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. 2020 Feb:232:119725.
doi: 10.1016/j.biomaterials.2019.119725. Epub 2019 Dec 27.

Porous bio-click microgel scaffolds control hMSC interactions and promote their secretory properties

Alexander S Caldwell  1 , Varsha V Rao  1 , Alyxandra C Golden  2 , Kristi S Anseth  3
Affiliations

Porous bio-click microgel scaffolds control hMSC interactions and promote their secretory properties

Alexander S Caldwell et al. Biomaterials. 2020 Feb.

Abstract

Human mesenchymal stem/stromal cells (hMSCs) are known to secrete numerous cytokines that signal to endogenous cells and aid in tissue regeneration. However, the role that biomaterial scaffolds can play in controlling hMSC secretory properties has been less explored. Here, microgels were co-assembled with hMSCs using three different microgel populations, with large (190 ± 100 μm), medium (110 ± 60 μm), and small (13 ± 6 μm) diameters, to create distinct porous environments that influenced hMSC clustering. Cells embedded in large diameter microgel networks resided in large clusters (~40 cells), compared to small clusters (~6 cells) observed in networks using medium diameter microgels and primarily single cells in small diameter microgel networks. Using a cytokine microarray, an overall increase in secretion was observed in scaffolds that promoted hMSC clustering, with over 60% of the measured cytokines most elevated in the large diameter microgel networks. N-cadherin interactions were identified as partially mediating these differences, so the microgel formulations were modified with an N-cadherin epitope, HAVDI, to mimic cell-cell interactions. Results revealed increased secretory properties for hMSCs in HAVDI functionalized scaffolds, even the non-clustered cells in small diameter microgel networks. Together, these results demonstrate opportunities for microgel-based scaffold systems for hMSC delivery and tailoring of porous materials properties to promote their secretory potential.

Keywords: Bio-click; HAVDI peptide; Mesenchymal stem/stromal cell; Microgels; Porous scaffolds; Secretome.

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Figures

Figure 1:
Figure 1:. Generation of varied porous scaffolds using clickable microgel building blocks.
(A) Clickable microgel building blocks were synthesized using an inverse suspension polymerization out of 8-arm poly(ethylene glycol) (PEG) functionalized dibenzylcyclooctyne (DBCO), 4-arm PEG-N3, and an azide functionalized cellularly adhesive peptide (GRGDS). During the polymerization shear was varied to create microgels with 190±100μm (left), 110±60μm (middle), and 13±6μm (right) mean particle diameters, termed large, medium, and small, respectively. (B) Microgel scaffolds were formed by co-assembling DBCO and N3 particles for each size group (190±100μm (left), 110±60μm (middle), and 13±6μm (right)). Particles were visualized via incorporation of an azide labeled AlexaFluor 647 dye (C). The resulting microgel scaffold structures were categorized by measuring the pore mean major axes lengths. Pore lengths correlated with the microgel diameter, with average lengths of 210±260μm (large diameter, left), 90±110μm (medium diameter, middle), and 13±12μm (small diameter, right).
Figure 2:
Figure 2:. Pore dimensions control human mesenchymal stem cell (hMSC) clustering in varied porous scaffolds.
(A) Images of hMSCs cultured in large (190±100μm) diameter (left), medium (110±60μm) diameter (middle), and small (13±6μm) diameter (right) microgel scaffolds for 72 hours. Cells stained for nuclei (blue, DAPI) and cytoplasm (green, Calcien) and particles shown via transmitted light. Scale bars = 100μm. (B) Percent of cells in a cluster in each microgel condition. Cell-cell interactions were quantified by measuring the average number of cells in a cluster (3 or more cells physically touching) in each condition. (C) Average number of cells in a cluster was also quantified for each condition. Average number of cells per cluster between the medium and small microgel scaffolds was not significantly different. Significance determined using a one-way ANOVA. All stars represent significance compared to large microgel condition. ****p<0.0001, ** p<0.01, # p<0.001 (compared to medium diameter).
Figure 3:
Figure 3:. hMSC secretory properties vary with scaffold porosity.
(A) Heatmap of cytokine expression of encapsulated hMSCs in large (190±100μm), medium (110±60μm), and small (13±6μm) diameter microgel networks. Red intensities represent high expression while blue intensities represent low or undetectable expression levels compared to control (cell media). Values were normalized to DNA content. (B) Cytokines that were most elevated in large (left, red), medium (middle, blue), and small microgel scaffolds (right, light blue). (C) List of regenerative factors that were most elevated in large (left, red), medium (middle, blue), and small microgel scaffolds (right, light blue).
Figure 4:
Figure 4:. N-cadherin interaction and expression increases with increased cell clustering.
(A) hMSCs in large (190±100μm) diameter microgel scaffolds (top, right) with highly clustered cells show more intense staining for N-cadherin punctate compared with smaller clusters in medium (110±60μm) diameter microgel scaffolds (middle, right) and largely single cells in small (13±6μm) diameter microgel scaffolds (bottom, right) Cells stained for nuclei (blue), N-cadherin (green), and F-actin (red). (B) Intensity quantification of the N-cadherin punctate. Stars represent significance relative to large microgel scaffolds. ** p<0.01, * p<0.05, n.s. – non-significant
Figure 5:
Figure 5:. Blocking N-cadherin interactions in microgel scaffolds decreases hMSC secretory properties.
(A) Log-fold change in cytokine secretion from hMSCs in large (190±100μm) diameter (red), medium (110±60μm) diameter (blue), and small (13±6μm) diameter (light blue) microgel scaffolds when cultured in the presence of an anti-N-cadherin antibody compared to their respective unmodified conditions. Negative fold change indicates a decreased in cytokine expression in the presence of blocking. (B) Principal component analysis of hMSC secretory profile of standard conditions (circles) and N-cadherin blocked conditions (diamonds). Colors correspond to conditions in (A) PC1 and PC2 explained 39.9% and 31.4% of the variance, respectively.
Figure 6 –
Figure 6 –. HAVDI inclusion in microgel scaffolds increases secretory phenotype of hMSCs.
(A) Heatmap of cytokine expression of encapsulated hMSCs in large (190±100μm), medium (110±60μm), and small (13±6μm) diameter microgel scaffolds with and without inclusion of the HAVDI peptide. (B) PCA analysis of hMSC secretory profile between large (red), medium (blue), and small (light blue) microgel scaffolds without (circles) and with HAVDI (X symbol). PC1 and PC2 explained 70.4% and 18.3% of the variance respectively. (C) ELISA quantification of VEGF, (D) GDNF, and (E) IGF-1. HAVDI conditions are represented by hashed bars. Stars represent significance relative to respective unmodified scaffolds. Overall significance (top bar) determined using a one-way ANOVA with multiple comparisons. ****p<0.0001, ** p<0.01, n.s. – non-significant
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References

    1. Trounson A. & Mcdonald C. Cell Stem Cell Stem Cell Therapies in Clinical Trials: Progress and Challenges. Stem Cell 17, 11–22 (2015). - PubMed
    1. Caplan AI & Correa D. The MSC: An Injury Drugstore. Cell Stem Cell 9, 11–15 (2011). - PMC - PubMed
    1. da Silva Meirelles L, Fontes AM, Covas DT & Caplan AI Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev. 20, 419–427 (2009). - PubMed
    1. Huebsch N. et al. Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation. Nat. Mater 14, 1269–77 (2015). - PMC - PubMed
    1. Engler AJ, Sen S, Sweeney HL & Discher DE Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 126, 677–689 (2006). - PubMed

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