Circulating microRNAs May Be Predictive of Degenerative Cervical Myelopathy.

STUDY DESIGN: Basic Science. OBJECTIVE: The objective of this study was to identify a unique serum profile of circulating miRNAs and inflammatory markers in patients with degenerative cervical myelopathy (DCM) compared to healthy controls (HC). SUMMARY OF BACKGROUND DATA: Currently, DCM is diagnosed with a combination of history, physical examination, and close correlation to advanced imaging. To date, no serum marker has been identified to be diagnostic of this condition. METHODS: Whole venous blood was collected from patients with DCM as well as healthy age- and sex-matched controls. miRNA was extracted from venous blood and a screening analysis was initially conducted to identify miRNA dysregulation in DCM patients. RT-qPCR was used to analyze the expression of 2 specific miRNAs based on screening analysis and literature review. Bioinformatics analysis was used to identify gene networks and potential targets of the miRNA. In addition, the serum inflammatory profile of DCM and HC groups was differentiated using a pro-inflammatory panel. RESULTS: Thirty-six patients were enrolled in the DCM group (36.1% male, 61.5±9.5 y) while 35 patients were enrolled in the HC group (31.4% male, 57.5±8.9 y). Of the 15 total miRNAs differentially expressed between DCM and HC groups, two were selected for further analysis: miR-223-3p (upregulated) and miR-451a (downregulated). Functional gene network analysis revealed the highest-ranking gene network was involved in neurological disease, while the most overexpressed miRNA in this network (miR-233-3p) was noted to have over 100 targets including CDKN1B and the insulin receptor. Serum cytokine analysis showed significant upregulation of several pro-inflammatory cytokines in the DCM cohort compared to the HC group. CONCLUSION: DCM patients demonstrated a set of unique circulating miRNAs in addition to a different serum inflammatory profile compared to HC. These miRNAs may potentially serve as targets for future therapeutic intervention or diagnostic/prognostic testing.

The Inhibitory Effect of NSAIDs and Opioids on Spinal Fusion: An Animal Model.

STUDY DESIGN: Translational research. OBJECTIVE: To evaluate the relative effects of NSAIDs, opioids, and a combination of the two on spinal fusion inhibition in a rodent model. SUMMARY OF BACKGROUND DATA: Nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids are common postoperative analgesic agents. Since NSAIDs inhibit the cyclooxygenase (COX) pathway, they are seldom prescribed following spinal fusion. Opioids may be given instead, but recent evidence suggests opioids also adversely affect spinal fusion quality and success. METHODS: Eighty male Sprague-Dawley rats underwent L4-5 posterior lumbar fusion and were given one of the following analgesia regimens: saline, morphine (6 mg/kg), ketorolac (4 mg/kg), or morphine (3 mg/kg) and ketorolac (2 mg/kg). Serum samples were drawn to evaluate systemic pro-osteoblastic cytokines and vascular endothelial growth factor-A (VEGF-A) levels, which were measured through enzyme-linked immunosorbent assays (ELISA). After six weeks, the rats were sacrificed, and the operated spinal segments underwent manual palpation, microCT, and histologic analysis. RESULTS: Manual palpation scores were significantly diminished in the opioid, NSAID, and multimodal groups when compared with control ( P <0.001). MicroCT fusion scores ( P <0.001) and fusion rates (control: 75% vs . NSAID: 35% vs . opioid: 0% vs . combination: 15%, P <0.001) were significantly diminished in the treatment groups. The bone volume (BV) to tissue volume (TV) ratio (BV/TV) ( P <0.001) and bone mineral density (BMD) ( P <0.001) were all lower in the treatment groups, with the opioid and combined groups having the lowest BMD. Although statistically insignificant ( P <0.09), the concentration of VEGF-A was greater in the control group compared with opioids, NSAIDs, and the combined group. CONCLUSION: Opioids and NSAIDs, both independently and combined, inhibited spinal fusion and caused inferior bony callus. Administration of opioids resulted in the lowest rate of spinal fusion. We propose this may be due to the inhibition of VEGF-A, which limits angiogenesis to the burgeoning fusion mass.

Inhibition of Neurogenic Inflammatory Pathways Associated with the Reduction in Discogenic Back Pain.

