Biological Performance of Cell-Laden MethacrylatedGellan Gum Hydrogels

Joana Silva-Correia1,2, Mariana B. Oliveira1,2, João F. Mano1,2, Joaquim M. Oliveira1,2, Rui L. Reis1,2

Acknowledgments:

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.

INTRODUCTION

CONCLUSIONS

MATERIALS AND METHODS

RESULTS AND DISCUSSION

13B´s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal

2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal

Tissue-engineered hydrogels hold great potential as nucleus pulposus substitutes (NP), as they replicate the native 3Denvironment1. Ionic- (iGG-MA) and photo-crosslinked (phGG-MA) methacrylated gellan gum hydrogels have been proposed asbiomaterials for supporting nucleus pulposus (NP) regeneration and/or repair2,3. These modified gellan gum-based hydrogelspossess adequate properties for being used as NP substitutes in acellular and cellular tissue engineering strategies, due toits ability to support cell viability, adequate mechanical properties, non-cytotoxicity and non-angiogenic capacity2-4.

References:1. J. Silva-Correia, et al., Biotechnol Adv, 2013, 31(8):1514-1531.2. J. Silva-Correia, et al., J Tissue Eng Regen Med, 2011, 5(6):e97-e107.3. J. Silva-Correia, et al., WO2011/119059, Priority date: 105030 26032010 PT.4. J. Silva-Correia, et al., Tissue Eng Part A, 2012, 18(11-12):1203-1212.

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

In this study, the mechanical stability and biocompatibility of these hydrogels were evaluated in vitro, by culturing human intervertebraldisc (hIVD) cells obtained from herniated patients within both hydrogels, for 2 hours up to 21 days. The isolated cells were previouslycharacterized by flow cytometry regarding the expression of markers specific for: NP (CD24 and HIF-1), adipose-derived stem cells ormesenchymal stem cells (CD29, CD73, CD90, CD105) and hematopoietic and endothelial lineages (CD34, CD45). The mechanical propertieswere investigated under dynamic mechanical analysis (DMA) after specific times of culturing. In addition, a Live/dead assay was employedto evaluate cell viability.

I. HYDROGELS PREPARATION III. IN VITRO STUDY

Figure 2. Fluorescence microscopy images of hIVD cells encapsulatedwithin ionic- (iGG-MA) and photo-crosslinked (phGG-MA) gellan gum-MA hydrogels, after 3, 14 and 21 d of culturing (data from 1 and 7 d notshown; green color – Calcein AM – corresponds to live cells and redcolor – PI – to dead cells.

Figure 3. Dynamic mechanical analysis of acellular and cell-loaded ionic- (iGG-MA) and photo-crosslinked (phGG-MA) gellangum-MA hydrogel discs after culturing for 3, 14 and 21 d (datafrom 2 h, 1 and 7 d not shown), showing the storage (E’) modulusand loss factor (tan δ) measured in PBS at 37ºC.

Table 1. Storage modulus (E’) of acellular and cell-loaded ionic-(iGG-MA) and photo-crosslinked (phGG-MA) gellan gum-MAhydrogels obtained by DMA at 1 Hz after different periods ofculturing (* indicates p < 0.05).

Time

E’ (x 104), Pa

iGG-MA phGG-MA iGG-MA + hIVDcells

phGG-MA + hIVD cells

2 h 8.30 ± 1.14 8.78 ± 1.66 7.58 ± 1.41 7.33 ± 1.53

1 d 7.90 ± 0.50 7.21 ± 1.14 8.26 ± 0.47 8.69 ± 1.40

3 d 7.84 ± 1.23 8.45 ± 0.30 8.17 ± 1.04 7.93 ± 0.57

7 d 9.50 ± 0.37 7.53 ± 0.90 9.30 ± 0.64 7.11 ± 0.36

14 d 8.58 ± 0.91 8.46 ± 1.12 9.64 ± 1.86 8.81 ± 0.16

21 d 8.04 ± 2.06 8.58 ± 0.42 11.6 ± 2.16* 9.84 ± 0.03

II. CELL ISOLATION AND CHARACTERIZATION

Figure 1. Analysis of markers expression on hIVD cells after isolationfrom a 51-year old female patient. Generally, hIVD cell populations werepositive for CD29, CD73, CD90 and CD105 and negative forhematopoietic markers such as CD45 and CD34. Differences were foundin the expression of NP-specific markers between thepopulations/passages analyzed. Results are shown for the populationused in the subsequent assays, which had an expression of 13.59% and0.5% for CD24 or HIF-1alpha respectively.

Both hydrogels enabled cellular encapsulation,and a good distribution of cells and viabilityuntil 21 d of culturing. The incorporation ofcells seems to have a positive effect(statistically significant) on the mechanicalproperties of the hydrogels, observed by anincrease in E’ value (in relation to acellularhydrogels) after longer culturing times (i.e., 21d; Figure 3 and Table 1).