Bone Morphogenetic Proteins for Nucleus Pulposus Regeneration.

Matrix production by nucleus pulposus (NP) cells, the cells residing in the center of the intervertebral disc, can be stimulated by growth factors. Bone morphogenetic proteins (BMPs) hold great promise. Although BMP2 and BMP7 have been used most frequently, other BMPs have also shown potential for NP regeneration. Heterodimers may be more potent than single homodimers, but it is not known whether combinations of homodimers would perform equally well. In this study, we compared BMP2, BMP4, BMP6, and BMP7, their combinations and heterodimers, for regeneration by human NP cells. The BMPs investigated induced variable matrix deposition by NP cells. BMP4 was the most potent, both in the final neotissue glysosaminoglycan content and incorporation efficiency. Heterodimers BMP2/6H and BMP2/7H were more potent than their respective homodimer combinations, but not the BMP4/7H heterodimer. The current results indicate that BMP4 might have a high potential for regeneration of the intervertebral disc. Moreover, the added value of BMP heterodimers over their respective homodimer BMP combinations depends on the BMP combination applied.

Association between Oncostatin M Expression and Inflammatory Phenotype in Experimental Arthritis Models and Osteoarthritis Patients.

Pro-inflammatory cytokines are considered to play a major role in osteoarthritis (OA), yet so far, the specific cytokines involved in the pathology of OA have not been identified. Oncostatin M (OSM) is a cytokine from the interleukin 6 (IL-6) family that has been shown to be elevated in synovial fluid of most rheumatoid arthritis (RA) patients, but only in a limited subset of OA patients. Little is known about OSM in the different joint tissues during OA and how its expression correlates with hallmarks of disease. Here, we mapped OSM expression in the joint tissues of two rat models of arthritis: an acute inflammatory model and an instability-induced osteoarthritic model. OSM expression was correlated with hallmarks of OA, namely cartilage damage, synovitis, and osteophyte formation. Reanalysis of an existing dataset on cytokine profiling of OA synovial fluid was performed to assess pattern differences between patients positive and negative for OSM. In the inflammatory model, OSM expression correlated with synovitis and osteophyte formation but not with cartilage damage. On the contrary, in the instability model of OA, an increase in synovitis, cartilage damage, and osteophyte formation was observed without changes in OSM expression. In line with these findings, synovial fluid of OA patients with detectable OSM contained higher levels of other inflammatory cytokines, namely interferon gamma (IFN-γ), IL-1α and tumor necrosis factor alpha (TNF-α), likely indicating a more inflammatory state. Taken together these data indicate OSM might play a prominent role in inflammatory phenotypes of OA.

No Effects of Hyperosmolar Culture Medium on Tissue Regeneration by Human Degenerated Nucleus Pulposus Cells Despite Upregulation Extracellular Matrix Genes.

STUDY DESIGN: An in vitro study using human degenerated nucleus pulposus cells. OBJECTIVE: To determine the effect of osmolality and different osmolytes on the regeneration by human nucleus pulposus cells through gene expression and extracellular matrix production. SUMMARY OF BACKGROUND DATA: Intervertebral disc (IVD) degeneration is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs due to diminished work absence and health care, and improve quality of life. The osmotic value of a disc decreases during degeneration due to loss of proteoglycans and might increase degeneration. It is known that gene expression of matrix genes of nucleus pulposus (NP) cells increases when cultured in hyperosmotic medium. Thus, increasing the osmolality of the disc might be beneficial for disc regeneration. METHODS: In the current study, isolated degenerated human NP cells were used in regeneration culture with medium of different osmolalities, adjusted with different osmolytes. NaCl, urea and sucrose. The cells were cultured for 28 days and expression of matrix genes and production of glycosaminoglycans and collagen II were measured. RESULTS: Gene expression for both collagen II and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production however, was not affected by increasing osmolality and was decreased when using urea and sucrose. Expression of genes for Col1A1, MMP13, and MMP14 decreased with increasing osmolality, whereas expression of LOXL2 and LOXL3 increased. Transient expression of TonEBP was found 6 hours after the start of culture, but not at later time points. CONCLUSION: Although expression of matrix genes is upregulated, hyperosmolality does not enhance matrix production by nucleus pulposus cells. Raising osmolality can potentially increase matrix production, but in itself is not sufficient to accomplish regeneration in the current in vitro culture system. LEVEL OF EVIDENCE: N /A.

