of cryoprotectants on ice crystal growth and use this property to realize separate control of the scaffold pore size and stiffness. Fibroblasts and macrophages are sensitive to both structural and mechanical properties of the gelatin scaffolds, particularly to pore sizes. Interestingly, macrophages within smaller and softer pores exhibit pro-inflammatory phenotype, whereas anti-inflammatory phenotype is induced by larger and stiffer pores. The structure-regulated cellular mechano-responsiveness is attributed to the physical confinement caused by pores or osmotic pressure. Finally, in vivo stimulation of endogenous fibroblasts and macrophages by implanted scaffolds produce mechano-responses similar to the corresponding cells in vitro, indicating that the physical properties of scaffolds can be leveraged to modulate tissue regeneration.

Highlights

Despite the wide applications, systematic mechanobiological investigation of 3D porous scaffolds has yet to be performed due to the lack of methodologies for decoupling the complex interplay between structural and mechanical properties
We discovered that the cryoprotectant DMSO can be used to control pore size through regulating ice crystal formation during fabrication of cryogelated 3D porous scaffolds (Fig. 1a)
We examined whether osmotic pressure-induced physical confinement influences cellular phenotypes in human dermal fibroblasts (HDFs) and bone marrow-derived macrophages (BMDMs) cultured in 3D gelatin scaffolds with large pore sizes (i.e., 80 μm)

Summary

Introduction

Systematic mechanobiological investigation of 3D porous scaffolds has yet to be performed due to the lack of methodologies for decoupling the complex interplay between structural and mechanical properties. The bulk stiffness of the resulting scaffolds following porogen removal tends to be significantly weakened[16], limiting their application in situations that require sufficient stiffness to support the defective area Together, these difficulties hinder the exploration of the mechano-responsiveness of cells in 3D porous bioscaffolds, which constitutes an obvious missing link in our understanding of cell–matrix interactions in mechanobiological studies. These difficulties hinder the exploration of the mechano-responsiveness of cells in 3D porous bioscaffolds, which constitutes an obvious missing link in our understanding of cell–matrix interactions in mechanobiological studies This missing link highlights the unmet need for 3D porous scaffold fabrication strategies that enable the precise and separate control of specific mechanical and structural properties. We explored in vivo cellular responses to these biophysically fine-tuned porous scaffolds after subcutaneous implantation, and our results highlight potential applications of the scaffolds in improving the therapeutic efficacy for regenerative medicine

Methods

Results

Conclusion