Chondroitin

Urine samples were defrosted at 4 °C and mixed well using a vortex mixer. A 400-μL aliquot of each sample was desalted by passing through a 3 kDa molecule weight cutoff (MWCO) spin column and washed twice with distilled water. A series of urine samples were also prepared for method validation by adding aqueous solutions containing varying amounts of standard CS and HS affording final concentrations of 50–500 ng/mL.
The casing tubes were replaced before 150 μL of digestion buffer (50 mM ammonium acetate containing 2 mM calcium chloride adjusted to pH 7.0) was added to the filter unit. Recombinant heparin lyase I, II, III (pH optima 7.0–7.5) and recombinant chondroitin lyase ABC (10 mU each, pH optimum 7.4) were added to each sample and mixed well. The samples were all placed in a water bath at 37 °C for 2 h, after which enzymatic digestion was terminated by removing the enzymes by centrifugation. Under these reaction conditions, these lyases could completely depolymerize their GAG substrates (in amounts of over 100 μg) into products containing each class of GAG disaccharides. The filter unit was washed twice with 100 μL distilled water and the filtrates, containing the disaccharide products, were dried via vacuum centrifuge and stored at –20 °C.
The dried samples were AMAC-labeled by adding 10 μL of 0.1 M AMAC in DMSO/acetic acid (17/3,V/V) incubating at room temperature for 10 min, followed by adding 10 μL of 1 M aqueous NaBH₃CN and incubating for 1 h at 45 °C. A mixture containing all 17-disaccharide standards prepared at 1250 ng/mL was similarly AMAC-labeled and used for each run as an external standard. After the AMAC-labeling reaction, the samples were centrifuged and each supernatant was recovered and an equal volume of DMSO:acetic acid:distilled water (17:3:20) was added to each. Samples were stored in a light-resistant container at room temperature until analyzed via LC-MS/MS.

Five different samples were prepared and were labeled by their components (C = chitosan, A = alginate, D = chondroitin, P = peptides): chitosan+alginate hydrogel (CA), chitosan + alginate + chondroitin hydrogel (CAD), peptide hydrogel (P), chitosan + alginate + peptide hydrogel (CAP), and chitosan + alginate + chondroitin + peptide hydrogel (CADP). Stock solutions of chitosan, alginate and chondroitin were made in phosphate buffered saline (PBS) at 1.5 % mass fraction. Chitosan solution also required the addition of 1 % mass fraction HCl in H₂O to dissolve. Stock solutions of both peptides were made in PBS at pH 7.4 at 4.54 % mass fraction (32 mmol/L). All sample preparation procedures and measurements were performed at 25 °C and the final pH for all samples was 7.4.
To prepare the CA network, stock solutions of chitosan and alginate were diluted to 0.5 % mass fraction by PBS, and equal volumes (1:1), 200 μL of each diluted solution were mixed together by simultaneous pipetting through a Y-shaped connector into the sealed cell of the rheometer. The measurements of gelation kinetics started immediately after mixing.
To prepare the CAD network, stock solutions of chitosan and alginate were diluted to 0.7 % mass fraction by PBS, and after mixing of 143 μL 0.7 % mass fraction chitosan with 143 μL 0.7 % mass fraction alginate in the rheometer cell through Y-shaped connector, the mixture was allowed to equilibrate for 2 hours (time necessary to build up of CA network estimated from the rheology experiments for CA network). Then 114 μL of chondroitin solution diluted to 0.28 % mass fraction (to get 7:1 chitosan/alginate to chondroitin weight ratio) was added to the mixture, immediately followed by rheological monitoring of the incorporation of chondroitin into the CA network resulting in the formation of CAD network.
To prepare the P network, stock solutions of individual KWK and EWE peptides were diluted to 2.27 % mass fraction (16 mmol/L) with PBS buffer at pH 7.4. Diluted solutions of the peptide modules were centrifuged separately for 10 min at 8,000 rpm, and 200 μL of each KWK and EWE peptides were mixed through Y-shaped connector in the sealed cell of rheometer, immediately followed by monitoring of the gelation process resulting in the P network.
The procedure for CAP network preparation was similar to the steps used for CAD networks described above. Stock solutions of chitosan and alginate were diluted to 1 % mass fraction, and 100 μL of each solution were mixed together in the sealed cell of the rheometer using a Y-shaped connector. After 2 hrs of equilibration (necessary to mature the CA network), pH of the network was measured and adjusted to 7.4 by addition of very small volumes (several μL) of concentrated NH₄OH solution. Then, 100 μL of each 4.54 % mass fraction (32 mmol/L) KWK and EWE peptides solution were mixed with the matured chitosan/alginate network. Rheological measurements of gelation kinetics were started immediately.
To prepare the CADP network, stock solutions of chitosan and alginate were diluted to 1.33 % mass fraction by PBS and 75 μL of each solution were mixed in the sealed cell of the rheometer using a Y-shaped connector. The resulting mixture was equilibrated for 2 hrs (to get mature CA network), then 50 μL of chondroitin solution diluted to 0.64 % mass fraction by PBS was added, and the sample was equilibrated for another 10–12 hrs (time necessary for maturation of CAD network estimated from the rheology experiments for CAD network). Then, 100 μL of each 4.54 % mass fraction (32 mmol/L) KWK and EWE peptides solution were mixed with the matured chitosan+alginate+chondroitin network. Rheological measurements of gelation kinetics were started immediately.
In all samples final concentrations for components in each sample were (in % mass fraction): chitosan (0.25 %), alginate (0.25 %), chondroitin (0.076 %), two peptide modules together (2.25 % or 16 mmol/L). During the equilibration procedures, mixtures in the sealed cell were covered with parafilm to prevent sample drying.