Chemical and physical factors to regenerate the nucleus pulposus

Abstract

Intervertebral disc (IVD) degeneration leading to chronic low back pain is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs related to health care expenditure and work absenteeism, and improve quality of life. The IVD is the flexible structure between our vertebral bodies that allows for movement of our spinal column. Degeneration is thought to start in the nucleus pulposus (NP), the gelatinous core of the IVD. The osmotic value of a disc decreases during degeneration due to the characteristic loss of proteoglycans. To understand how the osmolality affects NP cells, they were cultured with medium of different osmolalities, adjusted with different osmolytes: NaCl, urea and sucrose. Gene expression for both the NP extracellular matrix (ECM) components type II collagen and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production of these ECM components however, was not affected by increasing osmolality and was even decreased in the presence of urea or sucrose. Hydrogels are employed as NP cell carriers and can facilitate regeneration, either for clinical application or research into mechanisms of regeneration. We compared six different hydrogels based on natural polymers: alginate, agarose, fibrin, type II collagen, gelatin methacryloyl (gelMA), and hyaluronic acid–poly(ethylene glycol), and investigated the role of serum in the medium and of osmolarity during expansion or redifferentiation of NP cells. Agarose hydrogels seem to be the best option for in vitro culture of human NP cells, but for clinical application, type II collagen hydrogels may be better because they are more biocompatible. Culture in serum-free medium, necessary for clinical application, reduced the amount of proteoglycans produced during redifferentiation culture. Isolation and expansion of NP cells in high osmolarity medium increased regeneration, also in the absence of serum. High osmolality during redifferentiation culture again had no effect. One way hydrogels can influence regeneration is by their biomechanical properties. Stiffness sensing through the cell’s focal adhesions is believed to direct chondrogenesis, but the mechanisms involved are largely unknown. We compared focal adhesion formation and proteoglycan deposition by NP cells in a range of hydrogels. Using a focal adhesion kinase (FAK) inhibitor, we demonstrated that focal adhesion signaling is involved in the response of NP cells to stiffness of hydrogels that contain integrin binding sites (i.e. gelMA and type II collagen), but not to hydrogels deplete from integrin binding sites such as alginate and agarose, or CD44-binding hydrogels based on hyaluronic acid. NP cells can also be stimulated to regenerate by addition of growth factors or mesenchymal stromal cells (MSCs). Bone morphogenetic proteins (BMPs) are among the most promising growth factors for NP regeneration. It is not known which BMP, heterodimer or combination of homodimers would perform best for NP regeneration. Our results indicate that BMP4 might have the highest potential for regeneration of the intervertebral disc and can be applied together with MSCs. Moreover, the added value of BMP heterodimers over their respective homodimer BMP combinations depends on the BMP combination applied and the cells used.