Incorporation of aggrecan in interpenetrating network hydrogels to improve cellular performance for cartilage tissue engineering

Abstract

Interpenetrating network (IPN) hydrogels were recently introduced to the cartilage tissue engineering literature, with the approach of encapsulating cells in thermally gelling agarose that is then soaked in a poly(ethylene glycol) diacrylate (PEGDA) solution, which is then photopolymerized. These IPNs possess significantly enhanced mechanical performance desirable for cartilage regeneration, potentially allowing patients to return to weight-bearing activities quickly after surgical implantation. In an effort to improve cell viability and performance, inspiration was drawn from previous studies that have elicited positive chondrogenic responses to aggrecan, the proteoglycan largely responsible for the compressive stiffness of cartilage. Aggrecan was incorporated into the IPNs in conservative concentrations (40 μg/mL), and its effect was contrasted with the incorporation of chondroitin sulfate (CS), the primary glycosaminoglycan associated with aggrecan. Aggrecan was incorporated by physical entrapment within agarose and methacrylated CS was incorporated by copolymerization with PEGDA. The IPNs incorporating aggrecan or CS exhibited over 50% viability with encapsulated chondrocytes after 6 weeks. Both aggrecan and CS improved cell viability by 15.6% and 20%, respectively, relative to pure IPNs at 6 weeks culture time. In summary, we have introduced the novel approach of including a raw material from cartilage, namely aggrecan, to serve as a bioactive signal to cells encapsulated in IPN hydrogels for cartilage tissue engineering, which led to improved performance of encapsulated chondrocytes.

FIG. 1.

Schematic diagram showing formation of a bioactive interpenetrating network (IPN) gel containing chondroitin sulfate (CS) as a bioactive signal. Color images available online at www.liebertpub.com/tea

FIG. 2.

Schematic representation showing formation of agarose-poly(ethylene glycol) diacrylate (PEGDA) double network containing aggrecan as a bioactive signal. Color images available online at www.liebertpub.com/tea

FIG. 3.

Macroscopic views of IPNs gels: representative images of equilibrium swelled IPNs without (A) and with (B) encapsulated chondrocytes. Note the reduction in transparency with encapsulated chondrocytes. Scale bar=4 mm.

FIG. 4.

Spinning disc confocal microscope live/dead images of encapsulated chondrocytes in IPN at week 0 (A–C), week 3 (D–F), and week 6 (G–I) in pure IPN, IPN-aggrecan, and IPN-CS gels. Green (Calcein AM) dye indicates viable (live) cell populations, while red (ethidium homodimer) dye indicates dead cells. Note the improved cell viability emerging at 3 weeks and especially evident at 6 weeks in the CS and aggrecan IPNs relative to the pure IPN. Scale bar=50 μm. Color images available online at www.liebertpub.com/tea

FIG. 5.

Percent cell viability of encapsulated chondrocytes by image analysis mask statistics. Multiple confocal Z-scan series were performed on a representative sample in each group (mean±standard deviation). *Values statistically significant from week 0 (p<0.05 and n=3), while **values indicate significant differences from the control group (pure IPN) at that time point (p<0.05 and n=3).

FIG. 6.

The total DNA content within pure IPN, IPN-CS, and IPN-aggrecan gel constructs at 0, 3, and 6 weeks. Error bars represent mean±standard deviation. *Values statistically significant from week 0 time point, **values indicate statistically significant differences from the control at that time point, and ^#values indicate statistically significant differences between the groups at that time point (p<0.05, n=4).

FIG. 7.

(A) Total sulfated glycosaminoglycan (GAG) accumulated per IPN gel (construct). (B) Total GAG accumulated normalized to the DNA content. Values represent mean±standard deviation. The GAG content associated with incorporated CS and aggrecan was subtracted from the actual GAG value to provide the values reported here. *Values indicate statistically significant increase in GAG production vs. week 0 time point, **values indicate a statistically significant increase in GAG production from the control at that time point and ^#values indicate statistically significant differences among the groups at that time point (p<0.05, n=4).

FIG. 8.

(A) Total hydroxyproline accumulated per IPN (construct) at 0, 3, and 6 weeks. (B) The total accumulated hydroxyproline content normalized to the DNA content. Values represent mean±deviation. *Values indicate a statistically significant increase in hydroxyproline production vs. week 0 time point, **values indicate statistically significant differences from the control at that time point and ^#values indicate statistically significant differences among the groups at that time point (p<0.05, n=4).

FIG. 9.

Histology of representative sections of IPN gels encapsulated with chondrocytes at week 0 and 6: Hematoxylin and eosin (H&E) staining (A–F) and Safranin-O staining (G–L). Inset pictures showing the pericellular area. Both IPN-CS and IPN-aggrecan stained positive for incorporated CS. Scale bar=100 μm. Color images available online at www.liebertpub.com/tea