Intervertebral Disc Cells Research Articles

Intervertebral disc and endplate cell characterisation highlights annulus fibrosus cells as the most promising for tissue-specific disc degeneration therapy.

Degenerative processes of the intervertebral disc (IVD) and cartilaginous endplate lead to chronic spine pathologies. Several studies speculated on the intrinsic regenerative capacity of degenerated IVD related to the presence of local mesenchymal progenitors. However, a complete characterisation of the resident IVD cell populations, particularly that isolated from the endplate, is lacking. The purpose of the present study was to characterise the gene expression profiles of human nucleus pulposus (NPCs), annulus fibrosus (AFCs) and endplate (EPCs) cells, setting the basis for future studies aimed at identifying the most promising cells for regenerative purposes. Cells isolated from NP, AF and EP were analysed after in vitro expansion for their stemness ability, immunophenotype and gene profiles by large-scale microarray analysis. The three cell populations shared a similar clonogenic, adipogenic and osteogenic potential, as well as an immunophenotype with a pattern resembling that of mesenchymal stem cells. NPCs maintained the greatest chondrogenic potential and shared with EPCs the loss of proliferation capability during expansion. The largest number of selectively highly expressed stemness, chondrogenic/tissue-specific and surface genes was found in AFCs, thus representing the most promising source of tissue-specific expanded cells for the treatment of IVD degeneration.

Expression of Glial Cell Line-derived Neurotrophic Factor in the Human Intervertebral Disc.

Biochemical and immunohistochemical analyses by the human intervertebral disc (IVD) cells and tissues. To examine the expression of glial cell line-derived neurotrophic factor (GDNF) and its receptors, GDNF family receptor (GFR) α1 and rearranged during transfection (RET) in the human IVD cells and the tissues with the early and advanced stages of degeneration. The neurotrophin family, including nerve growth factor, has been reported to be expressed in the IVDs and plays a role in hyperalgesia and neuronal sensitization. Despite having properties similar to the nerve growth factor, the expression of GDNF in the IVD remains unknown. Human IVD cells were cultured in monolayer. Immunohistochemical analyses and western blotting were performed to examine the protein levels of GDNF and its receptors. To examine the effect of proinflammatory cytokines, cells were cultured in the presence of interleukin-1β (IL-1β). The immunohistochemical expression of these proteins was also evaluated using human IVD tissues with different stages of degeneration. Immunofluorescent reactivity against anti-GDNF, GFRα1, and RET antibodies was identified in human IVD cells. In protein extracts from IVD cells, those protein expressions were also identified by Western blot. IL-1β significantly stimulated the mRNA expression of GDNF compared with that of the control group. There was no significant effect of IL-1β on the mRNA expression of GFRα1 and RET. The percentage of GDNF-immunopositive cells in advanced degenerated discs was significantly higher than that in early degenerated discs, whereas those of GFRα1 and RET showed no significant differences. GDNF and its receptors were constitutively expressed in the human IVD cells. GDNF expression was significantly enhanced by proinflammatory stimuli, and in the microenvironment with advanced tissue degeneration. N/A.

Effects of IL-1β, IL-20, and BMP-2 on Intervertebral Disc Inflammation under Hypoxia.

Intervertebral disc (IVD) is an avascular tissue under hypoxic condition after adulthood. Our previous data showed that inflammatory cytokines (interleukin (IL)-1β), IL-20, and bone morphogenetic protein-2 (BMP-2) play important roles in the healing process after disc injury. In the current study, we investigated whether IL-1β, IL-20, or BMP-2 modulate the expression of pro-inflammatory cytokines, chemotaxis factor, and angiogenesis factor on IVD cells under hypoxia. IVD cells were isolated from patients with intervertebral disc herniation (HIVD) at the levels of L4–5 and L5–S1. We found that the expression of IL-1β, IL-20, BMP-2, hypoxia-inducible factor (HIF)-1α, IL-6, IL-8, angiogenetic factor (vascular endothelial growth factor (VEGF)), chemotactic factor (monocyte chemoattractant protein 1 (MCP-1)), and matrix metalloproteinase-3 (MMP-3) was upregulated in IVD cells under hypoxia conditions. In addition, IL-1β upregulated the expression of pro-inflammatory cytokines (IL-6 and IL-8), VEGF, MCP-1, and disc degradation factor (MMP-3) in IVD cells under hypoxia conditions. IL-20 upregulated MCP-1 and VEGF expression. BMP-2 also upregulated the expression of MCP-1, VEGF, and IL-8 in IVD cells under hypoxia conditions. Treatment with antibody against IL-1β decreased VEGF and MMP-3 expression, while treatment with IL-20 or BMP-2 antibodies decreased MCP-1, VEGF, and MMP-3 expression. Moreover, IL-1β modulated both the expression of IL-20 and BMP-2, but IL-20 only modulated BMP-2 either under a hypoxic or normoxic condition. Therefore, we concluded that the inflammation, chemotaxis, matrix degradation, and angiogenesis after disc herniation are influenced by the hypoxic condition and controlled by IL-1β, IL-20, and BMP-2.

