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.

Biophysical properties of extracellular matrix (ECM), such as matrix stiffness, viscoelasticity and matrix fibrous structure, are emerging as important factors that regulate progression of fibrosis and other chronic diseases. The biophysical properties of the ECM can be rapidly and profoundly regulated by crosslinking reactions in enzymatic or non-enzymatic manners, which further alter the cellular responses and drive disease progression. In-depth understandings of crosslinking reactions will be helpful to reveal the underlying mechanisms of fibrosis progression and put forward new therapeutic targets, whereas related reviews are still devoid. Here, we focus on the main crosslinking mechanisms that commonly exist in a plethora of chronic diseases (e.g. fibrosis, cancer, osteoarthritis) and summarize current understandings including the biochemical reaction, the effect on ECM properties, the influence on cellular behaviors, and related studies in disease model establishment. Potential pharmaceutical interventions targeting the crosslinking process and relevant clinical studies are also introduced. Limitations of pharmaceutical development may be due to the lack of systemic investigations related to the influence on crosslinking mechanism from micro to macro level, which are discussed in the last section. We also propose the unclarified questions regarding crosslinking mechanisms and potential challenges in crosslinking-targeted therapeutics development.

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