o-Vanillin Modulates Cell Phenotype and Extracellular Vesicles of Human Mesenchymal Stem Cells and Intervertebral Disc Cells.

Human mesenchymal stem cell (hMSC) and extracellular vesicle (EV) therapy is a promising treatment for discogenic low back pain (LBP). Although promising, major obstacles remain to be overcome. Cellular senescence reduces self-renewal and multipotent potentials, and the senescence-associated secretory phenotype creates an inflammatory environment negatively affecting tissue homeostasis. Reducing senescence could therefore improve regenerative approaches. Ortho-Vanillin (o-Vanillin) has senolytic activity and anti-inflammatory properties and could be a valuable supplement to MSC and EV therapy. Here, we used direct co-culture experiments to evaluate proteoglycan synthesis, inflammatory mediators, and senescent cells in the presence or absence of o-Vanillin. EV release and transfer between hMSCs and intervertebral disc cells (DCs) was examined, and the effect on hMSC differentiation and DC phenotype was evaluated in the presence and absence of o-Vanillin. This study demonstrates that o-Vanillin affects cell communication, enhances hMSC differentiation and improves DC phenotype. Co-cultures of DCs and hMSCs resulted in increased proteoglycan synthesis, a decreased number of senescent cells and decreased release of the cytokines IL6 and 8. Effects that were further enhanced by o-Vanillin. o-Vanillin profoundly increased EV release and/or uptake by hMSCs and DCs. DC markers were significantly upregulated in both cell types in response to conditioned media of o-Vanillin treated donor cells. Collectively, this study demonstrates that o-Vanillin affects hMSC and DC crosstalk and suggests that combining hMSCs and senolytic compounds may improve the outcome of cell supplementation and EV therapy for LBP.

Multiscale Regulation of the Intervertebral Disc: Achievements in Experimental, In Silico, and Regenerative Research.

Intervertebral disc (IVD) degeneration is a major risk factor of low back pain. It is defined by a progressive loss of the IVD structure and functionality, leading to severe impairments with restricted treatment options due to the highly demanding mechanical exposure of the IVD. Degenerative changes in the IVD usually increase with age but at an accelerated rate in some individuals. To understand the initiation and progression of this disease, it is crucial to identify key top-down and bottom-up regulations' processes, across the cell, tissue, and organ levels, in health and disease. Owing to unremitting investigation of experimental research, the comprehension of detailed cell signaling pathways and their effect on matrix turnover significantly rose. Likewise, in silico research substantially contributed to a holistic understanding of spatiotemporal effects and complex, multifactorial interactions within the IVD. Together with important achievements in the research of biomaterials, manifold promising approaches for regenerative treatment options were presented over the last years. This review provides an integrative analysis of the current knowledge about (1) the multiscale function and regulation of the IVD in health and disease, (2) the possible regenerative strategies, and (3) the in silico models that shall eventually support the development of advanced therapies.

Theoretical Explorations Generate New Hypotheses About the Role of the Cartilage Endplate in Early Intervertebral Disk Degeneration.

Altered cell nutrition in the intervertebral disk (IVD) is considered a main cause for disk degeneration (DD). The cartilage endplate (CEP) provides a major path for the diffusion of nutrients from the peripheral vasculature to the IVD nucleus pulposus (NP). In DD, sclerosis of the adjacent bony endplate is suggested to be responsible for decreased diffusion and disk cell nutrition. Yet, experimental evidence does not support this hypothesis. Hence, we evaluated how moderate CEP composition changes related to tissue degeneration can affect disk nutrition and cell viability. A novel composition-based permeability formulation was developed for the CEP, calibrated, validated, and used in a mechano-transport finite element IVD model. Fixed solute concentrations were applied at the outer surface of the annulus and the CEP, and three cycles of daily mechanical load were simulated. The CEP model indicated that CEP permeability increases with the degeneration/aging of the tissue, in accordance with recent measurements reported in the literature. Additionally, our results showed that CEP degeneration might be responsible for mechanical load-induced NP dehydration, which locally affects oxygen and lactate levels, and reduced glucose concentration by 16% in the NP-annulus transition zone. Remarkably, CEP degeneration was a condition sine-qua-non to provoke cell starvation and death, while simulating the effect of extracellular matrix depletion in DD. This theoretical study cast doubts about the paradigm that CEP calcification is needed to provoke cell starvation, and suggests an alternative path for DD whereby the early degradation of the CEP plays a key role.