STUDY DESIGN: Retrospective cohort study. PURPOSE: This study aimed to determine whether the initiation of anti-calcitonin gene-related peptide (CGRP inhibitor) medication therapy for migraines was also associated with improvements in back/neck pain, mobility, and function in a patient population with comorbid degenerative spinal disease and migraine. OVERVIEW OF LITERATURE: CGRP upregulates pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, brain-derived neurotrophic factor, and nerve growth factor in spinal spondylotic disease, which results in disc degeneration and sensitization of nociceptive neurons. Although CGRP inhibitors can quell neurogenic inflammation in migraines, their off-site efficacy as a therapeutic target for discogenic back/neck pain conditions remains unknown. METHODS: All adult patients diagnosed with spinal spondylosis and migraine treated with CGRP inhibitors at a single academic institution between 2017 and 2020 were retrospectively identified. Patient demographic and medical data, follow-up duration, migraine severity and frequency, spinal pain, functional status, and mobility before and after the administration of CGRP inhibitors were collected. Paired univariate analysis was conducted to determine significant changes in spinal pain, headache severity, and headache frequency before and after the administration of CGRP inhibitors. The correlation between changes in the spinal pain score and functional or mobility improvement was assessed with Spearman's rho. RESULTS: In total, 56 patients were included. The mean follow-up time after the administration of CGRP inhibitors was 123 days for spinal pain visits and 129 days for migraine visits. Back/neck pain decreased significantly (p <0.001) from 6.30 to 4.36 after starting CGRP inhibitor therapy for migraine control. As recorded in the spine follow-up notes, 25% of patients experienced a functional improvement in the activities of daily living, and 17.5% experienced mobility improvement while taking CGRP inhibitors. Change in back/ neck pain moderately correlated (ρ=-0.430) with functional improvement but was not correlated with mobility improvement (ρ=-0.052). CONCLUSIONS: Patients taking CGRP inhibitors for chronic migraines with comorbid degenerative spinal conditions experienced significant off-target reduction of back/neck pain.

Varenicline mitigates the increased risk of pseudarthrosis associated with nicotine.

BACKGROUND CONTEXT: High serum nicotine levels increase the risk of nonunion after spinal fusion. Varenicline, a pharmaceutical adjunct for smoking cessation, is a partial agonist designed to displace and outcompete nicotine at its receptor binding site, thereby limiting downstream activation. Given its mechanism, varenicline may have therapeutic benefits in mitigating nonunion for active smokers undergoing spinal fusion. PURPOSE: To compare fusion rate and fusion mass characteristics between cohorts receiving nicotine, varenicline, or concurrent nicotine and varenicline after lumbar fusion. STUDY DESIGN: Rodent noninstrumented spinal fusion model. METHODS: Sixty eight-week-old male Sprague-Dawley rats weighing approximately 300 grams underwent L4-5 posterolateral fusion (PLF) surgery. Four experimental groups (control: C, nicotine: N, varenicline: V, and combined: NV [nicotine and varenicline]) were included for analysis. Treatment groups received nicotine, varenicline, or a combination of nicotine and varenicline delivered through subcutaneous osmotic pumps beginning two weeks before surgery until the time of sacrifice at age 14 weeks. Manual palpation testing, microCT imaging, bone histomorphometry, and biomechanical testing were performed on harvested spinal fusion segments. RESULTS: Control (p=0.016) and combined (p=0.032) groups, when compared directly to the nicotine group, demonstrated significantly greater manual palpation scores. The fusion rate in the control (93.3%) and combined (93.3%) groups were significantly greater than that of the nicotine group (33.3%) (p=0.007, both). Biomechanical testing demonstrated greater Young's modulus of the fusion segment in the control (17.1 MPa) and combined groups (34.5 MPa) compared to the nicotine group (8.07 MPa) (p<0.001, both). MicroCT analysis demonstrated greater bone volume fraction (C:0.35 vs N:0.26 vs NV:0.33) (p<0.001, all) and bone mineral density (C:335 vs N:262 vs NV:328 mg Ha/cm3) (p<0.001, all) in the control and combined groups compared to the nicotine group. Histomorphometry demonstrated a greater mineral apposition rate in the combined group compared to the nicotine group (0.34 vs 0.24 µm/day, p=0.025). CONCLUSION: In a rodent spinal fusion model, varenicline mitigates the adverse effects of high nicotine serum levels on the rate and quality of spinal fusion. CLINICAL SIGNIFICANCE: These findings have the potential to significantly impact clinical practice guidelines and the use of pharmacotherapy for active nicotine users undergoing fusion surgery.

Do Inflammatory Cytokines Affect Patient Outcomes After ACDF?