Quantifying Biofield Therapy through Biophoton Emission in a Cellular Model

Biofield therapy has shown positive results over a broad range of pathology from preclinical research to human studies. However, biofield therapy investigation is limited by an inability to quantify the therapeutic effect. This study aimed to measure the effects Reiki had on mice intervertebral disc (IVD) cells compared with sham and to quantify Reiki by measuring photon emission. We treated mice IVD cells with ten-minute sessions of either Reiki or sham on three successive days. During treatment, we placed the cells in a specifically constructed box with an installed photomultiplier tube (PMT). Reiki significantly increased the photon emission of the cells post-treatment compared with Reiki pre-treatment and sham (p < 0.05). Real time PCR (RT PCR) showed an increase in collagen II and aggrecan (p < 0.05). We present a means to quantify biofield therapy by measuring the post-treatment photon emission. We concurrently demonstrate Reiki’s effect on the anabolic healing response.

Aging aggravates intervertebral disc degeneration by regulating transcription factors toward chondrogenesis.

Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.

Leptin receptor–expressing cells represent a distinct subpopulation of notochord-derived cells and are essential for disc homoeostasis

Background/objectiveIntervertebral disc degeneration (IDD) remains to be an intractable clinical challenge. Although IDD is characterised by loss of notochordal cells (NCs) and dysfunction of nucleus pulposus (NP) cells, little is known about the origin, heterogeneity, fate and maintenance of NCs and NP cells, which further stunts the therapeutic development. Thus, effective tools to spatially and temporally trace specific cell lineage and clarify cell functions in intervertebral disc (IVD) development and homoeostasis are urgently required. MethodsIn this study, NP specimens were obtained from 20 patients with degenerative disc disease or scoliosis. LepR-Cre mice was crossed with R26R-Tdtomato mice to generate LepR-Cre; R26R-Tdtomato mice, which enabled fate-mapping of NPs from embryo stage to late adult. LMNA G609G/G609G mice was used to determine the effect of premature-aging induced IDD on LepR NPs. X-ray imaging was used to measure lumber disc height of mice. ResultsHere, we provide the first evidence that the leptin receptor (LepR) is preferentially expressed in NCs at embryonic stages and notochord-derived cells in the postnatal IVD. By using R26R-Tdtomato fluorescent reporter mice, we systematically analysed the specificity of activity and targeting efficiency of leptin receptor-Cre (LepR-Cre) in IVD tissues from the embryonic stage E15.5 to 6-month-old LepR-Cre; Rosa26-Tdtomato (R26R-Tdtomato) mice. Specifically, LepR-Cre targets a distinct subpopulation of notochord-derived cells closely associated with disc homoeostasis. The percentage of LepR-expressing NP cells markedly decreases in the postnatal mouse IVD and, more importantly, in the human IVD with the progression of IDD. Moreover, both spine instability–induced and premature ageing–induced IDD mouse models display the phenotype of IDD with decreased percentage of LepR-expressing NP cells. These findings uncover a potential role of LepR-expressing notochord-derived cells in disc homoeostasis and open the gate for therapeutically targeting the NP cell subpopulation. ConclusionIn conclusion, our data prove LepR-Cre mice useful for mapping the fate of specific subpopulations of IVD cells and uncovering the underlying mechanisms of IDD. The translational potential of this articleThe translation potential of article is that we first identified LepR as a candidate marker of subpopulation of nucleus pulposus (NP) cells and provided LepR as a potential target for the treatment of intervertebral disc degeneration (IDD), which have certain profound significance.

