Speaker
Description
Introduction:
Modular tissue engineering exploits the 3D self-assembly of cells and building blocks to create larger tissue constructs with higher complexity and resolution.[1] However, current modular tissue engineering strategies have near-exclusively relied on static, non-responsive, micromaterials,[2] whereas functionality of native tissues is dictated by their inherently dynamic nature.[3] Here, we report on smart, dynamically tunable, microbuilding blocks of which the biochemical and biophysical properties can be altered via well-controlled secondary crosslinking strategies. The mechanical properties of the building blocks were modified post-synthesis by exploiting visible-light-induced secondary crosslinking of the free tyramines using ruthenium (Ru) and sodium persulfate (SPS) as initiators. Free biotins could be stepwise functionalized with biotinylated molecules of interest using competitive supramolecular complexation of avidin and biotin analogs.[4] The spatiotemporal control over mechanical and chemical properties of smart building blocks within living modular tissues provided a highly tunable, well-defined, and dynamic cellular microenvironment, which allowed for the in situ modification of (stem) cell behavior and fate.
Methodology:
Microbuilding block production and functionalization: Hydrogel precursor droplets composed of 5% (w/v) Dextran-Tyramine-Biotin (DexTAB; ~1 mM biotin) and 22 U/mL horseradish peroxidase in phosphate buffered saline (PBS) were emulsified in 2% (w/w) Pico-Surf 1 containing Novec 7500 Engineered Fluid using a microfluidic droplet generator with subsequent crosslinking on a separate controlled hydrogen peroxide (H2O2) supplementation.[5, 6]
Modular tissue engineering: RGD-functionalized microbuilding blocks were homogeneously co-seeded with cells into non-adherent 3% (w/v) agarose microwell chips at a density of ~50 units per microwell.
In situ stiffening: Building blocks within modular tissues were in situ stiffened by incubating them with 2.5 mM of SPS and 1 mM of Ru. Free radical crosslinking was induced using 60 seconds of visible light irradiation. Lineage commitment was visualized using histochemical staining, and imaged using confocal (fluorescence) microscopy, and analyzed using image analysis software.
Results:
Stepwise functionalization of the microbuilding blocks with c(RGD)fk initiated the self-assembly of cells and microbuilding blocks via integrin-mediated interactions. The cell-matrix interactions within cell-building block aggregates were optimized to steer stem cell fate towards stiffness-induced osteogenic interactions.
Furthermore, microbuilding blocks were tuned biophysically and biochemically in situ by secondary orthogonal crosslinking schemes. A visible-light-induced crosslinking, using Ru and SPS as initiators, allowed for on-demand control of building block stiffness. Stem cell fate toward osteogenic and adipogenic lineages was temporally steered by in situ tuning the building block stiffness within modular tissues. In situ biochemical control over cell-building block aggregates was shown by temporally endowing the building blocks with (desthio)biotinylated bone morphogenetic protein (BMP)7 neutralizing antibodies, which showed temporal control over the cellular response of a BMP7 reporter cell line.
Conclusion:
In conclusion, we developed the first biochemically, biophysically, and spatiotemporally controlled smart building blocks for modular tissue engineering. This allowed for the creation of highly tunable and defined cellular microenvironments, which more accurately resembled the dynamic microenvironment of cells in native tissues.
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