STUDY DESIGN: Prospective cohort study. OBJECTIVE: The aim was to determine the relationship between serum inflammatory mediators, preoperative cervical spine disease severity, and clinical outcomes after anterior cervical discectomy and fusion (ACDF). SUMMARY OF BACKGROUND DATA: Given the role of the inflammatory cascade in spinal degenerative disease, it has been hypothesized that inflammatory markers may serve as a predictor of patient outcomes after surgery. MATERIALS AND METHODS: All patients over age 18 who underwent ACDF for cervical spondylosis with associated radiculopathy and/or myelopathy between 2015 and 2017 from a single institution were prospectively recruited. Preoperative serum inflammatory markers including interleukin (IL)-6, IL-8, tumor necrosis factor-α, high-mobility group box-1 (HMGB1), and white blood cells were measured and correlated to patient demographics, surgical characteristics, duration of symptoms, previous opioid use, and preoperative and 1-year postoperative patient-reported outcomes measures (PROMs) including the neck disability index (NDI), visual analog scale neck pain, visual analog scale arm pain, and Physical and Mental Component Scores of the Short Form-12 (PCS and MCS, respectively) using spearman's rho coefficient. RESULTS: A total of 77 patients were enrolled with follow-up PROMs available for 62% (n=48) of patients at a minimum of 1-year after ACDF. The absolute concentrations of IL-6 and tumor necrosis factor-α were found to be weakly correlated with one another (ρ=0.479). Preoperative symptoms lasting <1-year were weakly correlated with elevation in HMGB1 (ρ=0.421). All other patient demographics exhibited negligible correlation with the preoperative inflammatory markers. Lower preoperative PCS (ρ=0.355) and higher preoperative NDI (ρ=0.336) were weakly correlated with elevated HMGB1. Lower MCS (ρ=0.395) and higher NDI (ρ=0.317) preoperatively were weakly correlated with elevated white blood cells. Postoperative improvement in MCS (ρ=0.306) and MCS recovery ratio (ρ=0.321) exhibited a weakly positive correlation with IL-6. CONCLUSION: Preoperative cytokine levels demonstrated minimal correlation with preoperative symptoms or clinical improvement, suggesting that profiling of patient cytokines has limited utility in predicting outcomes after ACDF. LEVEL OF EVIDENCE: Level III.

Functional Deficits in Mice Expressing Human Interleukin 8.

We showed previously that inflammatory mediators, including IL8, in intervertebral disc tissues from patients with discogenic back pain may play a key role in back pain. To investigate the molecular mechanism of IL8 signaling in back pain, we generated a mouse model that conditionally expresses human (h) IL8. We hypothesized that hIL8 levels affect mouse activity and function. Briefly, hIL8 cDNA was inserted into the pCALL2 plasmid, linearized, and injected into mouse embryos. Resulting pCALL2-hIL8 mice were then bred with GDF5-Cre mice to express the transgene in cartilage and intervertebral disc (IVD) tissues. Functional capacities including nest-making and other natural behaviors were measured. Both male and female mice expressing hIL8 showed lower nesting scores than did littermates that did not express hIL8 (n = 14 to 16 per group). At 28 wk of age, mice expressing hIL8 (n = 35) spent more time immobile and eating during each night than littermate controls (n = 33). Furthermore, hIL8-expressing mice traveled shorter distances and at a lower average speed than littermate controls. Thus, in an initial effort to investigate the relationship between this chemokine and mouse behavior, we have documented changes in normal activities in mice conditionally expressing hIL8.

Correlation of Early Outcomes and Intradiscal Interleukin-6 Expression in Lumbar Fusion Patients.

OBJECTIVE: To determine if there is correlation between intradiscal levels of interleukin-6 (IL-6) and early outcome measures in patients undergoing lumbar fusion for painful disc degeneration. METHODS: Intervertebral disc tissue was separated into annulus fibrosus/nucleus pulposus and cultured separately in vitro in serum-free medium (Opti-MEM). Conditioned media was collected after 48 hours. The concentration of IL-6 was quantified using enzyme-linked immunosorbent assay. Pearson correlation coefficients quantified relationships between IL-6 levels and pre- and postoperative visual analogue scale (VAS) back pain and Oswestry Disability Index (ODI), as well as change in VAS/ODI. RESULTS: Sixteen discs were harvested from 9 patients undergoing anterior lumbar interbody fusion (mean age, 47.4 years; range, 21-70 years). Mean preoperative and 6-month postoperative VAS were 8.1 and 3.7, respectively. Mean preoperative and postoperative ODI were 56.2 and 25.6, respectively. There were significant positive correlations between IL-6 expression and postoperative VAS (ρ = 0.38, p = 0.048) and ODI (ρ = 0.44, p = 0.02). No significant correlations were found between intradiscal IL-6 expression and preoperative VAS (ρ = -0.12, p = 0.54). Trends were seen associating IL-6 expression and change in VAS/ODI (ρ = -0.35 p = 0.067; ρ = -0.34, p = 0.08, respectively). A trend associated IL-6 and preoperative ODI (ρ = 0.36, p = 0.063). CONCLUSION: The direct association between IL-6 expression and VAS/ODI suggests patients with elevated intradiscal cytokine expression may have worse early outcomes than those with lower expression of IL-6 after surgery for symptomatic disc degeneration.

Bromodomain-containing protein 4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity in NP cells.

