Stimulation of intervertebral disc cells in alginate bead culture with bone morphogenetic protein 2 and/or L51P

Abstract

INTRODUCTION: In clinics, Bone Morphogenetic Protein 2 (BMP2) was applied for the support during spinal fusion. Further BMP2 was tested in IVD models and showed potential for IVD regeneration. The aim of this study is the investigation of BMP2 and the BMP2 analogue, L51P, on different cell types of the human IVD in 3D alginate beads, particularly their plasticity to undergo bone formation. METHODS: Human nucleus pulposus (NPC), annulus fibrosus (AFC) and cartilaginous endplate cells (CEPC) were each encapsulated in 1.2% alginate at a density of 4 Mio/mL. NPC, AFC, and CEPC beads were then cultured in α-MEM or osteogenic medium (OM) supplemented with 10% FBS and 100 ng/mL BMP2 and/or L51P for 21 days. Medium supplemented with cytokines was refreshed twice per week. Beads were snap frozen with liquid nitrogen after 7 days for mRNA analysis of Aggrecan (ACAN), Collagen type1 (COL1), Collagen type 2 (COL2) and runt-related transcription factor 2 (RUNX2) qPCR. Further, beads were stained with Alcian Blue after 21 days. RESULTS & DISCUSSION: ACAN expression was the highest up-regulated in IVD cells stimulated with OM and 100 ng/mL BMP2 and L51P compared to the negative control (basal medium only) in NPC, AFC and CEPC (mean ± SEM NP: 18.95 ± 15.65). The same was true for COL2 expression (NP: 72.47 ± 62.95). COL1 remained unaffected (N=2). CONCLUSIONS: In this study, we showed the trend of an increase in ACAN and COL2 gene expression in stimulated cells. Interestingly the co-treatment of BMP2 and L51P showed a cumulative effect towards an increased ECM production.