Hydrogel-based microspheres are commonly used for drug and cell delivery in regenerative medicine. Characterization of their physical and mechanical properties is important in monitoring their quality during fabrication and in predicting their performance upon injection. However, existing methods have limitations in measuring these micron-sized, soft and viscoelastic spherical structures. In this study, a protocol is developed to measure the elastic modulus of non-linear viscoelastic spheres by microplate compression, and is applied to collagen microspheres fabricated with or without cells. During the measurement, a microsphere is placed on a rigid surface and is compressed by a calibrated flexible microplate gripped to a rigid end. A step increase in the displacement rate of the rigid end of the flexible microplate is introduced and the reduced elastic modulus of the microsphere is calculated from the deformation response of the microsphere, using an equation derived in this study. The reduced elastic modulus of collagen microspheres with and without mesenchymal stem cells measured by this method was 9.1 kPa and 132 Pa, respectively.

Mechanical signal is important for regulating cellular activities, including proliferation, metabolism, matrix production, and orientation. Bioreactors with loading functions can be used to precondition cells in three-dimensional (3D) constructs so as to study the cellular responses to mechanical stimulation. However, full-scale bioreactor is not always an affordable option considering the high cost of equipment and the liter-sized medium with serum and growth factor supplements. In this study, a custom-built loading system was developed by coupling a conventional camera-equipped inverted research microscope with two micromanipulators. The system was programmed to deliver either cyclic compressive loading with different frequencies or static compressive loading for 1 week to investigate the cellular responses of human mesenchymal stem cells (hMSCs) entrapped in a 3D construct consists of reconstituted collagen fibers. Cellular properties, including their alignment, cytoskeleton, and cell metabolism, and properties of matrix molecules, such as collagen fiber alignment and glycosaminoglycan deposition, were evaluated. Using a MatLab-based image analysis program, reorientation of the entrapped cells from a random distribution to a preferred alignment along the loading direction in constructs with both static and cyclic compression has been demonstrated, but no such alignment was found in the free-floating controls. Fluorescent staining on filamentous actin cytoskeleton also confirmed the finding. Nevertheless, the collagen fiber meshwork entrapping the hMSCs remained randomly distributed, and no change in cellular metabolism and glycosaminoglycans production was noted. The current study provides a simple and affordable option toward setting up a mechanoregulation facility based on existing laboratory equipment and sheds new insights on the effect of mechanical loading on the alignment of hMSCs in 3D collagen constructs.