An imbalance between matrix synthesis and degradation is the hallmark of intervertebral disc degeneration while inflammatory cytokines contribute to the imbalance. Bromodomain and extra-terminal domain (BET) family is associated with the pathogenesis of inflammation, and inhibition of BRD4, a vital member of BET family, plays an anti-inflammatory role in many diseases. However, it remains elusive whether BRD4 plays a similar role in nucleus pulposus (NP) cells and participates in the pathogenesis of intervertebral disc degeneration. The present study aims to observe whether BRD4 inhibition regulates matrix metabolism by controlling autophagy and NLRP3 inflammasome activity. Besides, the relationship was investigated among nuclear factor κB (NF-κB) signaling, autophagy and NLRP3 inflammasome in NP cells. Here, real-time polymerase chain reaction, western blot analysis and adenoviral GFP-LC3 vector transduction in vitro were used, and it was revealed that BRD4 inhibition alleviated the matrix degradation and increased autophagy in the presence or absence of tumor necrosis factor α. Moreover, p65 knockdown or treatment with JQ1 and Bay11-7082 demonstrated that BRD4 inhibition attenuated NLRP3 inflammasome activity through NF-κB signaling, while autophagy inhibition by bafilomycin A1 promoted matrix degradation and NLRP3 inflammasome activity in NP cells. In addition, analysis of BRD4 messenger RNA expression in human NP tissues further verified the destructive function of BRD4. Simply, BRD4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity through NF-κB signaling in NP cells.

Circulating miR-155-5p as a Novel Biomarker of Lumbar Degenerative Disc Disease.

MINI: Circulating microRNAs provide an insight into current disease states. Comparing patients with degenerative disc disease to healthy controls, patients with disc disease were found to have significantly downregulated levels of miR-155-5p. This marker was found to be an accurate diagnostic predictor for the presence of degeneration (P = 0.006). STUDY DESIGN: Case-control study measuring differential gene expression of circulating microRNA (miRNA) in patients with degenerative disc disease (DDD). OBJECTIVE: To identify miRNA dysregulation in serum samples of patients with DDD compared to healthy controls (HC). SUMMARY OF BACKGROUND DATA: Early DDD can be a difficult diagnosis to make clinically, with lack of positive and specific findings on physical exam or advanced imaging. miRNAs are a class of molecules that act as gene regulators and have been shown to be dysregulated in local degenerative disc tissue. However, to date no studies have identified dysregulation of serum miRNA in patients with DDD. METHODS: Whole blood samples were obtained from 69 patients with DDD and 16 HC. Patient-reported outcomes were collected preoperatively and degree of DDD was classified using Pfirrmann grade on preoperative imaging. Differential gene expression analysis using a screening assay for several hundred miRNAs and further characterization for five specific miRNAs (miR-16-5p, miR-21-5p, miR-142-3p, miR-146a-5p, and miR-155-5p) was performed. In addition, a pro-inflammatory cytokine multiplex assay and bioinformatics analysis were done. RESULTS: The initial screening assay showed 13 miRNA molecules that were significantly dysregulated in DDD patients, with miR-155-5p showing significant downregulation (p = 0.027) and direct interactions with the pro-inflammatory cytokine IL-1ß, and the tumor suppressor genes p53 and BRAF. Analyzing the whole cohort, miR-155 showed an almost four-fold downregulation in DDD patients (-3.94-fold, P < 0.001) and was the sole miRNA that accurately predicted the presence of disc degeneration (P = 0.006). Downregulation of miR-155 also correlated with increased leg pain (P = 0.018), DDD (P = 0.006), and higher Pfirrmann grade (P = 0.039). On cytokine analysis, TNF-α (0.025) and IL-6 (P < 0.001) were significantly higher in DDD patients. CONCLUSION: Serum miR-155-5p is significantly downregulated in patients with DDD and may be a diagnostic marker for degenerative spinal disease. LEVEL OF EVIDENCE: N/A.

Case-control study measuring differential gene expression of circulating microRNA (miRNA) in patients with degenerative disc disease (DDD). To identify miRNA dysregulation in serum samples of patients with DDD compared to healthy controls (HC). Early DDD can be a difficult diagnosis to make clinically, with lack of positive and specific findings on physical exam or advanced imaging. miRNAs are a class of molecules that act as gene regulators and have been shown to be dysregulated in local degenerative disc tissue. However, to date no studies have identified dysregulation of serum miRNA in patients with DDD. Whole blood samples were obtained from 69 patients with DDD and 16 HC. Patient-reported outcomes were collected preoperatively and degree of DDD was classified using Pfirrmann grade on preoperative imaging. Differential gene expression analysis using a screening assay for several hundred miRNAs and further characterization for five specific miRNAs (miR-16-5p, miR-21-5p, miR-142-3p, miR-146a-5p, and miR-155-5p) was performed. In addition, a pro-inflammatory cytokine multiplex assay and bioinformatics analysis were done. The initial screening assay showed 13 miRNA molecules that were significantly dysregulated in DDD patients, with miR-155-5p showing significant downregulation (p = 0.027) and direct interactions with the pro-inflammatory cytokine IL-1ß, and the tumor suppressor genes p53 and BRAF. Analyzing the whole cohort, miR-155 showed an almost four-fold downregulation in DDD patients (−3.94-fold, P < 0.001) and was the sole miRNA that accurately predicted the presence of disc degeneration (P = 0.006). Downregulation of miR-155 also correlated with increased leg pain (P = 0.018), DDD (P = 0.006), and higher Pfirrmann grade (P = 0.039). On cytokine analysis, TNF-α (0.025) and IL-6 (P < 0.001) were significantly higher in DDD patients. Serum miR-155-5p is significantly downregulated in patients with DDD and may be a diagnostic marker for degenerative spinal disease. Level of Evidence: N/A.

