[Show abstract][Hide abstract] ABSTRACT: It has been shown that coculture of bone marrow-derived stromal cells (BMSCs) with intervertebral disc (IVD) nucleus pulposus (NP) cells significantly activates the biological characteristics of NP cells in animal models and in humans. We therefore predicted that activated NP cells would be a useful graft source for cellular transplantation therapy in the treatment of degenerative IVDs. However, the activation protocol is based on fresh isolation and activation of NP cells, which limits the timing of clinical application. Cell transplantation therapy could be offered to more patients than is now possible if activated NP cells could be transplanted as and when required by the condition of the patient. No study has investigated the effect of cryopreservation on NP cells after enzymatic isolation. We investigated the effects of cryopreservation of canine and human NP cells in both cell and tissue form before coculture with autologous BMSCs. Cell viability, proliferation, glycosaminoglycan production, aggrecan transcriptional activity, colony generation, and gene expression profile of the cells after cryopreservation and subsequent coculture were analyzed. The influence of cryopreservation on cell chromosomal abnormalities and tumorigenesis was also studied. The results showed that there were no clear differences between the noncryopreserved and cryopreserved cells in terms of cell viability, proliferation capacity, and capacity to synthesize extracellular matrix. Furthermore, the cells showed no apparent chromosomal abnormalities or tumorigenic ability and exhibited similar patterns of gene expression. These findings suggest that by using cryopreservation, it may be possible to transplant activated NP cells upon request for patients' needs.
[Show abstract][Hide abstract] ABSTRACT: Wnt/β-catenin (hereafter called Wnt) signaling is a key inducer and regulator of joint development, and is involved in the formation of bone and cartilage. We previously reported that Wnt signaling plays an essential role in the control of cell proliferation and cell senescence in intervertebral disc cells. In the present study, we provide evidence that the expression of c-myc, a key protein required for cell proliferation, is regulated by Wnt signaling. Our data also show that activation of Wnt signaling by LiCl, a Wnt signaling activator, leads to the suppression of c-myc promoter activity and expression. To ascertain whether Wnt signaling regulates the expression of c-myc, we measured both its transcript and protein expression. Following treatment with LiCl, c-myc expression was suppressed at both the mRNA and protein levels. In nucleus pulposus cells treated with c-myc, cell viability increased significantly, whereas treatment with a c-myc inhibitor decreased cell viability. Taken together, these results suggest that c-myc is an important factor that promotes the proliferation of nucleus pulposus cells. These findings provide new insight into the regulation and maintenance of cell proliferation in nucleus pulposus cells.
[Show abstract][Hide abstract] ABSTRACT: Degeneration of the lumbar intervertebral disc (IVD) is a cause of low back pain. In osteoarthritis patients, an increase in β-catenin accumulation has been reported. However, the molecular mechanisms involved in IVD remain unclear. In the present study, we examined the relationship of Wnt/β-catenin and transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signals in the IVDs. We found that treatment of nucleus pulposus (NP) cells with the Wnt/β-catenin activator lithium chloride (LiCl) results in the increased expression of β-catenin mRNA and protein, and cell proliferation is decreased due to the activation of the Wnt/β-catenin signals through the suppression of c-myc and cyclin-D1. In addition, T-cell-specific transcription factor (TCF) promoter activity was found to increase the following stimulation with LiCl alone, and was further increased when BMP2 was added, in comparison to the control group. We further observed the effects of treatment with PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway, on TCF promoter activity in NP cells. These effects were largely attenuated by PD98059. Moreover, when transfected IVDs were co-transfected with R-Smad expression plasmids, there was a significant decrease in TCF reporter activity. We thereafter evaluated the effects of increased Wnt/β-catenin activity on the transcriptional activity of the Smad binding element (SBE). As a result, LiCl suppressed the activity of SBE reporter activity. The present study demonstrates for the first time that there are opposing effects between the Wnt/β-catenin and TGF-β/BMP signals in IVDs, which is consistent with the Wnt/β-catenin signals contributing to the pathogenesis of IVD degeneration.