Degenerative disc disease (DDD) in 2017. Possible mechanism of action of ozone [abstract

PURPOSE: Show light on the biological mechanism of ozone in DDD.&#x0D; The increase in life expectancy in the population brings to the increase of diseases associated with aging: cancer, heart disease, diabetes and low back pain. DDD and osteoarthritis are the most common causes of chronic joint-related disability and disabling pain in adults. Aging of the organism is the result of accumulation of molecular and cellular alterations over time, leading to a physiological and functional decrease of tissues.&#x0D; &#x0D; ANALYSIS AND DISCUSSION: In the Intervertebral discs (IVDs), aging can be schematized in 3 phases: accumulation of damaged bio-molecules, aberrant cellular response to the lesion and loss of structure and biological function. This perspective focuses on considering that the aging mechanisms are senescence, inflammation and oxidative damage.&#x0D; The IVDs acquire fissures and neo-vascularisation, this causes that the resident cells that are accustomed to the low voltage of oxygen, are exposed to a greater tension of oxygen and therefore to the oxidative stress.&#x0D; During aging, IVD cells change from the anabolic to catabolic phenotype. There is increased necrosis, apoptosis, and cell senescence.&#x0D; The NF-kB signal is central in the inflammatory response, stress and injury. There is a chronic activation of NF-kb correlated with oxidative stress and degeneration.&#x0D; Symptomatic discs have high levels of pro-inflammatory cytokines that are considered targets of NF-kB.&#x0D; &#x0D; CONCLUSION: For many years attempts have been made to inhibit the action of NF-kb and other inflammatory targets to mitigate DDD without favourable results. However, ozone is the solution to this problem because it interferes precisely at the level of NF-kB avoiding its chronic activation.&#x0D; That is why, in DDD there are sustainable improvements on patients, despite the fact that the MRI images show no significant changes.

Moracin attenuates LPS-induced inflammation in nucleus pulposus cells via Nrf2/HO-1 and NF-κB/TGF-β pathway.

The present study was designed to investigate the protective effect of moracin on primary culture of nucleus pulposus cells in intervertebral disc and explore the underlying mechanism. Moracin treatment significantly inhibited the LPS-induced inflammatory cytokine accumulation (IL-1β, IL-6 and TNF-α) in nucleus pulposus cells. And moracin also dramatically decreased MDA activity, and increased the levels of SOD and CAT induced by LPS challenge. Moreover, the expressions of Nrf-2 and HO-1 were decreased and the protein levels of p-NF-κBp65, p-IκBα, p-smad-3 and TGF-β were increased by LPS challenge, which were significantly reversed after moracin treatments. Moracin treatments also decreased the levels of matrix degradation enzymes (MMP-3, MMP-13) as indicated by RT-PCR analysis. However, Nrf-2 knockdown abolished these protective effects of moracin. Together, our results demonstrated the ability of moracin to antagonize LPS-mediated inflammation in primary culture of nucleus pulposus in intervertebral disc by partly regulating the Nrf2/HO-1 and NF-κB/TGF-β pathway in nucleus pulposus cells.

Pulsed electromagnetic fields reduce acute inflammation in the injured rat-tail intervertebral disc.

Pro‐inflammatory cytokines are recognized contributors to intervertebral disc (IVD) degeneration and discogenic pain. We have recently reported the anti‐inflammatory effect of pulsed electromagnetic fields (PEMF) on IVD cells in vitro. Whether these potentially therapeutic effects are sufficiently potent to influence disc health in vivo has not been demonstrated. We report here the effect of PEMF on acute inflammation arising from a rat‐tail IVD injury model. Disc degeneration was induced by percutaneously stabbing the Co6‐7, Co7‐8, and Co8‐9 levels using a 20‐gauge needle. Seventy‐two (72) rats were divided into three groups: sham control, needle stab, needle stab+PEMF. Treated rats were exposed to PEMF immediately following surgery and for either 4 or 7 days (4 hr/d). Stab and PEMF effects were evaluated by measuring inflammatory cytokine gene expression (RT‐PCR) and protein levels (ELISA assay), anabolic and catabolic gene expression (RT‐PCR), and histologic changes. We observed in untreated animals that at day 7 after injury, inflammatory cytokines (interleukin [IL]‐6, tumor necrosis factor α, and IL‐1β) were significantly increased at both gene and protein levels (P < .05). Similarly, catabolic factors (MMP [metalloproteinases]‐2, MMP‐13 and the transcriptional factor NF‐kβ gene expression) were significantly increased (P < .05). At day 7, PEMF treatment significantly inhibited inflammatory cytokine gene and protein expression induced by needle stab injury (P < .05). At day 4, PEMF downregulated FGF‐1 and upregulated MMP‐2 compared to the stab‐only group. These data demonstrate that previously reported anti‐inflammatory effects of PEMF on disc cells carry over to the in vivo situation, suggesting potential therapeutic benefits. Though we observed an inhibitory effect of PEMF on acute inflammatory cytokine expression, a consistent effect was not observed for acute changes in disc histology and anabolic and catabolic factor expression. Therefore, these findings should be further investigated in studies of longer duration following needle‐stab injury.