The effect of capacitively coupled (CC) electrical stimulation on human disc nucleus pulposus cells and the relationship between CC and BMP-7.

PURPOSE: An in vitro study using human intervertebral disc nucleus pulposus cells to evaluate the effects of CC stimulation on disc-matrix macromolecule production. METHODS: Nucleus pulposus cells were cultured in alginate beads and treated with CC stimulation. The effect of BMP on CC stimulation of the cells was evaluated by applying a BMP blocker (noggin) or by applying additional BMP-7 to the culture. The mRNA levels of the disc extracellular matrix genes (collagen I, II, aggrecan) and BMPs were measured by real-time PCR. The protein levels of aggrecan, collagen II, and BMPs were determined by ELISAs and Western blots. Sulfated glycosaminoglycan (sGAG) content was assayed using the DMMB method. RESULTS: (1) CC stimulation upregulates the production of the disc-matrix macromolecular components: sGAG, aggrecan and collagen II; (2) CC stimulation increases the ratio of mRNA expression levels of collagen II to collagen I; (3) CC stimulation induces the expression of endogenous BMP-4 and BMP-7; (4) inhibition of BMP activity (using noggin) reduces CC-mediated upregulation of aggrecan and collagen II; (5) CC and BMP-7 act in synergy to increase the upregulation of disc-matrix macromolecules. CONCLUSION: CC stimulation upregulates the production of the intervertebral disc-matrix macromolecules aggrecan, collagen II, and sGAG by a mechanism involving BMPs. CC stimulation acts in synergy with BMP-7 to increase the upregulation of these disc-matrix macromolecules.

The matrikine N-acetylated proline-glycine-proline induces premature senescence of nucleus pulposus cells via CXCR1-dependent ROS accumulation and DNA damage and reinforces the destructive effect of these cells on homeostasis of intervertebral discs.

Intervertebral disc (IVD) cell senescence is a recognized mechanism of intervertebral disc degeneration (IDD). Elucidating the molecular mechanisms underlying disc cell senescence will contribute to understanding the pathogenesis of IDD. We previously reported that N-acetylated proline-glycine-proline (N-Ac-PGP), a matrikine, is involved in the process of IDD. However, its roles in IDD are not well understood. Here, using rat nucleus pulposus (NP) cells, we found that N-Ac-PGP induced premature senescence of NP cells by binding to CXCR1. N-Ac-PGP induced DNA damage and reactive oxygen species accumulation in NP cells, which resulted in activation of the p53-p21-Rb and p16-Rb pathways. Moreover, the RT2 profiler PCR array showed that N-Ac-PGP down-regulates the expression of antioxidant genes in NP cells, suggesting a decline in the antioxidants of NP cells. On the other hand, N-Ac-PGP up-regulated the expression of matrix catabolic genes and inflammatory genes in NP cells. Concomitantly, N-Ac-PGP reinforced the destructive effects of senescent NP cells on the homeostasis of the IVDs in vivo. Our study suggests that N-Ac-PGP plays critical roles in the pathogenesis of IDD through the induction of premature senescence of disc cells and via the activation of catabolic and inflammatory cascades in disc cells. N-Ac-PGP also deteriorates the redox environment of disc cells. Hence, N-Ac-PGP is a new potential therapeutic target for IDD.

Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways.

The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD.

Morphological similarities after compression trauma of bovine and human intervertebral discs: Do disc cells have a chance of surviving?

To study the behavior of bovine disc cells and changes in disc matrix following in vitro compression tests; to compare the findings to investigations on human intervertebral discs (IVD) after burst fracture of the cervical spine. Healthy IVDs (n = 21) from three bovine tails were studied at 6 and 12 h post-mortem, with 16 IVDs subjected to impact loading and five as unloaded controls. IVDs (n = 8) from patients with burst fractures were compared to the bovine compression group. Specimens were studied macroscopically, histologically, and ultrastructurally for healthy cells, balloon cells, and disc cell death (DCD). Annulus ruptures were seen in both post-trauma groups, with radial ruptures being present histologically in all loaded bovine discs. Balloon cells were found in some human IVDs and were induced in vitro in bovine loaded discs within a distinct range of absorbed energy. There was a positive correlation between DCD and absorbed energy in all compartments of bovine discs. Both species showed similar patterns of DCD in the different compartments. This study was able to show similarities between both species in cell morphologies and matrix damage. The survival of the disc after substantial compression trauma thus seems to remain highly questionable.

