An enzyme-sensitive PEG hydrogel based on aggrecan catabolism for cartilage tissue engineering

Abstract

A new cartilage-specific degradable hydrogel based on photoclickable thiol-ene poly(ethylene glycol) (PEG) hydrogels is presented. The hydrogel crosslinks are composed of the peptide, CRDTEGE-ARGSVIDRC, derived from the aggrecanase-cleavable site in aggrecan. This new hydrogel is evaluated for use in cartilage tissue engineering by encapsulating bovine chondrocytes from different cell sources (skeletally immature (juvenile) and mature (adult) donors and adult cells stimulated with proinflammatory lipopolysaccharide (LPS)) and culturing for 12 weeks. Regardless of cell source, a twofold decrease in compressive modulus is observed by 12 weeks, but without significant hydrogel swelling indicating limited bulk degradation. For juvenile cells, a connected matrix rich in aggrecan and collagen II, but minimal collagens I and X is observed. For adult cells, less matrix, but similar quality, is deposited. Aggrecanase activity is elevated, although without accelerating bulk hydrogel degradation. LPS further decreases matrix production, but does not affect aggrecanase activity. In contrast, matrix deposition in the nondegradable hydrogels consists of aggrecan and collagens I, II, and X, indicative of hypertrophic cartilage. Lastly, no inflammatory response in chondrocytes is observed by the aggrecanase-sensitive hydrogels. Overall, it is demonstrated that this new aggrecanase-sensitive hydrogel, which is degradable by chondrocytes and promotes a hyaline-like engineered cartilage, is promising for cartilage regeneration.

Keywords: biodegradation; cartilage tissue engineering; hydrogel; peptide; poly(ethylene glycol).

Figure 1

Schematic of hydrogel formation. (A) 8-arm PEG-amide-norbornene (8-armPEG-NB, 20 kDa) is crosslinked with (B) non-degradable PEG-dithiol (PEGdSH, 1000 Da) or (C) aggrecanase-sensitive peptide (CRDTEGE-ARGSVIDRC, 1767 Da) in the presence of photoinitiator and 365 nm light to create (D) a 3D crosslinked hydrogel network within which cells can be encapsulated. (E) Hydrogel degradability was confirmed after 7 hours by incubating acellular hydrogels in adult chondrocyte-conditioned medium (n = 3 – 4). (F) Bovine chondrocytes from three distinct cell sources were studied and included chondrocytes isolated from juvenile and adult donors and adult chondrocytes stimulated with pro-inflammatory lipopolysaccharide (LPS). Adult chondrocytes have been previously reported to exhibit higher catabolic activity compared to juvenile chondrocytes^[18] and LPS stimulation enhances catabolic activity in adult chondrocytes.^[40]

Figure 2

(A) Viability of encapsulated cells after 3 weeks of culture in non-degradable or enzymatically degradable hydrogels. Live cells are green, dead cells are red. Scale bars are 200 μm. (B) Cell number per construct. (C) Representative photographs of hydrogels taken at 12 weeks. Scale bars are 5 mm. (D) Hydrogel volume and (E) compressive modulus normalized to measurements from one day after encapsulation. The initial compressive modulus was similar for all conditions, 13 ± 3 kPa at day one. * indicates significant difference from degradable hydrogels (at same cell age). † indicates significant difference from adult cells (same hydrogel condition). # indicates significant difference from LPS condition (adult cells only) (p < 0.05). Error bars are standard deviation (n = 3).

Figure 3

(A) sGAG production per cell in the constructs at 12 weeks is shown by the (Image) bar and that which was released to the medium throughout 12 weeks is shown by the (Image) bar. The sum of the two represents on average the total sGAG production per cell over the entire 12 weeks. Letter groupings show statistical similarities (same letter) and differences (different letters) (p < 0.05). Top letters are for cumulative sGAG from medium and lower letters are for cumulative sGAG in constructs. Error bars are standard deviation (n = 3). (B) Representative images of histological staining for sulfated GAG (sGAG, orange-red) at weeks 6 and 12 with Safranin-O/Fast Green. Background proteins are blue, nuclei are dark blue-purple, scale bars are 50 μm. (C) Representative confocal microscopy images from immunohistochemistry staining for aggrecan (red) in hydrogels at weeks 6 and 12. Nuclei are blue, scale bars are 50 μm.

Figure 4

(A) Total collagen production per cell in the constructs at 12 weeks is shown by the (Image) bar and that which was released to the medium throughout 12 weeks is shown by the (Image) bar. The sum of the two represents on average the total collagen production per cell over the entire 12 weeks. Letter groupings show statistical similarities (same letter) and differences (different letters) (p < 0.05). Top letters are for total collagen from medium and lower letters are for total collagen in constructs. Error bars are standard deviation (n = 3). (B) Representative confocal microscopy images of immunohistochemical staining for collagen II (green) and (C) collagenase-generated collagen neoepitope C1,2C (green) in hydrogels at weeks 6 and 12. Nuclei are blue, scale bars are 50 μm.

Figure 5

(A) Representative confocal microscopy images of immunohistochemical staining for collagen I (green) and (B) collagen X (green) in hydrogels at weeks 6 and 12. Nuclei are blue, scale bars are 50 μm.

Figure 6

Activity of (A) aggrecanase-1 and (B) generic MMPs measured per cell within the constructs is shown by the (Image) bar and released to the culture medium is shown by the (Image) bar. The sum of the active enzyme in the constructs and released to the medium represents on average the total amount of active enzyme that was produced per cell over the entire 12 weeks. (C) Cumulative IL-6, shown as the additive quantity measured in the medium throughout 12 weeks. Letter groupings show statistical similarities (same letter) and differences (different letters) (p < 0.05). Top letters are for total activity in medium and lower letters are for total activity in constructs. Error bars are standard deviation (n = 3).