An Injectable Enzymatically Crosslinked Carboxymethylated Pullulan/Chondroitin Sulfate Hydrogel for Cartilage Tissue Engineering

Abstract

In this study, an enzymatically cross-linked injectable and biodegradable hydrogel system comprising carboxymethyl pullulan-tyramine (CMP-TA) and chondroitin sulfate-tyramine (CS-TA) conjugates was successfully developed under physiological conditions in the presence of both horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) for cartilage tissue engineering (CTTE). The HRP crosslinking method makes this injectable system feasible, minimally invasive and easily translatable for regenerative medicine applications. The physicochemical properties of the mechanically stable hydrogel system can be modulated by varying the weight ratio and concentration of polymer as well as the concentrations of crosslinking reagents. Additionally, the cellular behaviour of porcine auricular chondrocytes encapsulated into CMP-TA/CS-TA hydrogels demonstrates that the hydrogel system has a good cyto-compatibility. Specifically, compared to the CMP-TA hydrogel, these CMP-TA/CS-TA composite hydrogels have enhanced cell proliferation and increased cartilaginous ECM deposition, which significantly facilitate chondrogenesis. Furthermore, histological analysis indicates that the hydrogel system exhibits acceptable tissue compatibility by using a mouse subcutaneous implantation model. Overall, the novel injectable pullulan/chondroitin sulfate composite hydrogels presented here are expected to be useful biomaterial scaffold for regenerating cartilage tissue.

Figure 1

Synthesis of CMP, CMP-TA and CS-TA (A–C). (D) Hydrogel formation from CMP-TAs and CS-TAs via HRP-mediated crosslinking in the presence of H₂O₂.

Figure 2. Gelation time of CMP-TA/CS-TA hydrogels as a function of CMP-TA/CS-TA weight ratio, and the final concentrations of polymer, HRP and H₂O₂ were kept at 5 wt. %, 0.6 units/mL and 1 mM, respectively (n = 8, p < 0.01).

Figure 3

Storage moduli (G’) (A), Frequency sweep (B), Stress sweep (C) and Compressive modulus (n = 3, p < 0.01) (D) of CMP-TA/CS-TA hydrogels as a function of CMP-TA/CS-TA weight ratio, and the final concentrations of polymer, HRP and H₂O₂ were kept at 5 wt. %, 0.6 units/mL and 1 mM, respectively.

Figure 4

(A) The equilibrium swelling ratio (ESR) of CMP-TA/CS-TA hydrogels as a function of CMP-TA/CS TA weight ratio, and the final concentrations of polymer, HRP and H₂O₂ were kept at 5 wt. % , 4.8 units/mL and 5 mM, respectively (n = 6, p < 0.01). (B) Degradation of CMP-TA/CS-TA hydrogel as a function of CMP-TA/CS-TA weight ratio, and the final concentrations of polymer, HRP and H₂O₂ were kept at 5 wt. %, 4.8 units/mL and 5 mM, respectively (n = 6).

Figure 5

Live–dead assay showing chondrocytes encapsulated in CMP-TA/CS-TA hydrogels as a function of CMP-TA/CS-TA weight ratio of (A) 1/0, (B) 3/1 and (C) 1/1. The DNA content of CMP-TA/CS-TA hydrogels containing chondrocytes after culturing for 14 days (D) (n = 4, p < 0.05).

Figure 6. Quantitative RT-PCR of collagen type I, collagen type II and aggrecan by encapsulating chondrocytes into CMP-TA/CS-TA hydrogels after culturing for 14 days.

The gene expression of these markers was normalized to expression of the housekeeping gene β-actin (n = 3, p < 0.05).

Figure 7

Collagen type II (A), aggrecan (B) immunofluorescent staining and WB analysis (C,D) of CMP-TA/CS TA hydrogels as a function of CMP-TA/CS-TA weight ratio of 1/0 (A₁, B₁), 3/1 (A₂, B₂) and 1/1 (A₃, B₃) containing chondrocytes after culturing for 14 days.

Figure 8

Total collagen in CMP-TA/CS-TA hydrogels containing chondrocytes after culturing for 14 days (n = 4, p < 0.05) (A,B).

Figure 9

The implanted CMP-TA/CS-TA hydrogels of 1/0 (A), 3/1 (B) and 1/1 (C) on the backside of SD rats: the corresponding H&E staining was analyzed after subcutaneous implanting for 4 weeks. M represented residue of the implant and green arrow pointed to inflammatory cells.