Determination and comparison of specifics of nucleus pulposus cells of human intervertebral disc in alginate and chitosan-gelatin scaffolds

Abstract

Introduction: Low back pain is a major economical and social problem nowadays. Intervertebral disc herniation and central degeneration of disc are two major reasons of low back pain that occur because of structural impairment of disc. The intervertebral disc contains three parts as follows : Annulus fibrosus, transitional region, and nucleus pulposus, which forms the central nucleus of the disc. The reduction of cell count and extracellular matrix, especially in nucleus pulposus, causes disc degeneration. Different scaffolds (natural and synthetic) have been used for tissue repairing and regeneration of the intervertebral disc in tissue engineering. Most scaffolds have biodegradable and biocompatible characteristics and also prepare a fine condition for proliferation and migration of cells. In this study, proliferation of NP cells of human intervertebral disc compromised in Chitosan-gelatin scaffold with alginate scaffold was studied.

Materials and methods: NP cells derived from nucleus pulposus by collagenase enzymatic hydrolysis. They were derived from patients who undergoing open surgery for discectomy in the Isfahan Alzahra hospital. Chitosan was blended with gelatin and glutaraldehyde was used for cross linking the two polymers. Then, alginate scaffold was prepared. Cellular suspension with 1 × 10(5) transferred to each scaffold and cultured for 21 days. Cell viability and proliferation investigated by trypan blue and (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Scanning electron microscope (SEM) was used to assert the porosity and to survey structure of scaffold.

Results: MTT assay dem1onstrated that cell viability of third day had significant difference in contrast by first day in both scaffolds. Accordingly, there was a significant decreased in cellular viability from day 3 to 21. Results of the cell count showed a punctual elevation cell numbers for alginate scaffold but there was no similar result for chitosan-gelatin scaffold.

Conclusion: Alginate scaffold prepared a better condition for proliferation of NP cells in comparison with chitosan-gelatin scaffold. Results of this study suggest that alginate scaffold could be useful in in vivo studies and treatment.

Keywords: Alginate scaffold; NP cells; chitosan-gelatin scaffold; intervertebral disc; tissue enginiaring.

Figure 1

Light microscopic images of NP cells cultured on tissue culture dish. NP cells have polygonal (a) and fibroblastic morphology (b)(×60)

Figure 2

SEM Micrographs of porous chitosan-gelatin scaffold (a) surface (b) NP Cells grown on chitosan-gelatin scaffold for 1 days;note that the cells have spherical Morphology for 1 days in chitosan-gelatin scaffold (b) and alginate gel (e) and fobrobastic and elangated morphology of one NP cell for 3 days (d) cross-section of inner part (c)

Figure 3

Comparison of viability and proliferation of alginate and chitosan-gelatin scaffolds (^*: Significant difference between 3 and 14 days)

Figure 4

Comparison percent of alive NP cells in alginate and chitosan-gelatin Scaffolds. P<0.05