Comparing Hydrogels for Human Nucleus Pulposus Regeneration: Role of Osmolarity During Expansion.

Hydrogels can facilitate nucleus pulposus (NP) regeneration, either for clinical application or research into mechanisms of regeneration. However, many different hydrogels and culture conditions for human degenerated NP have been employed, making literature data difficult to compare. Therefore, we compared six different hydrogels of natural polymers and investigated the role of serum in the medium and of osmolarity during expansion or redifferentiation in an attempt to provide comparators for future studies. Human NP cells of Thompson grade III discs were cultured in alginate, agarose, fibrin, type II collagen, gelatin methacryloyl (gelMA), and hyaluronic acid-poly(ethylene glycol) hydrogels. Medium containing fetal bovine serum and a serum-free (SF) medium were compared in agarose, gelMA, and type II collagen hydrogels. Isolation and expansion of NP cells in low compared to high osmolarity medium were performed before culture in agarose and type II collagen hydrogels in media of varying osmolarity. NP cells in agarose produced the highest amounts of proteoglycans, followed by cells in type II collagen hydrogels. The absence of serum reduced the total amount of proteoglycans produced by the cells, although incorporation efficiency was higher in type II collagen hydrogels in the absence than in the presence of serum. Isolation and expansion of NP cells in high osmolarity medium improved proteoglycan production during culture in hydrogels, but variation in osmolarity during redifferentiation did not have any effect. 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, as opposed to agarose, it degrades in time. Although culture in SF medium reduces the amount of proteoglycans produced during redifferentiation culture, isolating and expanding the cells in high osmolarity medium can largely compensate for this loss.

Focal adhesion signaling affects regeneration by human nucleus pulposus cells in collagen- but not carbohydrate-based hydrogels.

Hydrogel-based 3D cell cultures are an emerging strategy for the regeneration of cartilage. In an attempt to regenerate dysfunctional intervertebral discs, nucleus pulposus (NP) cells can be cultured in hydrogels of various kinds and physical properties. Stiffness sensing through focal adhesions is believed to direct chondrogenesis, but the mechanisms by which this works are largely unknown. In this study we compared focal adhesion formation and glycosaminoglycan (GAG) 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 in hydrogels that contain integrin binding sites (i.e. methacrylated gelatin (gelMA) and type II collagen), but not in hydrogels deplete from integrin binding sites such as alginate and agarose, or CD44-binding hydrogels based on hyaluronic acid. As a result of FAK inhibition we observedenhanced proteoglycan production in gelMA, but decreased production in type II collagen hydrogels, which could be explained by alteration in cell fate as supported by the increase in the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARy). Furthermore, GAG deposition was inversely proportional to polymer concentration in integrin-binding gelMA, while no direct relationship was found for the non-integrin binding gels alginate and agarose. This corroborates our finding that focal adhesion formation plays an important role in NP cell response to its surrounding matrix. STATEMENT OF SIGNIFICANCE: Biomaterials are increasingly being investigated for regenerative medicine applications, including regeneration of the nucleus pulposus. Cells interact with their environment and are influenced by extracellular matrix or polymer properties. Insight in these interactions can improve regeneration and helps to understand degeneration processes. The role of focal adhesion formation in the regenerative response of nucleus pulposus cells is largely unknown. Therefore, the relation between materials, stiffness and focal adhesion formation is studied here.