Does Riluzole Influence Bone Formation?: An In Vitro Study of Human Mesenchymal Stromal Cells and Osteoblast.

STUDY DESIGN: A post-test design biological experiment. OBJECTIVE: The aim of this study was to evaluate the osteogenic effects of riluzole on human mesenchymal stromal cells and osteoblasts. SUMMARY OF BACKGROUND DATA: Riluzole may benefit patients with spinal cord injury (SCI) from a neurologic perspective, but little is known about riluzole's effect on bone formation, fracture healing, or osteogenesis. METHODS: Human mesenchymal stromal cells (hMSCs) and human osteoblasts (hOB) were obtained and isolated from healthy donors and cultured. The cells were treated with riluzole of different concentrations (50, 150, 450 ng/mL) for 1, 2, 3, and 4 weeks. Cytotoxicity was evaluated as was the induction of osteogenic differentiation of hMSCs. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and with Alizarin red staining. Osteogenic gene expression of type I collagen (Col1), ALP, osteocalcin (Ocn), Runx2, Sox9, Runx2/Sox9 ratio were measured by qRT-PCR. RESULTS: No cytotoxicity or increased proliferation was observed in bone marrow derived hMSCs and primary hOBs cultured with riluzole over 7 days. ALP activity was slightly increased in hMSCs after treatment for 2 weeks with riluzole 150 ng/mL and slightly upregulated by 150% (150 ng/mL) and 90% (450 ng/mL) in hMSCs at 3 weeks. In hOBs, ALP activity almost doubled after 2 weeks of culture with riluzole 150 ng/mL (P < 0.05). More pronounced 2.6-fold upregulation was noticed after 3 weeks of culture with riluzole at both 150 ng/mL (P = 0.05) and 450 ng/mL (P = 0.05). No significant influence of riluzole on the mRNA expression of osteocalcin (OCN) was observed. CONCLUSION: The effect of riluzole on bone formation is mixed; low-dose riluzole has no effect on the viability or function of either hMSCs or hOBs. The activity of ALP in both cell types is upregulated by high-dose riluzole, which may indicate that high-dose riluzole can increase osteogenic metabolism and subsequently accelerate bone healing process. However, at high concentrations, riluzole leads to a decrease in osteogenic gene expression, including Runx2 and type 1 collagen. LEVEL OF EVIDENCE: N/A.

MicroRNA-145 overexpression attenuates apoptosis and increases matrix synthesis in nucleus pulposus cells.

AIMS: Lower back pain is often associated with intervertebral disc degeneration (IDD), which results from a decrease in nucleus pulposus (NP) cells and an imbalance between the degradation and synthesis of extracellular matrix (ECM) components. Multiple microRNAs play crucial roles in the modulation of NP cell apoptosis and matrix degradation. miR-145 is an important microRNA related to degenerative diseases such as osteoarthritis. Here, the effect of miR-145 in IDD was elucidated. The aim of this study was to explore the role and mechanism of miR-145 in the apoptosis of NP cells and in matrix metabolism in NP cells. MATERIALS AND METHODS: Real-time PCR, western blotting and flow cytometry analysis were used to observe the effect of miR-145 on NP cell apoptosis in the absence or presence of oxidative stress. Cell transfection, loss-of-function experiments using an ADAM17 inhibitor or lentiviral shADAM17, immunofluorescence, real-time PCR and western blotting were performed to demonstrate the role and mechanism of miR-145 in NP cell matrix metabolism. KEY FINDINGS: miR-145 attenuated NP cell apoptosis in the absence and presence of oxidative stress. Moreover, miR-145 overexpression increased and miR-145 suppression decreased matrix synthesis. ADAM17, which is expressed in degenerative discs, is the target of miR-145. ADAM17 gene suppression with lentiviral shRNA or an inhibitor enhanced matrix synthesis in NP cells. In addition, siADAM17 reversed the matrix degradation induced by miR-145 inhibition. SIGNIFICANCE: miR-145 suppresses apoptosis and promotes ECM synthesis in NP cells. miR-145 is thus a potential therapeutic microRNA for IDD.

The local cytokine and growth factor response to recombinant human bone morphogenetic protein-2 (rhBMP-2) after spinal fusion.

