INTRODUCTION

Tissue-engineered hydrogels hold great potential as nucleus pulposus substitutes (NP) since it can mimic the native 3D environment¹. Methacrylated gellan gum (GG-MA) hydrogels have been reported as suitable matrices for NP regeneration², due to their remarkable biological and biomechanical properties. In this study, the biological performance and mechanical properties of cell-laden GG-MA hydrogels were evaluated in vitro.

EXPERIMENTAL METHODS

Human intervertebral disc (hIVD) cells were isolated from herniated patients submitted to surgery, following an enzymatic digestion-based method and expanded using standard culture conditions. Prior to cell encapsulation, hIVD cells were characterized by flow cytometry regarding the expression of markers specific for: NP (CD24 and HIF-1), adipose-derived stem cells or mesenchymal stem cells (CD29, CD73, CD90, CD105) and hematopoietic and endothelial lineages (CD34, CD45). The hIVD cells were then encapsulated at a cell density of 2x10⁶ cells/mL and cultured within ionic- (iGG-MA) and photo-crosslinked (phGG-MA) hydrogel discs, up to 21 d. Cell behaviour was studied in terms of viability (Live/dead assay) and morphology (DAPI/Texas red-Phalloidin staining), production of a cartilage-like tissue by histological staining and expression of typical chondrogenic markers and specific NP-markers through immunocytochemistry from 2 h until 21 d of culturing. The cell-laden constructs were also analyzed by dynamic mechanical analysis (DMA) after each culturing time.

RESULTS AND DISCUSSION

Different populations of hIVD cells, which were isolated from male/female patients (25-57 years) at different passages, were characterized by flow cytometry. Generally, hIVD cell populations were positive for CD29, CD73, CD90 and CD105 and negative for hematopoietic markers such as CD45 and CD34. Differences were found in the expression of NP-specific markers between the populations/passages analyzed. A population obtained from a 51-year old female patient, with an expression of 13% and 0.5% for CD24 or HIF-1alpha respectively, was selected for the subsequent assays.

Cell viability was evaluated in both cell-laden iGG-MA and phGG-MA constructs (Figure 1). It is possible to conclude from the microscopy images that both hydrogels enable cellular encapsulation, allowing for a good distribution of cells and support cell viability until 21 d of culturing. The effect of encapsulating hIVD cells on the mechanical performance of the hydrogels was evaluated by DMA. The incorporation of cells seems to have an immediate negative effect (not statistically significant) on the mechanical properties of the hydrogels, which is recovered after 1 d of culturing (data not shown). An increase in E’ value (in relation to acellular hydrogels) was observed only after longer culturing times (i.e., 21 d; Figure 2), which is consistent with the viability and production of extracellular matrix by hIVD cells, as demonstrated by the evaluation of biological performance.

CONCLUSION

This study showed that the iGG-MA and phGG-MA hydrogels are stable and non-cytotoxic in vitro and present adequate mechanical properties. The hydrogels supported hIVD cells encapsulation and viability, thus possessing promising properties for being tested in cellular-based tissue engineering strategies aimed to restore the functionality of NP.

REFERENCES

1. Silva-Correia J., et al., Biotechnol. Adv. 31:1514-1531, 2013

2. Silva-Correia J., et al., J. Tissue Eng. Regen. Med. 5:e97-e107, 2011

ACKNOWLEDGMENTS

The research leading to these results has received funding from the EU FP7/2007-2013 under grant agreement n° REGPOT-CT2012-316331-POLARIS and grant agreement n° NMP3-LA-2008-213904-DISC REGENERATION.