Injectable thermoreversible hyaluronan-based hydrogels for nucleus pulposus cell encapsulation

Abstract

Thermoreversible hydrogels have potential in spine research as they provide easy injectability and mild gelling mechanism (by physical cross-link). The purpose of this study was to assess the potential of thermoreversible hyaluronan-based hydrogels (HA-pNIPAM) (pNIPAM Mn = 10, 20, 35 × 10(3) g mol(-1)) as nucleus pulposus cells (NPC) carrier. Cytocompatibility (WST-1 assay), viability (trypan blue), morphology (toluidine blue), sulphated glycosaminoglycan synthesis (DMMB assay) and gene expression profile (real-time PCR) of bovine NPC cultured in HA-pNIPAM were followed for 1 week and compared to alginate gel bead cultures. The injectability and cell survival in a whole disc organ culture model were assessed up to day 7. All HA, HA-pNIPAM and their degradation products were cytocompatible to NPC. HA-pNIPAM hydrogels with no volume change upon gelling maintained NPC viability and characteristic rounded morphology. Glycosaminoglycan synthesis was similar in HA-pNIPAM and alginate gels. Following NPC expansion, both gels induced re-differentiation toward the NPC phenotype. Significant differences between the two gels were found for COLI, COLII, HAS1, HAS2 and ADAMTS4 but not for MMPs and TIMPs. Higher expression of hyaluronan synthases (HAS1, HAS2) and lower expression of COLI and COLII mRNA were noted in cells cultured in HA-pNIPAM (pNIPAM = 20 × 10(3)g mol(-1)). NPC suspension in HA-pNIPAM was injectable through a 22-G needle without loss of cell viability. Ex vivo, NPC viability was maintained in HA-pNIPAM for 1 week. A HA-pNIPAM composition suitable for nucleus pulposus repair that provides an injectable carrier for NPC, maintains their phenotype and promotes extracellular matrix generation was identified.