BACKGROUND CONTEXT: The systemic response regarding cytokine expression after the application of recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rat spinal fusion model has recently been defined, but the local response has not been defined. Defining the local cytokine and growth factor response at the fusion site will help explain the roles of these molecules in the fusion process, as well as that of rhBMP-2. Our hypothesis is that the application of rhBMP-2 to the fusion site will alter the local levels of cytokines and growth factors throughout the fusion process, in a manner that is different from the systemic response, given the tissue-specific effects of rhBMP-2. PURPOSE: The purpose of this study was to evaluate the local cytokine and growth factor response after the application of rhBMP-2 in a rat spinal fusion model. STUDY DESIGN/SETTING: This was a basic science animal model study. METHODS: This study was partially funded by a physician-sponsored grant from Medtronic. A total of 135 Wistar rats (age 8 weeks, weighing approximately 300-400 g) underwent L4-L5 posterolateral intertransverse fusion with demineralized bone graft (approximately 0.4-cm3 rat demineralized bone matrix [DBM] per side). In the first group, 10 µg of rhBMP-2 on an allograft collagen sponge (ACS) was added to the fusion site with approximately 0.4-cm3 rat DBM per side. In the second group, 100 µg of rhBMP-2 on an ACS was added to the fusion site with approximately 0.4-cm3 rat DBM per side, and the third experiment was the control group, which consisted of only an ACS plus 0.4-cm3 DBM per side. There were nine groups of five animals each per experiment. Each group was sacrificed at time points up to 4 weeks (1, 6, 24, and 48 hours, and 4, 7, 14, 21, and 28 days after surgery). At sacrifice, the DBM, transverse processes, and any new bone formed were harvested, immediately frozen in liquid nitrogen, and prepared for protein extraction. ELISA was performed to compare the levels of various cytokines (interleukin [IL]-1ß, tumor necrosis factor alpha, IL-6, IL-1RA [IL-1 receptor antagonist], IL-4, and IL-10) and growth factors (vascular endothelial growth factor [VEGF], endothelia growth factor [EGF], insulin-like growth factor-1 [IGF-1], platelet derived growth factor [PDGF], transforming growth factor beta [TGF-ß]) that are known to be involved in the fusion-fracture healing process. Fusion was evaluated on the rats sacrificed at 28 days by manual palpation and microcomputed tomography (microCT) by two independent observers. RESULTS: The expression of cytokines and growth factors varied throughout the fusion process at each time point. In the groups treated with rh-BMP-2, IL-6 and IL-1RA had higher expression in the early time points (1 and 6 hours). Tumor necrosis factor alpha demonstrated significantly lower expression in the groups treated with rhBMP-2 at Days 1, 2, and 4. At the early time points (1 and 6 hours), in the groups treated with rhBMP-2, all of the growth factors IGF-1, VEGF, platelet derived growth factor AB (PDGF-AB), TGF-ß had equal or lower expression compared with controls. At 24 hours, there was a peak in IGF-1, VEGF, and PDGF-AB. These growth factors then declined, with IGF-1 and PDGF-AB having a second peak at Day 7. At 4 weeks, all of the rhBMP-2-treated animals fused based on manual palpation and microCT. The control group had four of five rats fused based on manual palpation and two of five rats based on microCT. CONCLUSIONS: There is significant variability in the expression of cytokines throughout the fusion process after treatment with rhBMP-2. The inflammatory response appears to peak early (1 and 6 hours), followed by a significant decrease with rhBMP-2 treatment. However, the growth factor expression appears to be suppressed early (1 and 6 hours), followed by a peak at 24 hours, and a second peak at Day 7.

Are Modic changes associated with intervertebral disc cytokine profiles?

BACKGROUND CONTEXT: Degenerative changes including Modic changes (MCs) are commonly observed in patients with chronic low back pain. Although intervertebral disc (IVD) cytokine expression has been shown to be associated with low back pain, the cytokine profile for degenerative IVD with and without MC has not been compared. PURPOSE: This study aimed to evaluate the potential association between IVD cytokine expression and MCs. STUDY DESIGN: A laboratory study was carried out. METHODS: The IVD tissue samples from 10 patients with type II MCs and10 patients without MCs who underwent an anterior lumbar interbody and fusion for significant low back pain were collected. The expression levels of 42 cytokines were determined using a RayBio Human Cytokine Antibody Array 3 (RayBiotech Inc, Norcross, GA, USA) and the results were verified with enzyme-linked immunosorbent assay (ELISA). RESULTS: The cytokine array demonstrated a statistically significant increase in the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) (p=.001) and epithelial-derived neutrophil-activating peptide 78 (ENA-78) (p=.04), and a trend toward an increase in interleukin-1ß (IL-1ß) (p=.12) and tumor necrosis factor-α (TNF-α) (p=.22) in IVDs associated with type II MCs. These results were validated with ELISA which demonstrated a 3.85-fold increase in the GM-CSF level between IVDs with type II MCs compared with those without MCs (p=.03). Similarly there was a significant increase in the level of both ENA-78 (3.68-fold, p=.02) and IL-1ß (2.11-fold, p=.01) in IVDs with type II MCs. Lastly, there was a trend (p=.07) toward an increase in TNF-α in IVDs with type II MCs (4.4-fold). CONCLUSION: Intervertebral discs with type II MCs demonstrate a significant increase in IL-1ß, GM-CSF, and ENA-78, and there is a trend toward an increase in TNF-α. These results further strengthen the association between MCs and low back pain.

