Screening of hyaluronic acid–poly(ethylene glycol) composite hydrogels to support intervertebral disc cell biosynthesis using artificial neural network analysis

Hyaluronic acid (HA)–poly(ethylene glycol) (PEG) composite hydrogels have been widely studied for both cell delivery and soft tissue regeneration applications. A very broad range of physical and biological properties have been engineered into HA–PEG hydrogels that may differentially affect cellular “outcomes” of survival, synthesis and metabolism. The objective of this study was to rapidly screen multiple HA–PEG composite hydrogel formulations for an effect on matrix synthesis and behaviors of nucleus pulposus (NP) and annulus fibrosus (AF) cells of the intervertebral disc (IVD). A secondary objective was to apply artificial neural network analysis to identify relationships between HA–PEG composite hydrogel formulation parameters and biological outcome measures 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 vitro. Hydrogel mechanical properties ranged from 70 to 489 kPa depending on HA molecular weight, and measures of matrix synthesis, metabolite consumption and production and cell morphology were obtained to study relationships to hydrogel parameters. Results showed that NP and AF cell numbers were highest upon the HA–PEG hydrogels formed from the lower-molecular-weight HA, with evidence of higher sulfated glycosaminoglycan production also upon lower-HA-molecular-weight composite gels. All cells formed more multi-cell clusters upon any HA–PEG composite hydrogel as compared to gelatin substrates. Formulations were clustered into neurons based largely on their HA molecular weight, with few effects of PEG molecular weight observed on any measured parameters.