Collagen II in Native Nucleus Pulposus (NP) Tissue May Suppress COL2A1 Expression in NP Cells

Abstract

Introduction Biomimetic scaffolds have been implicated in tissue engineering. They have been shown superior in inducing stem cell differentiation or promoting native cell phenotype. We have investigated the use of native tissue as biomimetic scaffold in nucleus pulposus (NP) cell engineering for regeneration. We hypothesized that the NP phenotype can be better maintained or enhanced when NP cells are cultured on a scaffold derived from native NP tissues. In this study, we aimed to prepare freeze dried bovine NP sections as the substrate for NP cell attachment. We compared NP marker expression of cells cultured on these sections with cells on cell culture plastic. Experiments to study the effect of cell density and the effect of extracellular matrix proteins were also performed to understand the observed phenomenon. Materials and Methods Bovine NP tissue was isolated from bovine tail. Immunostaining and RT-qPCR were used to validate the NP markers chosen in this study (CDH2, KRT18, and KRT19). For preparing freeze dried NP sections, the NP tissue was snap frozen in liquid nitrogen, followed by cryosectioning and overnight freeze drying. NP cells were seeded on the freeze dried NP tissue sections in 12 well plates and wells without tissue were used as controls (Fig. 1A). RT-qPCR was performed on cells cultured for 11 days. To understand the effect of cell plating density, NP cells were plated at different cell densities (50, 100, and 200 thousand cells per well) in 12 well plates in the absence of tissue sections with low glucose DMEM or high glucose DMEM. RT-qPCR of ACAN, COL2A1, CDH2, KRT18 and KRT19 was performed on cells cultured for 2 days and 5 days. To study the effect of proteins on NP cells, NP cells were cultured on cell culture plates coated with different proteins (fibronectin, collagen I, collagen II) with culture wells without protein coating as controls. The RT-qPCR of ACAN, COL1A1, COL2A1, and CDH2 was performed on cells cultured for 3 days. Cell proliferation was estimated by Alamar blue assays at day 1, day 3, and day 7 in different cell culture medium (DMEM with 1% FBS, 10% FBS or 1X ITS). Results CDH2, KRT18, and KRT19 expression was higher in NP cells than AF cells at both protein and mRNA levels. Opposite to what we hypothesized, the expression of COL2A1 and CDH2 of cells on NP sections was lower compared with cells on plastic (Fig. 1B). Even though a same number of cells was seeded in the wells of cell culture plates with and without the sections, there was lower initial cell density on the tissue sections as only a portion of cells attached to the tissue sections while the rest attached to the plastic underneath. Thus, we also assessed the effect of cell density on NP marker expression separately. When the cells were cultured on plastic surface alone, CDH2 expression decreased with increasing cell seeding densities (Fig. 1C). This may partially explain why the CDH2 levels for cocultured cells had higher CDH2 expression compared with cells on plastic without coculturing with tissue sections. Interestingly, collagen II is one of the major extracellular matrix of NP but COL2A1 expression was lower when NP cells were cultured on collagen II coated surface compared with plastic (Fig. 1D). The cell proliferation was lower when cultured on fibronectin coating and collagen I coating compared with cells on plastic without any protein coating. Fig. 1 (A) Schematic showing the set up of the tissue section culture experiment in this study; (B) relative mRNA levels of cells cultured for 11 days in the set up shown in (A); (C) relative CDH2 mRNA levels of cells cultured for 5 days at different cell plating densities and; (D) relative COL2A1 mRNA levels of cells cultured for 3 days on different extracellular matrix proteins (FN: fibronection, COL1: collagen I, COL2: collagen II, P: plastic without protein coating). Conclusion Contrary to what we hypothesized, some NP marker expression of cells on NP sections was suppressed compared with cells directly cultured on plastic. It was shown that difference in cell density may affect CDH2 expression. This study also revealed that the use of collagen I or collagen II as substrate may reduce the COL2A1 mRNA expression in NP cells. Disclosure of Interest None declared