Spatial distribution of type II collagen gene expression in the mouse intervertebral disc.

Genetic tools such as the Cre‐Lox reporter system are powerful aids for tissue‐specific cell tracking. For example, it would be useful in examining intervertebral disc (IVD) cell populations in normal and diseased states. A Cre recombinase and its recognition site, loxP have been adapted from the bacteriophage for use in genetic manipulation. The reporter mice used here express the red fluorescent protein, tdTomato with flanking LoxP sites (Rosa26 TdTomato mice). We compared two different Collagen type II (Col2) promoter constructs that drive Cre‐recombinase expression in mice: (a) Col2‐Cre, which allows constitutive Cre‐recombinase expression under the control of the Col2 promoter/enhancer and (b) Col2‐CreER, which contains a shorter promoter/enhancer region than Col2‐Cre, but has human estrogen binding elements that bind tamoxifen, resulting in Cre‐recombinase expression. The goal of the study is to characterize Cre‐recombinase distribution pattern in Col2‐Cre and Col2‐CreER mice using tdTomato as reporter in the spine. The expression patterns of these two mice were further compared with Col2 gene expression in the native mouse NP and AF tissues by real‐time PCR. We crossed Col2‐Cre mice or Col2‐CreER mice with the tdTomato reporter mice, and compared the tdTomato expression patterns. Col2‐CreER/tdTomato mice were injected with tamoxifen at postnatal day 7 to activate the Cre‐recombinase. TdTomato in the constitutively active Col2‐Cre mice was detected in the nucleus pulposus (NP), the entire annulus fibrosus (AF), and in cartilaginous endplate and growth plate cells in the lower lumbar and coccygeal spine. In contrast, when Col2‐CreER activity was induced by tamoxifen at P7, tdTomato was limited to the inner AF, and was absent from the NP. We have described the differences in Col2 reporter gene expression, in Col2‐Cre/tdTomato and Col2‐Cre‐ER/tdTomato mouse IVD. The information provided here will help to guide future investigations of IVD biology.

Caspase-3 knockout inhibits intervertebral disc degeneration related to injury but accelerates degeneration related to aging

Approximately 40% of people under 30 and over 90% of people 55 or older suffer from moderate-to-severe levels of degenerative intervertebral disc (IVD) disease in their lumbar spines. Surgical treatments are sometimes effective; however, the treatment of back pain related to IVD degeneration is still a challenge; therefore, new treatments are necessary. Apoptosis may be important in IVD degeneration because suppressing cell apoptosis inside the IVD inhibits degeneration. Caspase-3, the primary effector of apoptosis, may be a key treatment target. We analyzed caspase-3’s role in two different types of IVD degeneration using caspase-3 knockout (Casp-3 KO) mice. Casp-3 KO delayed IVD degeneration in the injury-induced model but accelerated it in the age-induced model. Our results suggest that this is due to different pathological mechanisms of these two types of IVD degeneration. Apoptosis was suppressed in the IVD cells of Casp-3 KO mice, but cellular senescence was enhanced. This would explain why the Casp-3 KO was effective against injury-induced, but not age-related, IVD degeneration. Our results suggest that short-term caspase-3 inhibition could be used to treat injury-induced IVD degeneration.

