Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells

Abstract

Background: Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. While the underlying mechanism remains unknown, this observation could be due to incomplete removal of NP cells (NPCs) that secrete factors preventing disc calcification, such as bone morphogenetic protein (BMP) antagonists including noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family.

Methods: Monolayer human bone marrow-derived mesenchymal stem cells (MSCs) were cocultured withNPCs and annulus fibrosus cells (AFCs) embedded in alginate for 21 days. At the end of coculture, MSCs were stained for mineral deposition by alizarin red, and relative expression of bone-related genes [Runt-related transcription factor 2, (RUNX2), Osteopontin (OPN), and Alkaline phosphatase (ALP)] and ALP activity were analyzed. Relative expression of three BMP antagonists, chordin (CHRD), gremlin (GREM1), and noggin (NOG), was determined in primary human NPCs and AFCs. These cells were also stained for Gremlin and Noggin by immunocytochemistry.

Results: Alizarin red staining showed that MSC osteogenesis in monolayer cultures was inhibited by coculture with NPCs or AFCs. ALP activity and RT-PCR analyses confirmed these results and demonstrated inhibition of osteogenesis of MSC in the presence of disc cells. NOG was significantly up-regulated in MSCs after coculture. Relative gene expression of intervertebral disc (IVD) cells showed higher expression of GREM1 in NPCs than in AFCs.

Conclusions: We show that primary IVD cells inhibit osteogenesis of MSCs. BMP inhibitors NOG, GREM1 and CHRD were expressed in IVD cells. GREM1 appears to be differentially expressed in NPCs and AFCs. Our results have implications for the design and development of treatments for non-union in spinal fusion.

Fig. 1

Design of coculture experiments with human bone marrow-derived mesenchymal stem cells (MSCs) and intervertebral disc cells without cell contact. Four experimental groups were cultured for 21 days. Controls were MSCs cultured in osteogenic medium (positive control) and MSCs cultured in growth medium (negative control). The experimental groups were cocultures of MSCs with either nucleus pulposus cells (NPCs) or annulus fibrosus cells (AFCs)

Fig. 2

Osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs) in coculture with nucleus pulposus cells (NPCs) and annulus fibrosus cells (AFCs). a Alizarin red staining. Macroscopic (top row) and microscopic images (bottom row, ×10 magnification) showed reduced alizarin staining of MSCs in coculture with NPCs or AFCs compared with the positive control. Scale bar 100 μm. b Alkaline phosphatase (ALP) activity of MSCs after 21 days (n = 5) showed an increase under all osteogenic conditions compared with the negative control. ALP activity was reduced in cocultures of MSCs with NPCs and similar levels were found between cocultures of MSCs with AFCs and the positive control. c Relative gene expression of osteogenic markers (ALP, RUNX2 and OPN) in MSCs in response to the presence of intervertebral disc (IVD) cells in alginate beads (n = 5). Cocultures with IVD cells showed reduced gene expression of these osteogenic markers compared with the positive control. Furthermore, a significant increase in gene expression of NOG was observed under the osteogenic condition: *p <0.05, **p <0.01, ***p <0.001, and ^# p <0.0001

Fig. 3

Expression of bone morphogenic protein antagonists Chordin, Gremlin, and Noggin in intervertebral disc (IVD) cells. a Immunocytochemistry of GREM1 and NOG in monolayers of passage 1 nucleus pulposus cells (NPCs) (a), annulus fibrosus cells (AFCs) (b) and (c) mixed human IVD cells. GREM1 and NOG were both expressed in NPCs, whereas minimal expression of NOG, but not GREM1, was found in AFCs. GREM1 and NOG were highly expressed in mixed cultures of NPCs and AFCs. d Quantification of BMP antagonist gene expression in NPCs relative to AFCs (n = 8 donors) at passage 1. NPC monolayer culture showed no differences in CHRD or NOG expression compared with NPCs and AFCs. GREM1 expression was 2-fold higher in NPCs than in AFCs. DAPI 4',6-diamidino-2-phenylindole