Vernengo, A. J. | Search bioRxiv

bioRxiv2024

Effect of molecular weight of tyramine-modified hyaluronan on polarization state of peripheral blood mononuclear cells-derived macrophages

The immunomodulatory properties of hyaluronan and its derivatives are key to their use in medicine and tissue engineering. In this work we evaluated the capability of soluble tyramine-modified hyaluronan (THA) of two molecular weights (low Mw=280 kDa and high Mw=1640 kDa) for polarization of THP-1 and peripheral blood mononuclear cells (PBMCs)-derived macrophages (M{Phi}s). We demonstrate the polarization effects of the supplemented THA by flow cytometry and multiplex ELISA for the THP-1 derived M{Phi}s and by semi-automated image analysis from confocal microscopy, immunofluorescent staining utilising CD68 and CD206 surface markers, RT-qPCR gene expression analysis, as well as using the enzyme-linked immunosorbent assay (ELISA) for PBMCs-derived M{Phi}s. Our data indicate that supplementation with LMW THA drives changes in THP-1 derived M{Phi}s towards a pro-inflammatory M1-like phenotype, whereas supplementation with the HMW THA leads to a more mixed profile with some features of both M1 and M2 phenotypes, suggesting either a heterogeneous population or a transitional state. For cells directly sourced from human patients, PMBCs-derived M{Phi}s, results exhibit a higher degree of variability, pointing out a differential regulation of factors including IL-10 and CD206 between the two cell sources. While human primary cells add to the clinical relevance, donor diversity introduces wider variability in the dataset, preventing drawing strong conclusions. Nevertheless, the M{Phi}s profiles observed in THP-1 derived cells for treatments with LMW and HMW THA are generally consistent with what might be expected for the treatment with non-modified hyaluronans of respective molecular weights, confirming the known association holds true for the chemically tyramine-modified hyaluronan. We stipulate that these responses will provide basis for more accurate in vivo representation and translational immunomodulatory guidance for the use of THA-based biomaterials to a wider biomaterials and tissue engineering communities. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/575241v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@899d95org.highwire.dtl.DTLVardef@c8b80aorg.highwire.dtl.DTLVardef@1aad98dorg.highwire.dtl.DTLVardef@1b0ce60_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Biofabrication of development-inspired scaffolds for regeneration of the annulus fibrosus macro- and microarchitecture

Annulus fibrosus (AF) tissue engineering is a promising strategy for repairing the degenerated intervertebral disc (IVD) and a research area that could benefit from improved tissue models to drive translation. AF tissue is composed of concentric layers of aligned collagen bundles arranged in an angle-ply pattern, an architecture which is challenging to recapitulate with current scaffold design strategies. In response to this need, we developed a strategy to print 3D scaffolds that induce cell and tissue organization into oriented patterns mimicking the AF. Polycaprolactone (PCL) was printed in an angle-ply macroarchitecture possessing microscale aligned topographical cues. The topography was achieved by extrusion through custom-designed printer nozzles which were either round or possessing circumferential sinusoidal peaks. Whereas the round nozzle produced extruded filaments with a slight uniaxial texture, patterned nozzles with peak heights of 60 or 120 m produced grooves, 10.87 {+/-} 3.09 m or 17.77 {+/-} 4.91 m wide, respectively. Bone marrow derived mesenchymal stem cells (BM-MSCs) cultured on the scaffolds for four weeks exhibited similar degrees of alignment within {+/-} 10 {degrees} of the printing direction and upregulation of outer AF markers (COL1, COL12, SFRP, MKX, MCAM, SCX and TAGLN), with no statistically significant differences as a function of topography. Interestingly, the grooves generated by the patterned nozzles induced longitudinal end-to-end alignment of cells, capturing the arrangement of cells during fibrillogenesis. In contrast, topography produced from the round nozzle induced a continuous web of elongated cells without end-to-end alignment. Extracellular collagen I, decorin and fibromodulin were detected in patterns closely following cellular organization. Taken together, we present a single-step biofabrication strategy to induce anisotropic cellular alignments in x-, y-, and z-space, with potential application as an in vitro model for studying AF tissue morphogenesis and growth.

bioengineering↗