Expression and activity of hyaluronidases HYAL-1, HYAL-2 and HYAL-3 in the human intervertebral disc

PurposeHyaluronic acid plays an essential role in water retention of the intervertebral disc (IVD) and thus provides flexibility and shock absorbance in the spine. Hyaluronic acid gets degraded by hyaluronidases (HYALs), and some of the resulting fragments were previously shown to induce an inflammatory and catabolic response in human IVD cells. However, no data currently exist on the expression and activity of HYALs in IVD health and disease.MethodsGene expression, protein expression and activity of HYALs were determined in human IVD biopsies with different degrees of degeneration (n = 50 total). Furthermore, freshly isolated human IVD cells (n = 23 total) were stimulated with IL-1β, TNF-α or H2O2, followed by analysis of HYAL-1, HYAL-2 and HYAL-3 gene expression.ResultsGene expression of HYAL-1 and protein expression of HYAL-2 significantly increased in moderate/severe disc samples when compared to samples with no or low IVD degeneration. HYAL activity was not significantly increased due to high donor–donor variation, but seemed overall higher in the moderate/severe group. An inflammatory environment, as seen during IVD disease, did not affect HYAL-1, HYAL-2 or HYAL-3 expression, whereas exposure to oxidative stress (100 µM H2O2) upregulated HYAL-2 expression relative to untreated controls.ConclusionAlthough HYAL-1, HYAL-2 and HYAL-3 are all expressed in the IVD, HYAL-2 seems to have the highest pathophysiological relevance. Nonetheless, further studies will be needed to comprehensively elucidate its significance and to determine its potential as a therapeutic target.Graphic abstractThese slides can be retrieved under Electronic Supplementary Material.

Pulsed Electromagnetic Fields Reduce Interleukin-6 Expression in Intervertebral Disc Cells Via Nuclear Factor-κβ and Mitogen-Activated Protein Kinase p38 Pathways.

This is an in vitro study of bovine disc cells exposed to pulsed electromagnetic fields. The purpose of the present study was to investigate whether pulsed electromagnetic fields (PEMF) effects on the expression of interleukin-6 (IL-6) expression is mediated by two known inflammation regulators, nuclear factor-κB (NF-κβ) and phosphorylated mitogen-activated protein kinase p38 (p38-MAPK) signaling pathways SUMMARY OF BACKGROUND DATA.: Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Increasing evidence showed that PEMF, a noninvasive biophysical stimulation, can have physiologically beneficial effects on inflammation and tissue repair. Our previous research shows that PEMF treatment can reduce IL-6 expression by intervertebral disc cells. However, the underlying mechanisms of PEMF action are yet to be uncovered. Intervertebral disc nuclear pulposus cells were challenged with interleukin-1α (IL-1α) (for mimicking inflammatory microenvironment) and treated with PEMF simultaneously up to 4 hours. Cells were then collected for NF-κβ and phosphorylated p38-MAPK protein detection with Western blot. Additionally, the RelA (p65) subunit of NF-κβ was examined with immunostaining for assessment of NF-κβ activation. As expected, Western blot results showed that both NF-κβ and phosphorylated p38 expression were significantly increased by IL-1α treatment. This induction was significantly inhibited to control condition levels by PEMF treatment. Immunostaining demonstrated similar trends, that PEMF treatment reduced the NF-κβ activation induced by IL-1α exposure. Our data indicate that the previously-reported inhibitory effect of PEMF treatment on disc inflammation is mediated by NF-κβ and phosphorylated p38-MAPK signaling pathways. These results further establish PEMFs anti-inflammatory activity, and may inform potential future clinical uses for management of inflammation associated with disc degeneration. N/A.

Lactate Efflux From Intervertebral Disc Cells Is Required for Maintenance of Spine Health.

Maintenance of glycolytic metabolism is postulated to be required for health of the spinal column. In the hypoxic tissues of the intervertebral disc and glycolytic cells of vertebral bone, glucose is metabolized into pyruvate for ATP generation and reduced to lactate to sustain redox balance. The rise in intracellular H+ /lactate concentrations are balanced by plasma-membrane monocarboxylate transporters (MCTs). Using MCT4 null mice and human tissue samples, complemented with genetic and metabolic approaches, we determine that H+ /lactate efflux is critical for maintenance of disc and vertebral bone health. Mechanistically, MCT4 maintains glycolytic and tricarboxylic acid (TCA) cycle flux and intracellular pH homeostasis in the nucleus pulposus compartment of the disc, where hypoxia-inducible factor 1α (HIF-1α) directly activates an intronic enhancer in SLC16A3. Ultimately, our results provide support for research into lactate as a diagnostic biomarker for chronic, painful, disc degeneration. © 2019 American Society for Bone and Mineral Research.

