Abstract
Introduction
Thermoreversible hyaluronan (HA) based hydrogels represent a promising carrier for IVD repair because of the mild gel formation allowing for encapsulation of viable cells and biological stimulation of cells by HA.1,2 Additionally, polyurethane (PU) foams can provide elasticity, which is of key importance for the restoration of IVD mechanical function, as the latter experiences repeated loading during the daily human activities. Finally, the addition of relevant cells may promote a faster restoration of the IVD function. Therefore, the aim of this study was to compare the single and combined effect of PU particles and HA hydrogel on the proliferation and differentiation of human IVD and mesenchymal stem cells.
Materials and Methods
Human bone marrow stromal cells (hMSCs) or human disk cells (hIVD cells) (P2-P4) were seeded on PU particles; after 12 hours, the seeded particles were suspended in either a 12% w/v thermoreversible hyaluronan-poly(N-isopropylacrylamide) hydrogel (HA-pNIPAM)2 or in a fibrinogen/thrombin solution.3 Each sample contained 50 µL hydrogel, 2.5 mg PU particles, and 2.5 × 105 cells. Controls included hMSC (or hIVD cells) seeded HA-pNIPAM, hMSC (or hIVD cells) seeded fibrin, and hMSC (or hIVD cells) seeded PU. After gelling for 1 hour at 37°C, samples were cultured in chondrogenic medium (DMEM high glucose, 1% ITS, 130 µM ascorbic acid, 100 nM dexamethasone and 10 ng/mL TGF-β1) for one week and analyzed for DNA (by Picogreen), sulfated glycosaminoglycan (GAG) (by 1,9 dimethylmethylene blue), mRNA expression, and histology (toluidine blue) at day 3 and 7. Three independent experiments were performed in triplicates for each group and cell type. After confirming the normality of data distribution and exclusion of the outliers, data were analyzed with a univariate general linear model and post hoc Bonferroni test (p < 0.05).
Results
In the MSC cultures, a slight DNA increase was observed between day 3 and 7 for both PU/HA-pNIPAM and PU/fibrin samples. At day 7, the highest amounts of DNA were found for the PU/fibrin group, followed by fibrin and PU groups (Fig. 1a). The amount of DNA was higher in the PU group than PU/HA-pNIPAM and HA-pNIPAM, suggesting a lower cell proliferation in the presence of the thermoreversible hydrogel. A similar DNA profile was obtained when hIVD cells were used instead of hMSCs. A significantly increased amount of GAG was observed in hMSC-seeded PU/HA-pNIPAM samples (Fig.1B) and there was an increase of GAG from day 3 to day 7. All other groups had a much lower amount of glycosaminoglycans and there was only a slight increase of GAG from day 3 to day 7. The GAG retention (GAG in sample divided by the sum of GAG in the sample and GAG released in the medium) at day 7 was comprised between 50% and 80% for all groups. Disk cells produced significantly less GAG in the PU/HA-pNIPAM system compared to hMSC, indicating that MSCs were more responsive to the combination of these two biomaterials (Fig. 1B). Moreover, the GAG retention at day 7 was lower with human IVD cells (20–60%) compared to hMSCs in all groups. This suggests that an early and higher GAG accumulation is associated to a better GAG retention in the hydrogel. This finding may be related to the fact that while MSCs were obtained from patients undergoing various types of surgeries, IVD cells were obtained from (degenerate) disks of patients undergoing total IVD replacement. The gene expression profile confirmed MSC chondrogenic differentiation with a strong upregulation of collagen type II and, to a lower extent, of aggrecan and SOX9 (compared to cells collected on the day of seeding) without significant differences between the two gels. Histological sections revealed cell migration from PU particles to the hydrogels.
Mean and standard deviation of DNA (a) and sulfated glycosaminoglycans (GAG) (b) in HA-pNIPAM and fibrin, with and without the addition of PU particles after 1 week of culture; * p < 0.05 relative to PU/Fibrin, o p < 0.05 relative to fibrin.
Conclusion
Human disk cells from degenerating disks synthesized less GAG than hMSCs, indicating that MSCs may represent a suitable cell type for the biological repair of IVDs. A synergic effect for MSC differentiation was observed when a thermoreversible hyaluronan-based hydrogel was combined with polyurethane particles.
Yes
None declared
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