Abstract
BackgroundClinical 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.MethodsMonolayer 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.ResultsAlizarin 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.ConclusionsWe 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.
Highlights
Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification
Histology of calcium deposition mesenchymal stem cells (MSCs) grown in osteogenic medium had the greatest calcium deposition (Fig. 2a), which served as the positive control
NOG gene expression was significantly increased in osteogenic cultures compared with the negative control (p = 0.042, negative vs positive controls; p = 0.001, negative control vs MSC-NP cells (NPCs) cocultures; p >0.0001, negative control vs MSC-annulus fibrosus cells (AFCs) cocultures)
Summary
Clinical observations indicate that the presence of nucleus pulposus (NP) tissue during spinal fusion hinders the rate of disc ossification. IVD cells secrete bone morphogenetic protein (BMP)-binding proteins, such as NOG, gremlin (GREM1), and chordin (CHRD), to block BMP signaling that originates from the vertebral bodies to provide a BMP-reduced zone and prevent ossification of the disc [8, 9]. The expression of these BMP antagonists in IVD cells is important for normal formation of the disc and plays a role in its segmental pattern formation [7, 10]. There is limited information on the expression of the BMP antagonists in human IVDs
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