Application of Cytokines of the Bone Morphogenetic Protein (BMP) Family in Spinal Fusion - Effects on the Bone, Intervertebral Disc and Mesenchymal Stromal Cells.

Low back pain is a prevalent socio-economic burden and is often associated with damaged or degenerated intervertebral discs (IVDs). When conservative therapy fails, removal of the IVD (discectomy), followed by intersomatic spinal fusion, is currently the standard practice in clinics. The remaining space is filled with an intersomatic device (cage) and with bone substitutes to achieve disc height compensation and bone fusion. As a complication, in up to 30% of cases, spinal non-fusions result in a painful pseudoarthrosis. Bone morphogenetic proteins (BMPs) have been clinically applied with varied outcomes. Several members of the BMP family, such as BMP2, BMP4, BMP6, BMP7, and BMP9, are known to induce osteogenesis. Questions remain on why hyper-physiological doses of BMPs do not show beneficial effects in certain patients. In this respect, BMP antagonists secreted by mesenchymal cells, which might interfere with or block the action of BMPs, have drawn research attention as possible targets for the enhancement of spinal fusion or the prevention of non-unions. Examples of these antagonists are noggin, gremlin1 and 2, chordin, follistatin, BMP3, and twisted gastrulation. In this review, we discuss current evidence of the osteogenic effects of several members of the BMP family on osteoblasts, IVD cells, and mesenchymal stromal cells. We consider in vitro and in vivo studies performed in human, mouse, rat, and rabbit related to BMP and BMP antagonists in the last two decades. We give insights into the effects that BMP have on the ossification of the spine. Furthermore, the benefits, pitfalls, and possible safety concerns using these cytokines for the improvement of spinal fusion are discussed.

Reciprocal Regulation of TRPS1 and miR-221 in Intervertebral Disc Cells.

Intervertebral disc (IVD), a moderately moving joint located between the vertebrae, has a limited capacity for self-repair, and treating injured intervertebral discs remains a major challenge. The development of innovative therapies to reverse IVD degeneration relies primarily on the discovery of key molecules that, occupying critical points of regulatory mechanisms, can be proposed as potential intradiscal injectable biological agents. This study aimed to elucidate the underlying mechanism of the reciprocal regulation of two genes differently involved in IVD homeostasis, the miR-221 microRNA and the TRPS1 transcription factor. Human lumbar IVD tissue samples and IVD primary cells were used to specifically evaluate gene expression and perform functional analysis including the luciferase gene reporter assay, chromatin immunoprecipitation, cell transfection with hTRPS1 overexpression vector and antagomiR-221. A high-level expression of TRPS1 was significantly associated with a lower pathological stage, and TRPS1 overexpression strongly decreased miR-221 expression, while increasing the chondrogenic phenotype and markers of antioxidant defense and stemness. Additionally, TRPS1 was able to repress miR-221 expression by associating with its promoter and miR-221 negatively control TRPS1 expression by targeting the TRPS1-3′UTR gene. As a whole, these results suggest that, in IVD cells, a double-negative feedback loop between a potent chondrogenic differentiation suppressor (miR-221) and a regulator of axial skeleton development (TRPS1) exists. Our hypothesis is that the hostile degenerated IVD microenvironment may be counteracted by regenerative/reparative strategies aimed at maintaining or stimulating high levels of TRPS1 expression through inhibition of one of its negative regulators such as miR-221.

Electrospray-Based Microencapsulation of Epigallocatechin 3-Gallate for Local Delivery into the Intervertebral Disc.

