Glucosamine sulfate

Mouse monoclonal anti-heparan sulfate (HS) antibody (10E4 epitope, AMS Biotechnology, Abingdon, UK) was used as a primary antibody, and polyclonal goat anti-mouse antibody Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA) as a secondary antibody for immunofluorescence staining. Heparinase 1, fucoidan, glucosamine sulfate, Brefeldin A and Wortmannin were ordered from Sigma-Aldrich (Munich, Germany). U0126 was purchased from Cell Signalling Technology Inc., Danver, MA, USA. W146 was purchased from Cayman Chemical, Ann Arbor, MI, USA. ECX was kindly provided by Microvascular Health Solutions, Salt Lake City, UT, USA, and was composed as follows (per capsule, in total 750.75 mg): fucoidan (85%) 106.25 mg, antioxidants (SOD, catalase, polyphenols) 120 mg, glucosamine sulfate 375 mg, hyaluronic acid (1800–3000 kDa) 17.5 mg, microcrystalline cellulose 130 mg, silicon dioxide 2 mg. ECX was dissolved in DMSO, centrifuged, and the supernatant was sterile filtered and diluted with HEPES buffer as indicated in the results and figure legends.
Concentrations and incubation times of heparinase I, albumin, W146 (we used 5 µM, half the dose specified in the literature, since the cells became detached from the bottom of the dish in a 10 µM solution), Wortmannin, U0126 and Brefeldin A were adopted from previous studies [27 (link),35 (link),38 (link),58 (link),59 (link),60 (link)].

Morphine hydrochloride and naloxone were purchased from Darupakhsh Co., (Iran). Glucosamine sulfate was obtained from Sigma Chemical Co., (USA). All drugs were dissolved in normal saline.

Analytical grade Chloroform of Merck and analytical grade Methanol of Sigma Andrich were used as organic solvents for the formulation of transfersome and liposomes. Absolute Hydrochloric acid (Merck) and Sodium Hydroxide (Sigma-Aldrich, Germany) were used for the quantitative analysis of Glucosamine sulfate as per official compendia method. Triethylamine procured from Merck was used for the formulation of hydrogel. Eugenol was purchased from Sigma Scientific Pvt limited. Phosphate Buffer saline (PBS) solution having PH. 7.0 was made from PBS tablets purchased from Sigma Scientific. Distilled water was taken from pharmaceutics lab, Department of Pharmacy, Riphah International University (Islamabad, Pakistan).
For the formulation of transfersome, Phospholipid 90G was generously gifted by Lipoid Germany, Glucosamine sulfate was gifted from Polyline ChemPharma Peshawar, Polyoxyethylene (20) sorbitan monooleate (Tween 80) and sorbitan monostearate (Span 60) were procured from Merck. For the induction of osteoarthritis in knee joint of rabbits l-Cysteine and Papain were procured from Sigma Adrich Germany. All the chemicals were of pure analytical grade.