The BMP2-variant L51P Enhances the Osteogenic Differentiation of Human Mesenchymal Stromal Cells in the Presence of Intervertebral Disc Cells

Abstract

Introduction Discectomy and spinal fusion represents the gold standard treatment for spinal surgery to relieve pain. Fusion can be hindered, however, for yet unknown reasons that lead to non-fusions with pseudo-arthrosis. We previously showed that the intervertebral disc (IVD)-derived cells hinder the ossification process of human bone marrow-derived stromal cells (hMSC).1 Within this study, we hypothesized that BMP-antagonists secreted by IVD cells are the factors responsible for such inhibition and this can be reversed by addition of L51P. L51P is an engineered BMP2 variant that has been recently demonstrated to be a generic antagonist of a variety of BMP-inhibitors that control osteoinduction of bone.2,3 Material and Methods The experimental work was ethically approved and written consent of patients was obtained. hMSCs, primary nucleus pulposus (NPC) and annulus fibrosus cells (AFC) ( N = 6) were obtained from patients undergoing spinal surgery, isolated and expanded in monolayer cultures up to passage 3. IVD cells were seeded in 1.2% alginate beads (4M/mL) and separated by culture inserts from MSCs in a co-culture (CC)-set-up. The allogenic CCs were paired in 11 repeated experiments. MSCs were kept in 1: control medium (±alginate beads), 2: osteogenic medium+NPC (±100ng/mL L51P) and 3: osteogenic medium+AFC (±L51P) for 21 days. Relative gene expression of bone-related markers and of BMP antagonists such as noggin and members of the DAN (differential screening selected gene aberrative in neuroblastoma) family were quantified with qPCR, and histological staining for calcium deposition and Alkaline Phosphatase (ALP) assay were performed. The endogenous expression of the BMP-antagonists in IVD cells (passage 1) was evaluated by immunohistochemistry. Results Osteogenesis of MSCs was hindered as shown by reduced alizarin red staining in the presence of NPC and AFC. However, L51P added to CCs of MSCs with either NPC or AFC induced mineralization by blocking the activity of the IVD cell's secreted BMP-antagonists. It was noted that L51P caused a general reduction in ALP activity in all experimental groups. ALP activity was significantly up-regulated in positive control, CCNPC, and in CCAFC+L51P relative to negative controls, suggesting osteogenesis in these groups. CCNPC+L51P was significantly different from positive control but not CCNPC+L51P and CCAFC+L51P suggesting a reduction of the ALP activity. For the relative gene expression of potential BMP inhibitors (i.e., chordin, gremlin, noggin and TWSG-1), RNA and protein level using qPCR and immunohistochemistry confirmed expression of these BMP-antagonists inside the cells. Conclusion Alizarin red staining revealed an inhibition of the osteogenic differentiation of MSCs in CC with NPC or AFC. L51P could rescue osteogenesis of MSCs in the presence of NPC and AFC. Addition of L51P caused a general reduction in the ALP activity after 21 days of culture. L51P could thus be an attractive therapeutic treatment for spinal fusion, where it could enhance bone formation in the presence of NP and AF tissue. Acknowledgments This project was funded by two projects of the Swiss National Science Foundation grant number #IZK0Z3_154384 and #310030_153411. Eva Roth assisted in the biochemical assays and Hans-Jörg Sebald, provided the BMP2 variant L51P. References Chan SCW, Benneker LM, Heini P, Gantenbein B. An in vitro investigation into bone inhibition in non-unions caused by intervertebral disc cells. In: Proceedings of Biospine 5; Berlin, Germany, 2015 Albers CE, Hofstetter W, Sebald HJ, Sebald W, Siebenrock KA, Klenke FM. L51P - A BMP2 variant with osteoinductive activity via inhibition of Noggin. Bone 2012;51(3):401–406 Khattab HM, Ono M, Sonoyama W, et al. The BMP2 antagonist inhibitor L51P enhances the osteogenic potential of BMP2 by simultaneous and delayed synergism. Bone 2014;69:165–173