Locally delivered anti-inflammatory compounds can restore the homeostasis of the degenerated intervertebral disc (IVD). With beneficial effects on IVD cells, epigallocatechin 3-gallate (EGCG) is a promising therapeutic candidate. However, EGCG is prone to rapid degradation and/or depletion. Therefore, the purpose of this study was to develop a method for controlled EGCG delivery in the degenerated IVD. Primary IVD cells were isolated from human donors undergoing IVD surgeries. EGCG was encapsulated into microparticles by electrospraying of glutaraldehyde-crosslinked gelatin. The resulting particles were characterized in terms of cytocompatibility and anti-inflammatory activity, and combined with a thermoresponsive carrier to produce an injectable EGCG delivery system. Subsequently, electrospraying was scaled up using the industrial NANOSPIDER™ technology. The produced EGCG microparticles reduced the expression of inflammatory (IL-6, IL-8, COX-2) and catabolic (MMP1, MMP3, MMP13) mediators in pro-inflammatory 3D cell cultures. Combining the EGCG microparticles with the carrier showed a trend towards modulating EGCG activity/release. Electrospray upscaling was achieved, leading to particles with homogenous spherical morphologies. In conclusion, electrospray-based encapsulation of EGCG resulted in cytocompatible microparticles that preserved the activity of EGCG and showed the potential to control EGCG release, thus favoring IVD health by downregulating local inflammation. Future studies will focus on further exploring the biological activity of the developed delivery system for potential clinical use.

168. Non-viral transfection of human intervertebral disc cells with developmental factors induces reprogramming to a healthy anti-catabolic/inflammatory phenotype with enhanced extracellular matrix accumulation

BACKGROUND CONTEXT Intervertebral disc (IVD) degeneration is a significant contributor to low back pain (LBP) and is characterized by a decrease in proteoglycan synthesis, increased catabolism, secretion of inflammatory factors and neurovascular invasion. Although viral gene therapies have been proposed for LBP treatment associated with IVD degeneration, there are concerns due to potential mutagenesis and unwarranted immune responses. To overcome these limitations, nonviral electroporation approaches have been developed. Transcription factor, Brachyury (T) is expressed in the developing notochord and associated with a healthy immature nucleus pulposus (NP) and thus it is a promising factor to reprogram degenerate IVD cells to halt degeneration and promote repair PURPOSE To examine effects of Ttransfection on human NP cells ex-vivo. We hypothesize effective delivery of T can reprogram degenerate IVD cells into healthy cells with increased proteoglycan and decreased inflammatory, catabolic and pain factors. STUDY DESIGN/SETTING 1) Cells isolated from patient and cadaveric human NP tissue. 2) Bulk electroporated with T3) 3D cultured over 4 weeks in agarose gels. PATIENT SAMPLE N=5 samples from patients undergoing microdiscectomy (painful, 19-70yo) and N=5 cadaveric samples(nonpainful, 19-58yo). IRB: 2015H0385 OUTCOME MEASURES Gene expression (RT-qPCR) for healthy NP phenotypic markers, inflammatory/pain markers, matrix genes, and matrix cleaving enzymes, proteoglycan protein (GAG) (dimethylmethylene blue assay), cell viability (calcein/ethidium staining). METHODS Cells were enzymatically isolated from NP tissue and expanded. Subsequently, nonpainful (nPT) andpainful (PT) NP cells were electroporated with a plasmids encoding for T, or an empty vector as a SHAM control. The transfected cells were then expanded in disc cell media and seeded in 3D agarose constructs. Outcome measures were examined at day 0, week 2 and week 4. Statistical Mann-Whitney Tests at α=0.05 were used. RESULTS Cell viability remained high for all groups. Gene expression: healthy marker T was overexpressed and maintained for 4 weeks while KRT19 was significantly increased in nPT and PT cells compared to SHAM controls. Inflammatory cytokines IL-1β and IL6 decreased at 2 weeks for nPT cells and over time for PT cells. Pain marker NGF expression showed significant decrease at week 2 for PT cells and over time in nPT cells. Matrix gene ACAN was increased at 2 weeks in nPT and PT cells while MMP13 was significantly decreased in nPT cells at all time points and at week 4 for PT cells. GAG content was significantly increased in PT groups at 2 weeks compared to SHAM and observed to a lesser extent at week 4. CONCLUSIONS This study demonstrates: (i) sustained cell viability post electroporation of IVD cells, (ii) high expression of T maintained over 4 weeks, and (iii) potential of T to reprogram degenerate human NP cells to a healthy phenotype (up regulation of T and KRT19) with down regulation of inflammatory (IL-1B, 1L6), pain (NGF), and catabolic (MMP12) markers on the gene level with increase matrix synthesis at the protein level (GAG). This is the first study to demonstrate successful reprogramming of diseased human NP cells into a healthy phenotype using nonviral transfection and is a potential therapy to LBP. Future work will use novel electroporation technique, tissue nanotransfection, which has shown to reprogram and rescue diseased tissue. This allows for high efficiency delivery of factors into patient cells while bypassing caveats of viral methods to stimulate IVD repair FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Tension induces intervertebral disc degeneration via endoplasmic reticulum stress-mediated autophagy.

