A versatile pH sensitive chondroitin sulfate-PEG tissue adhesive and hydrogel

Abstract

We developed a chondroitin sulfate-polyethylene glycol (CS-PEG) adhesive hydrogel with numerous potential biomedical applications. The carboxyl groups on chondroitin sulfate (CS) chains were functionalized with N-hydroxysuccinimide (NHS) to yield chondroitin sulfate succinimidyl succinate (CS-NHS). Following purification, the CS-NHS molecule can react with primary amines to form amide bonds. Hence, using six arm polyethylene glycol amine PEG-(NH2)6 as a crosslinker we formed a hydrogel which was covalently bound to proteins in tissue via amide bonds. By varying the initial pH of the precursor solutions, the hydrogel stiffness, swelling properties, and kinetics of gelation could be controlled. The sealing/adhesive strength could also be modified by varying the damping and storage modulus properties of the material. The adhesive strength of the material with cartilage tissue was shown to be ten times higher than that of fibrin glue. Cells encapsulated or in direct contact with the material remained viable and metabolically active. Furthermore, CS-PEG material produced minimal inflammatory response when implanted subcutaneously in a rat model and enzymatic degradation was demonstrated in vitro. This work establishes an adhesive hydrogel derived from biological and synthetic components with potential application in wound healing and regenerative medicine.

Figure 1

CS-NHS reacts with primary amines of both PEG-(NH₂)₆ and proteins of tissue to form a covalently bound hydrogel to tissue. (A-D) Solutions of PEG-(NH₂)₆ (colored with blue dye) and CS-NHS (clear solution) are mixed to form a hydrogel. (E) The same hydrogel without dye (star) in a cartilage defect after 11 days of swelling in PBS. The NHS activated carboxyl groups of CS-NHS (F) react with the primary amines of PEG-(NH₂)₆ (G) and the primary amines of proteins in tissue (H, reactive groups circled). The new bonds that form are amide bonds. (H) The material in contact with tissue before (left) and after (right) it has reacted with primary amines. NHS = N-Hydroxysuccinimide; PEG = Poly(ethylene glycol); CS = Chondroitin Sulfate.

Figure 2

Effects of PEG-(NH₂)₆ solution pH, polymer ratios and synthesis conditions on mechanical, swelling and gelation properties of 10% (w/v) CS-PEG hydrogels. For panels A-C, 50% (% dry weight) CS-NHS and 50% (% dry weight) PEG-(NH₂)₆ were used. (A) The tangent modulus increases as pH decreases. Also, if humidity is decreased during the EtOH washing steps of the synthesis, the modulus consistently increases. (B) The swelling ratios as a function of pH show the opposite trend than that of the moduli. (C) As pH decreases, the time it takes the components to form a hydrogel increases. (D-F) when the concentration of CS to PEG is varied, there is a significant decrease in modulus at lower concentrations of CS-NHS, but it has little effect on swelling ratio and gelation time. Values are reported in mean ± SD with a sample size of 5 per treatment for tangent moduli and swelling ratio experiments, and a sample size of 3 for gelation time experiments. *p<0.05, **p<0.01, ***p<0.001.

Figure 3

Effect of CS-PEG hydrogels with varying gelation times (GT) and polymer ratios on cytotoxicity to chondrocytes. (A) Live/Dead analysis of chondrocytes following one day of encapsulation in hydrogels with varying gelation times. For all gelation times, most of the cells are alive (green) with very few cells dead (red). (B) Chondrocytes were exposed to CS-PEG hydrogels with varying CS-NHS content and viability was quantified using the WST-1 reagent. Values are normalized to controls (monolayer of cells without exposure to hydrogel) and are reported in mean ± SD for a sample size of 4 per treatment. No significant differences was observed between the treatments and the control.

Figure 4

Swelling, kinetics of gelation, sealing and adhesive properties of the CS-PEG hydrogel adhesive. (A) There is an inverse relationship between gelation time and temperature. (B,C) Varying the crosslinking density and the osmolarity of the swelling medium has little effect on when swelling equilibrium is reached. In all cases, swelling equilibrium is achieved within five and a half hours. (C) When ddH2O is used instead of PBS, swelling equilibrium is again reached within five and a half hours even though the gels swell twice as much. (D-F) Effects of HA thickener on the burst pressure, storage modulus and damping properties of the hydrogels. (G) The glued cartilage pieces were pulled apart using a Bose ELF 3200 Mechanical Tester. (H) Adhesive strengths of CS-PEG and fibrin glue were significantly different and were an order of magnitude apart. Values are reported in mean ± SD with a sample size of 5 per treatment except for gelation time experiments which had a sample size of 3. *p<0.05, **p<0.01, ***p<0.001.

Figure 5

Biological activity of CS-PEG gels. Fifteen hours after incubation CS-PEG gels in buffer (A) without and (B) with chondroitinase ABC. (C) Within 30 hours, most of the gel had been degraded by the enzyme. (D) H&E staining of CS-PEG and PEG gels after subcutaneous injection in a rat model. In each section, the gels are marked with a star. CS-PEG = chondroitin sulfate-poly(ethylene glycol) gel. Values are reported in mean ± SD with a sample size of 3 per treatment.

Scheme 1

Synthesis of CS-NHS.