Metabolic Labeling to Probe the Spatiotemporal Accumulation of Matrix at the Chondrocyte–Hydrogel Interface

Abstract

Hydrogels are engineered with biochemical and biophysical signals to recreate aspects of the native microenvironment and to control cellular functions such as differentiation and matrix deposition. This deposited matrix accumulates within the pericellular space and likely affects the interactions between encapsulated cells and the engineered hydrogel; however, there has been little work to study the spatiotemporal evolution of matrix at this interface. To address this, metabolic labeling is employed to visualize the temporal and spatial positioning of nascent proteins and proteoglycans deposited by chondrocytes. Within covalently crosslinked hyaluronic acid hydrogels, chondrocytes deposit nascent proteins and proteoglycans in the pericellular space within 1 d after encapsulation. The accumulation of this matrix, as measured by an increase in matrix thickness during culture, depends on the initial hydrogel crosslink density with decreased thicknesses for more crosslinked hydrogels. Encapsulated fluorescent beads are used to monitor the hydrogel location and indicate that the emerging nascent matrix physically displaces the hydrogel from the cell membrane with extended culture. These findings suggest that secreted matrix increasingly masks the presentation of engineered hydrogel cues and may have implications for the design of hydrogels in tissue engineering and regenerative medicine.

Success Criteria for Preclinical Testing of Cell-Instructive Hydrogels for Tendon Regeneration

“…Beyond the provisional synthetic matrix of hydrogels, ECM secreted by cells, such as collagen type-I, is a tunable factor within engineered hydrogels and can influence hydrogel success [61][62][63] . In this work, we observed collagen type-I in the pericellular matrix of hMSCs in 3D culture with or without mfCMPs yet with significantly increased cell elongation in the presence of assembled mfCMPs, representing a cellular response to mfCMPs that may be a function of both ECM composition and structure.…”

“…In this work, we observed collagen type-I in the pericellular matrix of hMSCs in 3D culture with or without mfCMPs yet with significantly increased cell elongation in the presence of assembled mfCMPs, representing a cellular response to mfCMPs that may be a function of both ECM composition and structure. Bio-orthogonal labeling of hydrogels may be useful to determine the spatiotemporal effects of the cell-secreted ECM in hydrogels 62 in vitro and in preclinical models in vivo 64,65 .…”

The role of pregnancy-specific glycoproteins on trophoblast motility in three-dimensional gelatin hydrogels

“…Cell motility can be strongly influenced by dynamic remodeling of the extracellular matrix. We investigated bulk nascent protein production of encapsulated Swan71 trophoblast spheroids for days 1 through 3 of culture via a recently described metabolic labeling approach (utilizing cell media supplemented with the methionine-analog AHA) (Loebel et al, 2020). We observed significant nascent protein production as well as accumulation in close proximity to trophoblast cells in three-dimensional hydrogels ( Figure 5).…”

“…Nascent protein staining was performed as previously described by Loebel et al (Dieterich et al, 2007;Loebel et al, 2020;Loebel et al, 2019). Starting on day 1 of culture, encapsulated spheroids were incubated with the methionine analog azidohomoalanine (Click-iT AHA;…”

Complementary techniques to analyse pericellular matrix formation by human MSC within hyaluronic acid hydrogels

“…of each pixel along the radii. 6,33,34 In this way, mean pixel intensity was calculated at every 0.082 mm step from the cell membrane and these values summed to calculate fluorescence intensity on a per cell basis. The distance at which secreted matrix extended from the cell membrane was calculated likewise on a per cell basis by identifying the distance in radii at which fluorescence signal was no different than background.…”