Glucocorticoid Mechanism of Inhibition of the Inflammatory Cells in Lumbar Intervertebral Disc Cells Stimulated by TNF-alpha Production of Nuclear Factor-kappaB / 대한정형외과연구학회지

PURPOSE: To analyze the action mechanism of NF-kappaB, IkappaB-alpha and effect of the Dexamethasone (DEXA) in mediating this inflammation, after stimulating cultured herniated intervertebral disc cells with TNF-alpha. MATERIALS AND METHODS: After cultured human intervertebral disc cells passaged three times, they were divided into four groups: A control group (A), DEXA treatment group (B), TNF-alpha treated group (C), TNF-alpha and DEXA were treated at the same time (D). IL-6 and IL-1beta gene expression were measured with semi-quantitative RT-PCR. Western blot analysis was performed to measure protein expression of IkappaB-alpha in the above groups for 10 minutes, 1 hour, 2 hours. In addition, in order to explain the mechanism of NF-kappaB nuclear binding for each group, the nuclear amount of NF-kappaB binding in the nucleus is measured by EMSA. RESULTS: In RT-PCR, expression of IL-6 and IL-1beta was greatest in group C, followed by group D, group A. IkappaB-alpha expression of the group treated with DEXA was not detected in Western blot results within 10 minutes. However, if stimulated by TNF-alpha, the DEXA was not inhibited of IkappaB-alpha concentration. After 1 hour and 2 hours, IkappaB-alpha levels were expressed by cells autonomously (autoregulatory induction). EMSA results expression levels in nuclear protein was maintained in accordance with protein expression. CONCLUSIONS: Our study shows that DEXA inhibits the production of mediators such as inflammatory IL-6 and IL-1beta, however, may not inhibit the transcription of NF-kappaB stimulated by TNF-alpha.

Mesenchymanl Stem Cell Based Intradiscal Gene Therapy: Therapeutic Implication in Degenerative Disc Disease / 대한척추외과학회지

STUDY DESIGN: In-vitro experiments using human mesenchymal stem cells (MSCs), intervertebral disc (IVD) cells and type 5 adenovirus/transforming growth factor-beta1 construct (Ad/TGF-beta1). OBJECTIVES: To determine the effect of MSC-based gene therapy for matrix regeneration of IVD cells. SUMMARY OF LITERATURE REVIEW: MSCs are known to be multipotent in tissue regeneration. In degeneration of IVD, cellular replacement with genetic modification other than that of IVD cells may prove an enhanced mechanism for the regeneration of MATERIALS AND METHODS: MSCs and IVD cells were cultured and an adenovirus construct containing TGF-beta1 cDNA (Ad/TGF-beta1) was also produced. In the first step, the MSCs were transduced with Ad/TGF-beta1, then mixed with IVD cells in various proportions and three dimensionally cultured. [methyl-(3)H]Thymidine and [(35)S]Sulfur incorporation for DNA and proteoglycan synthesis, respectively, were measured. RT-PCR was performed to assess the aggrecan and collagen types I and II mRNA RESULTS: Mixed cultures of MSC and IVD cells showed relatively similar amounts of newly synthesized proteoglycan compared with cultures of IVD cells only. In mixed cultures transduced with Ad/TGF-beta1, there were significant decreases in newly synthesized proteoglycan with increasing the proportions of MSCs, which was also found with the aggrecan and collagen type II mRNA expressions. However, the collagen type I mRNA expression increased with increased proportions of MSCs transduced with Ad/TGF-beta1. CONCLUSION: Cell therapy with MSCs and IVD cells provided a mechanism for cellular augmentation. However, MSC-based gene therapy coupled with IVD cells did not maintain a chondrogenic phenotype.

Double Plate Occipitocervical Fusion After Failed Posterior Fusion C 1-2 With Wiring / 대한척추외과학회지

A 31-year-old female complained of neck pain and limitation in neck motion. She had a 3 month history of treatment with Halovest at another hospital for a fracture of the odontoid process due to a car accident. The patient complained of persistent pain and limitation in neck motion following the cessation of Halovest. A dynamic radiograph demonstrated instability on C1-2 and she underwent a posterior cervical fusion with wiring. A wound infection developed, and loosening of the wire and lysis of the posterior arch at C1-2 were seen on a follow up plain radiograph 2 months postoperatively. She was transferred to our hospital where she underwent occipitocervical fusion with a double plate after control of the infection. There were rigid fixations of the plate and bone union on a follow up radiograph 24 months postoperatively.