The Effect of Substance P on an Intervertebral Disc Rat Organ Culture Model.

STUDY DESIGN: Laboratory study. OBJECTIVE: Evaluate the effect of substance P (SP) on an intervertebral disc rat organ culture model. SUMMARY OF BACKGROUND DATA: Monolayer cell experiments have demonstrated that exposure intervertebral disc tissue cells to SP leads to upregulation in inflammatory cytokine expression; however, this has not been evaluated in a more complex organ culture model. METHODS: Forty-eight intervertebral discs from eight rats were used in an organ culture model. Intervertebral discs were divided into three groups: control, SP-treated group, and a group treated with an SP antagonist followed by SP. Cytokine antibody array was used to quantify expression patterns, which were confirmed using ELISA and real-time polymerase chain reaction. RESULTS: The cytokine array demonstrated a 3.40 ±â€Š 0.59-fold increase in interleukin 6 (IL-6) expression in the SP group (P = 0.004), and the effect of SP was mitigated by the SP antagonist (P = 0.03). These results were verified as ELISA demonstrated a significant difference in the IL-6 level between the control group and SP group (0.73 vs. 5.80 ng/mL, P < 0.001), and there was a significant difference in the IL-6 level between the SP and the SP antagonist group (5.80 vs. 4.02 ng/mL, P = 0.01). Similarly, the real-time polymerase chain reaction demonstrated that the discs treated with SP had a 4.77-fold increase in IL-6 levels (P = 0.01) compared to controls, and a significantly greater increase in IL-6 levels between the intervertebral discs in the SP group and those in the SP antagonist group versus control (4.77 vs. 1.57, P = 0.02). CONCLUSION: SP lead to the activation of an inflammatory pathway by increasing expression of IL-6 in an intervertebral disc organ culture model. These results provide evidence that SP may be an important factor in the link between intervertebral disc degeneration and low back pain. LEVEL OF EVIDENCE: N/A.

Intervertebral Disc Cells Produce Interleukins Found in Patients with Back Pain.

OBJECTIVE: To examine the link between cytokines in intervertebral disc (IVD) tissues and axial back pain. DESIGN: In vitro study with human IVD cells cultured from cadaveric donors and annulus fibrosus (AF) tissues from patients. RESULTS: Cultured nucleus pulposus (NP) and AF cells were stimulated with interleukin (IL)-1ß. IL-8 and IL-7 gene expression was analyzed using real-time polymerase chain reaction. IL-8 protein was quantified by enzyme-linked immunosorbent assay. After IL-1ß stimulation, IL-8 gene expression increased 26,541 fold in NP cells and 22,429 fold in AF cells, whereas protein released by the NP and AF cells increased 2,389- and 1,784-fold, respectively. IL-7 gene expression increased 3.3-fold in NP cells (P < 0.05).Cytokine profiles in AF tissues collected from patients undergoing surgery for back pain (painful group) or scoliosis (controls) were compared by cytokine array. IL-8 protein in the AF tissues from patients with back pain was 1.81-fold of that in controls. IL-7 and IL-10 in AF tissues from the painful group were 6.87 and 4.63 times greater than the corresponding values in controls, respectively (P < 0.05). CONCLUSION: Inflammatory mediators found in AF tissues from patients with discogenic back pain are likely produced by IVD cells and may play a key role in back pain.

Forelimb contractures and abnormal tendon collagen fibrillogenesis in fibulin-4 null mice.

Fibulin-4 is an extracellular matrix glycoprotein essential for elastic fiber formation. Mice deficient in fibulin-4 die perinatally because of severe pulmonary and vascular defects associated with the lack of intact elastic fibers. Patients with fibulin-4 mutations demonstrate similar defects, and a significant number die shortly after birth or in early childhood from cardiopulmonary failure. The patients also demonstrate skeletal and other systemic connective tissue abnormalities, including joint laxity and flexion contractures of the wrist. A fibulin-4 null mouse strain was generated and used to analyze the roles of fibulin-4 in tendon fibrillogenesis. This mouse model displayed bilateral forelimb contractures, in addition to pulmonary and cardiovascular defects. The forelimb and hindlimb tendons exhibited disruption in collagen fibrillogenesis in the absence of fibulin-4 as analyzed by transmission electron microscopy. Fewer fibrils were assembled, and fibrils were disorganized compared with wild-type controls. The organization of developing tenocytes and compartmentalization of the extracellular space was also disrupted. Fibulin-4 was co-localized with fibrillin-1 and fibrillin-2 in limb tendons by using immunofluorescence microscopy. Thus, fibulin-4 seems to play a role in regulating tendon collagen fibrillogenesis, in addition to its essential function in elastogenesis.

Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

STUDY DESIGN: Laboratory study. OBJECTIVE: To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SUMMARY OF BACKGROUND DATA: SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. METHODS: Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1ß, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. RESULTS: We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1ß, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. CONCLUSION: NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. LEVEL OF EVIDENCE: 4.

Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration: Novel HIF-1-mediated regulation of aquaporins in NP cells.

Objectives of this study were to investigate whether AQP1 and AQP5 expression is altered during intervertebral disc degeneration and if hypoxia and HIF-1 regulate their expression in NP cells. AQP expression was measured in human tissues from different degenerative grades; regulation by hypoxia and HIF-1 was studied using promoter analysis and gain- and loss-of-function experiments. We show that both AQPs are expressed in the disc and that mRNA and protein levels decline with human disease severity. Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs. Surprisingly, hypoxia failed to induce promoter activity or expression of either AQP. While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities. Stable HIF-1α suppression significantly decreased mRNA and protein levels of both AQPs, but HIF-1α failed to induce AQP levels following accumulation. Together, our results demonstrate that AQP1 and AQP5 expression is sensitive to human disc degeneration and that HIF-1α uniquely maintains basal expression of both AQPs in NP cells, independent of oxemic tension and HIF-1 binding to promoter HREs. Diminished HIF-1 activity during degeneration may suppress AQP levels in NP cells, compromising their ability to respond to extracellular osmolarity changes.

Fibronectin splice variation in human knee cartilage, meniscus and synovial membrane: observations in osteoarthritic knee.

Fibronectin (FN) is a widely expressed molecule that can participate in development of osteoarthritis (OA) affecting cartilage, meniscus, and synovial membrane (SM). The alternatively spliced isoforms of FN in joint tissues other than cartilage have not been extensively studied previously. The present study compares FN splice variation in patients with varying degrees of osteoarthritic change. Joint tissues were collected from asymptomatic donors and patients undergoing arthroscopic procedures. Total RNA was amplified by PCR using primers flanking alternatively spliced Extra Domain A (EDA), Extra Domain B (EDB) and Variable (V) regions. EDB(+) , EDB(-) and EDA(-) and V(+) variants were present in all joint tissues, while the EDA(+) variant was rarely detected. Expression levels of EDB(+) and EDV(+) variants were similar in cartilage, synovium, and meniscal tissues. Synovial expression of V(+) FN in arthroscopy patients varied with degree of cartilage degeneration. Two V(-) isoforms, previously identified in cartilage, were also present in SM and meniscus. Fibronectin splicing in meniscus and SM bears striking resemblance to that of cartilage. Expression levels of synovial V(+) FN varied with degree of cartilage degeneration. V(+) FN should be investigated as a potential biomarker of disease stage or progression in larger populations.

Xylosyltransferase-1 expression is refractory to inhibition by the inflammatory cytokines tumor necrosis factor α and IL-1ß in nucleus pulposus cells: novel regulation by AP-1, Sp1, and Sp3.

We investigated whether expression of xylosyltransferase-1 (XT-1), a key enzyme in glycosaminoglycan biosynthesis, is responsive to disk degeneration and to inhibition by the inflammatory cytokines tumor necrosis factor α and IL-1ß in nucleus pulposus (NP) cells. Analysis of human NP tissues showed that XT-1 expression is unaffected by degeneration severity; XT-1 and Jun, Fos, and Sp1 mRNA were positively correlated. Cytokines failed to inhibit XT-1 promoter activity and expression. However, cytokines decreased activity of XT-1 promoters containing deletion and mutation of the -730/-723 bp AP-1 motif, prompting us to investigate the role of AP-1 and Sp1/Sp3 in the regulation of XT-1 in healthy NP cells. Overexpression and suppression of AP-1 modulated XT-1 promoter activity. Likewise, treatment with the Sp1 inhibitors WP631 and mithramycin A or cotransfection with the plasmid DN-Sp1 decreased XT-1 promoter activity. Inhibitors of AP-1 and Sp1 and stable knockdown of Sp1 and Sp3 resulted in decreased XT-1 expression in NP cells. Genomic chromatin immunoprecipitation analysis showed AP-1 binding to motifs located at -730/-723 bp and -684/-677 bp and Sp1 binding to -227/-217 bp and -124/-114 bp in XT-1 promoter. These results suggest that XT-1 expression is refractory to the disease process and to inhibition by inflammatory cytokines and that signaling through AP-1, Sp1, and Sp3 is important in the maintenance of XT-1 levels in NP cells.