Research ArticleHEALTH AND MEDICINE

Combinatorial screening of biochemical and physical signals for phenotypic regulation of stem cell–based cartilage tissue engineering

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Science Advances  22 May 2020:
Vol. 6, no. 21, eaaz5913
DOI: 10.1126/sciadv.aaz5913
  • Fig. 1 Stem cell–based cartilage repair strategy is based on engineered multicomponent biomaterials and a combinatorial system for screening of combinatorial physical and biochemical cues in cartilage microenvironments.

    (A) Schematic depicting key factors guiding chondrogenic differentiation of hMSCs. (B) Chemical synthesis scheme of hybrid of photo–cross-linkable PEG/OMA hydrogels. (C) Schematic showing six layers (i.e., TMSPA-treated glass slides with hydrogel arrays, caps to maintain gel height, main chamber, pistons with different heights, membrane with pillars, and pressure chamber) comprising the combinatorial high-throughput system. A representative photograph showing a combinatorial system for screening combinatorial cues (Photo credit: J.L., University of California, Los Angeles) (please also see fig. S1, F and G). Scale bar, 20 mm. (D) Schematic illustrations of cross-sectional view of the combinatorial system. (E) Compressive modulus of PEG/OMA hydrogels (8, 10, and 12% PEG/OMA) with or without cells on day 0 (n = 5) (One-way ANOVA with Tukey’s significant difference post hoc test; *P < 0.05 and ***P < 0.005 compared with 8% without cells). (F) Time profile of hydrogel degradation without compression for 21 days (n = 5). (One-way ANOVA with Tukey’s significant difference post hoc test; *P < 0.05 compared with 10% without cell group, **P < 0.05 compared with 12% with cell group, ***P < 0.005 compared with 8% with cells, and ****P < 0.005 compared with 12% without cell group at day 0.) (G) Relative DNA content of cells in PEG/OMA hydrogels with compositions of 8 and 12%, TGF-β1 (10 ng/ml), and RGD conjugation under 0 or 40% cyclic compression (n = 6). (H) Representative DAPI/F-actin images of hMSCs cultured for 7 days in 8 and 12% PEG/OMA gels with or without 40% cyclic compression and quantification of cell spreading at days 1, 3, and 7 (n = 3). Scale bar, 300 μm (inset: 100 μm).

  • Fig. 2 Cross-linking density of PEG/OMA that could guide different levels of both degradation and stiffness regulates expression level of markers associated with articular or hypertrophic chondrogenesis of hMSCs.

    (A) Representative 3D confocal images of hMSCs stained with collagen II in different combinations of parameters. (B) Quantified heat maps of collagen II and aggrecan markers for cells encapsulated in the hydrogel microarrays cultured with combinations of all the factors for 21 days (n = 4). All data were normalized by the condition, 10% PEG/OMA without the presence of RGD, compressive stain, and TGF-β1 supplement. (C) Surface plot displaying the effect of the two variables on chondrogenic marker expression (collagen II) of hMSCs when other two factors are fixed. (D) Representative 3D confocal images of hMSCs stained with representative osteogenic marker (Runx2) in different combinations of parameters. (E) Quantified heat maps of Runx2 for cells encapsulated in the hydrogel microarrays cultured with combinations of all the factors for 21 days (n = 4). (F) Plot of measured immunofluorescence intensity data to define the role of Runx2 in lineage specification of hMSCs. Data were selected randomly (n = 1000). a.u., arbitrary unit. (G) Surface plot displaying the effect of the variables composition and strain on collagen II, aggrecan, Runx2 expression of hMSCs with other factors (with RGD and 10 ng/ml TGF-β1 supplement) fixed. Scale bars, 100 μm.

  • Fig. 3 Engineered multicomponent biomaterials regulate hypertrophic chondrogenesis of hMSCs through YAP-dependent mechanotransduction and WNT signaling pathway.

    (A) Representative 3D confocal images of hMSCs cultured in different combinations of parameters for 7 days and stained with YAP. (B) Quantified heat map and surface plot of measured immunofluorescence nuclear intensity of YAP at days 1, 3, and 7 (n = 4). (C) Quantified heat map and surface plot of YAP for cells encapsulated in the hydrogel microarrays cultured with combinations of all the factors for 21 days (n = 4). (D) Plots of LR model coefficients across the parameters regulating expression levels of articular (collagen II and aggrecan) and hypertrophic (Runx2 and YAP) cartilage markers. (E) Quantified heat map of scores based on positive correlation of articular (collagen II and aggrecan) and negative correlation of hypertrophic (Runx2 and YAP) cartilage marker expression levels for the best chondrogenic metrics on day 21. (F) Representative 3D confocal images of cells stained with aggrecan and Runx2 when treated with an inhibitor of the WNT signaling pathway for the 8% PEG/OMA condition or of the YAP signaling pathway for the 12% PEG/OMA condition. Relative expression of aggrecan and Runx2 markers for cells cultured in (G) 8% PEG/OMA hydrogels with or without WNT inhibitor or (H) 12% PEG/OMA hydrogels with or without YAP inhibitor, demonstrating possible pathways that promote hypertrophy in articular-like or hypertrophic-like chondrogenesis (n = 4). (P values were obtained on the basis of one-way ANOVA with Tukey’s post hoc testing *P < 0.05, **P < 0.005, and ***P < 0.0005 compared with the DMSO condition.) (I) Proposed pathway for mechanical stimuli guiding articular or hypertrophic chondrogenesis of hMSCs. Scale bars, 100 μm.

  • Fig. 4 hMSCs cultured in high-ranked conditions for the prevention of hypertrophic chondrogenesis show higher potency for cartilage repair in vitro and in vivo.

    (A) Quantification of GAG/DNA and ALP/DNA for 21 days in culture of hMSCs in RGD-conjugated PEG/OMA gels supplemented with TGF-β1 under 40% compression compared with those cultured without compression (n = 6). (B) Toluidine blue O staining after treatment with threshold-based imaging analysis. (C) Heat map of real-time PCR gene expression results associated with articular or hypertrophic chondrogenic differentiation for cells cultured in the combinatorial bioreactor with modulation of mechanical stimuli for 21 days (n = 6 for Col2a1, ACAN, Sox9, and ALP & n = 3 for Col1a1, Col10a1, OCN, and OSX). Quantification of (D) GAG/DNA and (E) ALP/DNA for cells cultured for 21 days with or without WNT and YAP inhibitors and transplanted in vivo for 7 or 21 days after the in vitro pretreatment (n = 6). (F) Real-time PCR quantification of gene expression associated with chondrogenic (Col2a1, ACAN, and Sox9) or osteogenic (ALP, Col1a1, Col10a1, OCN, and OSX) differentiation for cells before and after transplantation in vivo (n = 3 to 5, please see fig. S9D). (*P < 0.05, **P < 0.005, and ***P < 0.0005 based on one-way ANOVA with Tukey’s post hoc testing). Scale bar, 100 μm.

Supplementary Materials

  • Supplementary Materials

    Combinatorial screening of biochemical and physical signals for phenotypic regulation of stem cell–based cartilage tissue engineering

    Junmin Lee, Oju Jeon, Ming Kong, Amr A. Abdeen, Jung-Youn Shin, Ha Neul Lee, Yu Bin Lee, Wujin Sun, Praveen Bandaru, Daniel S. Alt, KangJu Lee, Han-Jun Kim, Sang Jin Lee, Somali Chaterji, Su Ryon Shin, Eben Alsberg, Ali Khademhosseini

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