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

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.

at or above 1.2 unit/mL (Fig. S4A). From the frequency sweep experiments, it was found that, with the exception of the hydrogel formed using 0.1 unit/mL HRP, the rest of hydrogels was elastic and the G' was not dependent on the frequency (Fig. S4B). In addition, the fracture stress of the hydrogel of the lowest HRP concentration was about 125 Pa, however, the fracture stress values of the rest of hydrogels were about 400 Pa in the stress sweeps ( Fig. S4D) concentration of 25 mM, the G' of the rest of hydrogels remained independent of the frequency (Fig. S5B), demonstrating that these hydrogels displayed a predominantly elastic behavior. The fracture stress reached to a maximum at H 2 O 2 concentration of 5 mM in the stress sweeps (Fig. S5D). Moreover, the variation tendency of the fracture stress of the hydrogels was consistent with the G' in time sweeps. In contrast to HRP concentration, the stiffness of hydrogel very depended on H 2 O 2 concentration. These results were in good agreement with previous reports of the hydrogel systems using the same enzymatic oxidation reaction 3b, 3d . Fig. S6 shown that polymer concentration had an influence on the rheological behavior of CMP-TA/CS-TA (1/1) hydrogel at fixed HRP concentration of 0.6 unit/mL and H 2 O 2 concentration of 1 mM. It was noteworthy that the increase in polymer concentration led to higher G' and fracture stress as a result of higher amounts of TA moieties participating in the crosslinking reaction ( Fig. S6A and D). Furthermore, the G' was almost insensitive to the oscillatory frequency within the range of 3-10% (Fig. S6B).

Synthesis and characterization of carboxymethyl pullulan-tyramine and chondroitin sulfate-tyramine:
Carboxymethyl pullulan (CMP) was synthesized beforehand by the reaction of pullulan with ClCH 3 COONa in the presence of sodium hydroxide as previously described method 5 . CMP-TA and CS-TA were synthesized by the coupling reaction of amine groups of TA to carboxylic acid groups of CMP and CS using EDC/NHS activation. 4,6 The degree of substitution (DS) and molecular weight of CMP-TA and CS-TA were analyzed using 1 Table S1.

In vitro degradation:
The CMP-TA/CS-TA hydrogels were by lyophilized and accurately recorded initial weight Unit/mL and 5 mM, respectively. The cell seeding density was about 5.0×10 6 cells/mL. After gelation, the cell-laden constructs were cultured in chondrocyte specific medium at 37 o C in a humidified atmosphere containing 5 % CO 2 for 14 days, and the medium was replaced every two days. Quantitative Real-time PCR analysis: Total RNA was extracted from the cell-laden constructs using Trizol reagent (Sigma) following the standard protocols. Subsequently, RNA was reverse transcribed into cDNA using revert aid first strand cDNA Synthesis Kit (Thermo) according to the manufacturer's recommendations.

Cell viability and proliferation:
Quantitative Real-time PCR (RT-PCR) was performed using Fast Start Universal SYBR Green Master (Roch) in a 7300 RT-PCR System (ABI), with β -actin as reference genes. The expression levels of targets gene were then calculated as 2 -ΔΔ t as previously described. 8 The primer sequences were listed in Table S2.
Matrix production: The secretion of collagen type II and aggrecan by chondrocytes was evaluated by immunofluorescent staining and Western-blot (WB) as previously reported. 9 In addition, total collagen content was determined using the hydroxyproline assay in which hydroxyproline makes up 12.5 % of collagen as previously reported. [7] Samples were extrapolated against hydroxyproline standards between 0 and 200 µg/mL. All values were corrected for the background staining of gels without cells and normalized to the dry gel mass or DNA content.
In vivo subcutaneous implant of the hydrogels: A mouse subcutaneous implant model was used to evaluate the tissue compatibility. Following 4 weeks of implantation, SD rats were humanely euthanized via carbon dioxide inhalation. Retrieved implants with surrounding tissues were fixed in 10 % paraformaldehyde solution. The fixed samples were then dehydrated, embedded in paraffin and stained with hematoxylin and eosin (H&E). All animals received humane care in compliance with protocols approved by Shanghai Jiao Tong University Animal Research Committee.
Statistical analysis: Data was expressed as mean ±standard deviations (SD). Statistical significance was determined by single-factor analysis of variance followed by Tukey's post hoc analysis. Analysis was performed using the SPSS 13.0 statistical software. Statistical significance was set at p < 0.01 or p < 0.05.