Molecular Biological and Pathological Aspects of Intercostal Muscles and Intervertebral Discs in Adolescent Idiopathic Scoliosis in Korea / 대한척추외과학회지

STUDY DESIGN: A molecular biological study of intercostal muscles and intervertebral disc cells of Korean scoliosis patients. OBJECTIVES: To study the pathological results of intercostal muscles and molecular biological activity of intervertebral disc cells of the scoliotic major curve in Korean patients. SUMMARY OF LITERATURE REVIEW : The cause of idiopathic scoliosis has been investigated in terms of many parameters. Although, molecular biological studies of intercostal muscles and intervertebral disc cells have been performed in foreign countries, few studies have been conducted in Korea. MATERIALS AND METHODS: Ten patients, one male and nine female, who underwent thoracoscopic surgery were reviewed. The age range was 13 to 23 years old. Intercostal muscles were taken from the portal site of the major curve (1x1 cm sized). Ten tissues were stained with H/E and ATPase immunohistochemical staining. An appropriate amount of intervertebral disc was taken from the major curve of three scoliotic patients and each concentration of collagen type I, II, GAG gene and proteoglycan synthesis activity was measured. The results were compared with those of grade 0 and grade II degenerative change on each MRI. RESULTS: The intercostal muscle of scoliotic patients showed 60.4+/-8.4% in type I muscle fiber and 39.6+/-8.8% in type II-A. These results were not different from those of previous studies. The size of muscle fiber was 48-65 microns, which was slightly smaller than the absolute value, but the difference was not statistically significant. The amount of produced proteoglycans was slightly higher in the intervertebral disc cells of scoliotic patients, the total amount of collagen was significantly lower and there was a difference in the production of type II collagen. CONCLUSIONS: The intercostal muscles were not affected by the muscle of scoliotic patients and there was no molecular biological significant difference between control and scoliotic patients. We can assume that scoliosis was not caused by problems of intervertebral disc or intercostal muscles.

Change of TypeI and TypeII Collagen Biosynthesis by Growth Factors in Cultured Cells Isolated from Rabbit Intervertebral Disc / 대한정형외과학회잡지

Growth factors influencing the function of chondrocytes are insulin-like growth factor I(IGF-I), basic fibroblast growth factor(bFGF), transforming growth factor-beta1(TGF-beta1), and epidermal growth factor(EGF). To find out the role of four kinds of growth factors in the biosynthesis of type I and II collagen represented as the phenotype of the disc cells, we cultured the disc cells isolated from rabbit intervertebral discs primarily and then checked cell proliferation, the expression of type I and II procollagen mRNA, and the immunohistochemical stains with type I and II collagen antibodies during in vitro culture in the maintenance medium containing low serum concentration with adding four kinds of growth factors. The results are as follows. FBS(10% Fetal bovine serum) group showed the highest cell proliferation potential. EGF and TGF groups showed remarkable cell proliferation, but there was no significant difference in IGF and FGF groups comparing to control group. A partial clone that encodes the rabbit type II procollagen C-propeptide region(RbCo12A1) was successfully isolated by reverse transcription-polymerase chain reaction using total RNA extracted from articular chondrocytes of rabbits. The identity of the cDNA clone was confirmed by DNA sequencing of the polymerase chain reaction products. A comparison of human al(II) cDNA sequence showed high sequence homology(83.6%). Type I procollagen mRNA expressed highly in EGF group. FGF, IGF, and TGF groups showed no significant expression comparing to control group. FBS group showed lower expression than control group. Type II procollagen expression was increased with passage of time, so at Day 10 it was the highest in all groups. Control group showed the highest expression among 6 experimental groups. The expression of type II procollagen in FGF and TGF groups was slightly lower than that of control. EGF and IGF groups showed markedly decreased expression comparing to control group. That in FBS group was the lowest, so it was three times lower than control group. In immunohistochemical stains with type I collagen, there was no difference among control, FBS, and EGF groups. FGF, IGF, and TGF groups showed increased positivity on stain comparing to control group, but the positivity didnt exceed 10%. For type II collagen, EGF and FGF groups showed decreased positivity, but there was no significant difference in FBS, IGF, and TGF groups comparing to control group. On the basis of this study, it may be concluded that TGF-pl showed the possibility of regeneration or delay the degeneration process of the intervertebral disc through the contribution to the stimulatory effects of cell proliferation and the synthesis of type II collagen. For the clinical use of this, more studies about the combination effects with FBS or other kinds of growth factors and finding out the ideal concentration about TGF-pl will be needed.