Characterization of the chondrocyte secretome in photoclickable poly(ethylene glycol) hydrogels

Abstract

Poly(ethylene glycol) (PEG) hydrogels are highly tunable platforms that are promising cell delivery vehicles for chondrocytes and cartilage tissue engineering. In addition to characterizing the type of extracellular matrix (ECM) that forms, understanding the types of proteins that are secreted by encapsulated cells may be important. Thus, the objectives for this study were to characterize the secretome of chondrocytes encapsulated in PEG hydrogels and determine whether the secretome varies as a function of hydrogel stiffness and culture condition. Bovine chondrocytes were encapsulated in photoclickable PEG hydrogels with a compressive modulus of 8 and 46 kPa and cultured under free swelling or dynamic compressive loading conditions. Cartilage ECM deposition was assessed by biochemical assays and immunohistochemistry. The conditioned medium was analyzed by liquid chromatography-tandem mass spectrometry. Chondrocytes maintained their phenotype within the hydrogels and deposited cartilage-specific ECM that increased over time and included aggrecan and collagens II and VI. Analysis of the secretome revealed a total of 64 proteins, which were largely similar among all experimental conditions. The identified proteins have diverse functions such as biological regulation, response to stress, and collagen fibril organization. Notably, many of the proteins important to the assembly of a collagen-rich cartilage ECM were identified and included collagen types II(α1), VI (α1, α2, and α3), IX (α1), XI (α1 and α2), and biglycan. In addition, many of the other identified proteins have been reported to be present within cell-secreted exosomes. In summary, chondrocytes encapsulated within photoclickable PEG hydrogels secrete many types of proteins that diffuse out of the hydrogel and which have diverse functions, but which are largely preserved across different hydrogel culture environments. Biotechnol. Bioeng. 2017;114: 2096-2108. © 2017 Wiley Periodicals, Inc.

Keywords: cartilage tissue engineering; chondrocyte; hydrogel; poly(ethylene glycol); secretome.

Figure 1

Analysis of cell-laden photoclickable PEG hydrogels for soft and stiff hydrogels cultured under free swelling or dynamic loading conditions as a function of culture time. A) Mass swelling ratio of cell-laden hydrogels defined as swollen mass / dry mass as an indicator of water content. B) Tangent compressive modulus of cell-laden hydrogels. C) Total DNA content per hydrogel. D) Sulfated glycosaminoglycan (sGAG) content per DNA. E) Total collagen content per DNA. Immunohistochemical staining for F) aggrecan, G) collagen II, and H) collagen VI at day 9. Scale bars are 20 μm. Semi-quantitative image analysis for I) aggrecan, J) collagen II, K) and collagen VI. Data is taken from 3–4 images of 2–3 constructs per condition. Data are presented as mean with standard deviation (n=2–3) as error bars. P-values are presented for a one-way ANOVA or after post-hoc analysis.

Figure 2

Venn diagrams showing the overlap of proteins identified with mass spectrometry for different comparisons of A) soft hydrogel versus stiff hydrogel regardless of culture condition, B) free swelling (FS) versus dynamic loading (DL) culture conditions regardless of hydrogel, C) soft hydrogel under FS versus DL culture conditions, D) stiff hydrogel under FS versus DL culture conditions, E) FS for soft hydrogel versus stiff hydrogel, F) DL soft hydrogel versus stiff hydrogel. Uniquely identified proteins are listed next to the Venn diagram in each panel.

Figure 3

Intensity plots (arbitrary units) of ECM and ECM related proteins for A) COL2A1, B) COL6A1, C) COL6A2, D) COL6A3, E) COL9A1, F) COL11A1, G) COL11A2, H) COL5A1, I) COL3A1, J) biglycan, K) lumican, and L) SERPINH1. Data are shown for soft, free swelling hydrogels (white bar), soft, dynamic loading hydrogels (gray bar), stiff, free swelling hydrogels (black bar), and stiff, dynamic loading hydrogels (black stripe bar). Data are presented as mean with standard deviation as error bars. P-values are presented for a one-way ANOVA.

Figure 4

The protein-protein interaction (PPI) network shown for both hydrogels (soft and stiff) and culture conditions (free swelling and dynamic loading) combined.