Microparticle-mediated sequestration of cell-secreted proteins to modulate chondrocytic differentiation

Abstract

In this work, we present a proof-of-principle set of experiments in which heparin-based microparticles are shown to modulate cellular differentiation through binding of cell-secreted protein. Unlike existing systems that rely on expensive protein with limited half-lives to elicit changes in cellular behavior, this technique focuses on temporal modulation of cell-generated proteins. This technique also provides a biomaterials-based method that can be used to further identify sequestered proteins of interest. Thus, this work indicates that glycosaminoglycan-based biomaterial approaches could be used as substitutes or additions to traditional methods for modulating and identifying the cell-secreted proteins involved in directing cellular behavior.

LEM domain–containing protein 3 antagonizes TGFβ–SMAD2/3 signaling in a stiffness-dependent manner in both the nucleus and cytosol

“…Purified fragments were separated by SDS-PAGE on a 4 -12% BisTris gel in 1ϫ MES buffer and visualized by SimplyBlue staining (ThermoFisher Scientific). In gel digestion, nano-LC-MS/MS, and peptide identification was performed as described previously (82) with the following modifications. Reverse-phase chromatography was performed using an in-house packed column (40 cm long ϫ 75 m inner diameter ϫ 360 outer diameter, Dr. Maisch GmbH ReproSil-Pur 120 C18-AQ 1.9-m beads) and a 120-min gradient.…”

Integration of Hydrogel Microparticles With Three-Dimensional Liver Progenitor Cell Spheroids

“…After examining the effects of unmodified hydrogel microparticles, we sought to investigate if the surface functionalization of the microparticles could further influence the differentiation process (Benoit et al, 2008;Lutolf et al, 2009). In particular, we conjugated the glycosaminoglycan heparin to the PEG microparticles due to its known ability to bind and sequester growth factors (Rinker et al, 2018). This was achieved by the addition of biotinylated heparin to the microparticles via the incorporation of acrylate-PEG-biotin into the PEG microparticles during fabrication, and the post-particle fabrication treatment with streptavidin, to establish a streptavidin bridge (Figures 5A,B).…”

“…Heparin has been demonstrated to bind a broad range of growth factors, and our results demonstrate that at higher concentrations of TGFβ1 there was an increase in the expression of a marker of biliary differentiation that has been previously established to be induced by TGFβ1 treatment. Consequently, we hypothesize that the heparin presented by the modified PEG microparticles may sequester TGFβ1 in the spheroid, and thereby facilitate an increased local concentration of TGFβ1, which subsequently leads to an enhancement of biliary differentiation (Rinker et al, 2018). In future efforts, to mitigate some of the variations observed across distinct microparticle culture experiments, a change in the particle formation to select for more narrow range of particle sizes, or the incorporation of defined microparticle surface modification that could enable a better standardization in regards to microparticle spheroidal integration, may help to control these variations.…”

“…Coupling the physical effects of microparticles with growth factors in human mesenchymal stem cell spheroids can tune chondrogenesis (Ravindran et al, 2011;Ansboro et al, 2014;Goude et al, 2014). Further surface functionalization of the hydrogel can also be used to sequester proteins within the spheroid (Rinker et al, 2018).…”

Microgels: Modular, tunable constructs for tissue regeneration

“…Heparinbased microparticles have also been used to investigate the sequestration of cell-secreted proteins and the consequent controlled effect on the differentiation of cells in the localized area. [58] Omitting the need for introduction of exogenous proteins but instead regulating the naturally produced proteins is a unique and novel step for tissue engineering.…”