Hyaluronic acid (HA) poly(ethylene glycol) (PEG) composite hydrogels have been widely studied for both cell delivery and soft tissue regeneration applications. for each cell type of the IVD. Eight different hydrogels were developed from preparations of thiolated HA (HA-SH) and PEG vinylsulfone (PEG-VS) macromers and used as substrates for NP and AF cell culture in culture. Under these conditions important features of cell morphology proliferation matrix synthesis and metabolite consumption and production can be assessed that will reveal and reflect cell-matrix interactions. A secondary objective was to apply ANN analysis in order to identify relationships between HA-PEG composite hydrogel formulation parameters and biological outcome measures for each cell type of the IVD. Overall this work has revealed how different cell populations interact with these HA-PEG composite hydrogels and also identified a set of HA-PEG hydrogels that can be supportive of IVD cells in culture. 2 Materials and Methods 2.1 Materials Hyaluronic acid (HA) was purchased from Beta Pharma (MW = 637 kDa determined by viscosity measurement [59] Branford CT). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) N-hydroxysuccinimide (NHS) dithiothreitol (DTT) calcium hydride and divinylsulfone (DVS) were purchased from Sigma-Aldrich (St. Louis MO). Cystamine dichloride phosphorus pentoxide and water (ultrapure TG 100713 HPLC Grade) were purchase from VWR International LLC (Radnor PA). All 4-arm-polyethylene glycols (4-arm-PEGs MW = 20 kDa PDI = 1.02 and MW = 40 kDa PDI = 1.02) were purchased from Jenkem Technology USA (Allen TX). 2.2 Preparation of thiolated hyaluronic acid Pulsed-ultrasonication [60 61 was used to produce low molecular weight HAs from commercially-available HA as described here. HA solutions (6.25 mg/mL) in ultrapure water were degassed with bubbling N2 (30 minutes) and aliquots were exposed to pulsed ultrasound for 120 20 or 5 minutes (13 ml 8.7 W/cm2 1 on/1s off 6 °C Vibracell Model VCX500 12.8 mm tip probe Sonics and Materials Inc. Newton CT). After sonication the solution was passed through a nylon syringe filter (pore size=0.45 μm) to yield low molecular weight HAs (~27 kDa 59 kDa or 98 kDa as determined by viscometry [49]) and the filtrate was freeze-dried as white foam (Figure 1 (a)). TG 100713 Figure 1 Schematics for synthesis of HA-SH from HA (a) 4 based on 4-arm-PEG (b) and formation of the HA-PEG composite hydrogel via the Michael addition reaction between HA-SH and 4-arm-PEG-VS TG 100713 (c). Thiolated HAs were prepared by a two-step procedure [54 TG 100713 62 First to a stirring solution of low molecular weight HA (27 kDa 742 mg in 70 mL ultra-pure water) was added EDCI (1.45 g) and the stirring was continued at room TG 100713 temperature for 10 minutes under N2. NHS (0.87 g) was then added and the pH value of the solution was adjusted to 4.5 by addition of 4 M HCl. After 2 hours cystamine dichloride (1.70 g) was added. The reaction mixture was kept under N2 and stirred at room temperature for 2 days then dialyzed exhaustively against NaCl solution (4 g/L) and water for 2 days. The resulting dialysate was freeze-dried to yield white foam and then dissolved in H2O (70 mL) at the start of the modification. DTT (11.5 g) was added and the pH value of the solution was adjusted to 8.2. The reaction mixture was stirred (2 days room temperature) under N2 and the pH value adjusted to 5 by adding 4 M HCl. Following the reaction the solution was first dialyzed against NaCl/HCl (NaCl: 4 g/L pH = 5) followed by exhaustive dialysis against diluted HCl solution (pH = 5). After dialysis Rabbit Polyclonal to AKT1/3. the solution pH was altered to 7 with the addition of 1 M NaOH and freeze-dried to produce a thiolated hyaluronic acidity product (HA-SH-1 produce = 81%). 1H-NMR (D2O; 400MHz Varian spectrophotometer): δ 4.3-4.5 (HA anomeric proton) 3 (HA band protons) 2.6 (NH-= 20 0 6 g 2.4 mmol-OH) was dissolved in anhydrous TG 100713 dichloromethane (90 mL) as well as the mix was stirred under argon for thirty minutes. NaH (303 mg) was suspended in anhydrous DCM (30 mL) as well as the suspension system was added in to the flask dropwise and stirred for just one hour. DVS (14.1 g 120 mmol) was then dissolved in anhydrous dichloromethane (60 mL) as well as the suspension of PEG and NaH was put into the DVS solution dropwise over one hour. The response mix was after that stirred for 3 times under argon at night neutralized with acetic acidity (pH = 7) filtered and focused. The final item was purified by precipitation in frosty Et2O double yielding a white natural powder of vinylsulfone functionalized 4-arm-poly(ethylene glycol) (4-arm-PEG-VS-20kDa: MW=20.5 kDa 82 produce). The final end group.