Background: Intervertebral disc degeneration is a common degenerative disease. The present study aimed to explore the role and mechanism of tension-induced endoplasmic reticulum stress in intervertebral disc degeneration.Methods: Intervertebral disc degeneration models of SD rat were analyzed for apoptosis, the expression of Poly(ADP-ribose) polymerase (PARP), Caspase-12, Caspase-3, LC3, Beclin-1 and CHOP using immunohistochemistry, qPCR and Western blot analysis. Annulus fibrosus cells of intervertebral disc were isolated, subjected to cyclic deformation stress and analyzed for ROS and apoptosis, lysosome activity and expression of genes. The cells were knockdown with siRNA or treated with endoplasmic reticulum stress inhibitor 4-PBA and assayed for ROS, apoptosis, lysosome activity and gene expression.Results: Compared with the controls, intervertebral disc degeneration was observed through X-rays examinations and HS staining. Apoptosis and expression of PARP, Caspase-12, Caspase-3, LC3, Beclin-1 and CHOP were significantly increased in the intervertebral disc tissue of the models. In mechanic mimic experiments, the primary annulus fibrosus cells were subjected to 18% cyclic deformation, ROS and apoptosis as well as the activity of lysosome were increased. Similarly, the expression of PARP, Caspase-12, Caspase-3, LC3, Beclin-1 and CHOP was also increased significantly after deformation treatment. On other hand, when the cells were treated with 9 mM 4-PBA and/or CHOP-siRNA4, the apoptosis rate, ROS level, lysosome activity and expression of PARP, Caspase-12, Caspase-3, LC3, Beclin-1 and CHOP were significantly reduced.Conclusions: Autophagy reaction mediated by endoplasmic reticulum stress plays important rale in tension-induced intervertebral disc degeneration. Intervertebral disc degeneration likely results from interactions between autophagy, apoptosis and reticulum stress, and is ROS-dependent.

Mesenchymal Stem Cell Homing Into Intervertebral Discs Enhances the Tie2-positive Progenitor Cell Population, Prevents Cell Death, and Induces a Proliferative Response.

Experimental study with human mesenchymal stem cells (MSCs) and intervertebral disc (IVD) tissue samples. This study aimed to characterize the effect of MSC homing on the Tie2-positive IVD progenitor cell population, IVD cell survival, and proliferation. Homing of human MSCs has been described as potential alternative to MSC injection, aiming to enhance the regenerative capacity of the IVD. IVD cells expressing Tie2 (also known as CD202b or Angiopoietin-1 receptor TEK tyrosine kinase) represent a progenitor cell population with discogenic differentiation potential. However, the fraction of Tie2-positive progenitor cells decreases with aging and degree of IVD degeneration, resulting in a potential loss of the IVD's regenerative capacity. Human MSCs, isolated from vertebral bone marrow aspirates, were labeled and seeded onto the endplate of bovine IVDs and human IVD tissue. Following MSC migration for 5 days, IVD cells were isolated by tissue digestion. The fractions of Tie2-positive, dead, apoptotic, and proliferative IVD cells were evaluated by flow cytometry and compared to untreated IVDs. For human IVDs, 3 groups were investigated: nondegenerated (organ donors), IVDs of patients suffering from spinal trauma, and degenerative IVD tissue samples. MSC homing enhanced the fraction of Tie2-positive IVD cells in bovine and human IVD samples. Furthermore, a proliferative response and lower fraction of dead cells were observed after MSC homing in both bovine and human IVD tissues. Our findings indicate that MSC homing enhances the survival and regenerative capability of IVD cells, which may be mediated by intercellular communication. MSC homing could represent a potential treatment strategy to prevent the onset of the degenerative cascade in IVDs at risk such as IVDs adjacent to a fused segment or IVDs after herniation. N/A.