Injectable thermoreversible hyaluronan-based hydrogels for nucleus pulposus cell encapsulation

Abstract

Introduction: 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.

Materials and methods: 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.

Results: 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.

Conclusion: 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.

Scheme 1

Molecular structure of HA-pNIPAM

Fig. 1

Influence of HA and HA-pNIPAM compositions (HA-pNIPAM10, HA-pNIPAM20 and HA-pNIPAM35) and their degradation products with constant HA concentration (100 μg/mL) and variable hyaluronidase (Hase) concentrations (0–1,000 U/mL) on nucleus pulposus cell number at 48 h as measured by WST-1 assay and normalized against positive control (cells with medium only): viability median, quartile, maximum and minimum; *p < 0.05 versus 0 U/mL Hase, **p < 0.05 versus 100 U/mL Hase, ***p < 0.05 versus 500 U/mL Hase, + p < 0.05 versus HA with equivalent concentration of HAse

Fig. 2

Representative images of calcein AM/ethidium homodimer 1 (live-dead) staining of nucleus pulposus cells cultured in HA-pNIPAM20 (a–c), HA-pNIPAM35 (d–f) and alginate (g–i) hydrogels for 1 (a, d, g), 3 (b, e, h) and 7 (c, f, i) days; green living cell, red dead cell. Scale bar 200 μm

Fig. 3

Cell viability of nucleus pulposus cells assessed by trypan blue exclusion method after 1, 3 and 7 days of culture in HA-pNIPAM hydrogels (HA-pNIPAM20 and HA-pNIPAM35); #p < 0.05 versus HA-pNIPAM20, *p < 0.05 versus HA-pNIPAM35 day 1. Cell viability was maintained in HA-pNIPAM20 hydrogel, while it dropped significantly in HA-pNIPAM35 hydrogel

Fig. 4

Representative images of toluidine blue staining of nucleus pulposus cells cultured for 7 days in a HA-pNIPAM20 hydrogel, b HA-pNIPAM35 hydrogel and c alginate gel. Scale bar 50 μm. The characteristic rounded morphology of nucleus pulposus cells was maintained in HA-pNIPAM20 and alginate hydrogels, while cells appeared stretched in HA-pNIPAM35 composition

Fig. 5

Total sulphated glycosaminoglycan (GAG) content in the HA-pNIPAM20 hydrogel and alginate gel (constructs and media) relative to the amount of DNA; average and standard deviations are reported. An increase in GAG/DNA was found during the 7 days of culture without significant differences between the two gels

Fig. 6

Gene expression profile of nucleus pulposus cells (NPC) cultured for 1 week in HA-pNIPAM20 or alginate gel. Real-time PCR data were normalized to house-keeping gene (18s rRNA) and are presented relative to nucleus pulposus cells subcultured to P1; viability median, quartile, maximum and minimum, *p < 0.05 versus alginate. Compared to NPC in alginate gels, NPC in HA-pNIPAM20 hydrogel showed significant down-regulation of COL1 and COL2 and up-regulation of HAS1, HAS2 and ADAMTS4

Fig. 7

Representative images of a nucleus pulposus cavity created in bovine intervertebral disc with endplates by punching (punch diameter 3.5 mm, length 7 mm) and red PKH26 labelled nucleus pulposus cells after recovery and staining with calcein AM at b day 1 and c day 7 of culture in HA-pNIPAM20 hydrogel; yellow delivered living cell, green living cell from the surrounding tissue, red dead cell. Scale barsa 10 mm, b–c 200 μm. Cell viability was maintained for at least 1 week, although a decrease in cell density was observed

Fig. 8

Representative images of red PKH26 labelled nucleus pulposus cells (NPC) recovered and stained with calcein AM 24 h after injection through a 22-G needle of a NPC suspension in a HA-pNIPAM20 and b alginate gel; yellow living cell, red dead cell. Scale bar 200 μm. Cell viability after injection was higher in HA-pNIPAM20 hydrogel than in alginate gels