Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture

Abstract

Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p < 0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells. Keywords: bone morphogenetic protein 13; human intervertebral disc; low back pain; cell migration; intervertebral disc degeneration Chronic low back pain is strongly associated with intervertebral disc (IVD) degeneration. IVD consists of a central gelatinous nucleus pulposus (NP), surrounded circumferentially by annulus fibrosus (AF) and separated from the vertebral bodies by cartilaginous endplates (EPs). During IVD degeneration, cellular viability is decreased and the gel-like composition of NP becomes fibrous. Additionally, there is increasing irregularity and bifurcation of the lamellar structure of AF. 1Biological therapies aim to retard IVD degeneration and re-establish its biological function by restoring cellular viability and extracellular matrix (ECM). Growth factors such as bone morphogenetic proteins (BMPs), members of transforming growth factor beta (TGF-b) superfamily, play a critical role in stimulation of ECM formation and maintenance of cell numbers. The effect of BMPs on degenerate IVDs in vitro and in vivo has been extensively studied, 2 however, the role of BMP-13 in disc regeneration has not been explored. BMP-13, the human homologue of murine growth and differentiation factor 6, 3 has been highly associated with the stimulation of ECM production. BMP-13 induced aggrecan gene expression and proteoglycan (PG) synthesis in mouse mesenchymal cell line 4 and human articular chondrocytes.5 BMP-13 also stimulated gene expression of collagen II in mouse mesenchymal cell line 4 and collagen synthesis in juvenile bovine articular cartilage explants. 6 Moreover, Zhang et al.have demonstrated t...

Cartilage Derived Morphogenetic Protein-2 Induces Cell Migration and Its Chondrogenic Potential in C28/I2 Cells

“…These data are reinforced by other investigations in our laboratory, which have demonstrated similar patterns of GAG production in human IVD cells when cultured with CDMP-2. 32 This, conversely, validates our study, as the results derived from the cell line appear to resemble the effect CDMP-2 has on endogenous disc cells. Our results are further supported by previous reports of enhanced proteoglycan production in response to in vivo CDMP-2 injection into injured ovine IVD.…”

Therapeutic potential of growth differentiation factors in the treatment of degenerative disc diseases

“…Through adenoviral‐mediated overexpression studies, Zhang et al compared the effects overexpressing various BMP family molecules on ECM accumulation in bovine AF cells and demonstrated GDF6 overexpression induced one of the most significant PG and collagen production responses . In 3D alginate bead cultures of human NP and AF cells, GDF6 supplementation increased both PG and collagen production . Interestingly, at high concentrations (800 ng/mL) GDF6 was also shown to act as a potent chemoattractant for NP cells with similar responses observed to potent chemoattractants, such as fetal calf serum (FCS) .…”

“…This potential interaction of GDFs and proinflammatory molecules hints at the presence of a natural equilibrium that is dysregulated during degeneration, though this relationship appears to be complex. In vitro, GDF6 increases ECM production in degenerate NP cells, while in vivo GDF6 treatment prevented ECM degradation in a sheep model . GDF5 has been shown to directly inhibit the expression of MMP‐13 and ADAMTS4 in human chondrocytes and expression of other proinflammatory markers such as TNF‐α, IL‐1β, and prostaglandinE2 (PGE2) in murine NP cells .…”

Microparticles for controlled growth differentiation factor 6 delivery to direct adipose stem cell‐based nucleus pulposus regeneration

“…One growth factor recently proposed for NP regeneration is growth differentiation factor 6 (GDF6), a member of the bone morphogenetic protein (BMP) family that plays important developmental roles in the formation of cartilaginous tissues, including the IVD (Clendenning & Mortlock, 2012;Settle et al, 2003;Tassabehji et al, 2008;Wei et al, 2016). GDF6 expression has been linked to anti-ageing effects in multiple tissues (Hisamatsu, Ohno-Oishi, Nakamura, Mabuchi, & Naka-Kaneda, 2016) and, whereas studies conducted in our laboratory and others do not suggest GDF6 expression decreases with degeneration (Gulati, Chung, Wei, & Diwan, 2015; Le Maitre, Freemont, & Hoyland, 2009), preclinical studies have demonstrated that injection of GDF6 into experimentally induced rabbit and ovine annular puncture models attenuates pro-inflammatory molecular changes and prevents progression of IVD degeneration (Miyazaki et al, 2018;Shen et al, 2009). However, the hypocellular nature of the tissue and the aberrant phenotype of degenerate NP cells suggests that growth factor therapy alone may be insufficient to promote regeneration of human IVD.…”