Identification of extracellular matrix proteins in plasma as a potential biomarker for intervertebral disc degeneration.

PURPOSE: Recently, there has been significant focus on extracellular matrix proteolysis due to its importance in the pathological progression of intervertebral disc degeneration (IVDD). The present study investigates the circulating levels of extracellular matrix proteins in the plasma of IVDD and determines their potential relevance as biomarkers in disc degeneration. METHODS: Global proteomic analysis was performed in the plasma samples of 10 healthy volunteers (HV) and 10 diseased subjects (DS) after depletion of highly abundant proteins such as albumin and IgG. RESULTS: We identified 144 and 135 matrix-associated proteins in plasma samples from healthy volunteers (HV) and patients with disc degeneration (DS), respectively. Among these, 49 of the matrix-associated proteins were identical to the proteins found in intervertebral disc (IVD) tissues retrieved from the in-house library. Applying stringent parameters, we selected 28 proteins, with 26 present in DS and 21 in HV. 19 proteins were found common between the groups, two of which-aggrecan (ACAN) and fibulin 1 (FBLN1) - showed statistically significant differences. Specifically, ACAN was up-regulated and FBLN1 was down-regulated in the DS-plasma. In particular, DS-plasma exhibited specific expression of collagen type 2a1 (COL2A1), native to the nucleus pulposus. CONCLUSION: The distinct presence of collagen type 2a1 and the elevated expression of aggrecan in IVDD plasma may serve as the basis for the development of a potential biomarker for monitoring the progression of disc degeneration.

Risk predictive score and cord morphology classification for intraoperative neuromonitoring alerts in kyphosis surgery.

BACKGROUND: Intraoperative neuromonitoring (IONM) alert is one of the worrying events of kyphosis corrective surgery, which can result in a postoperative neurological deficit. To our knowledge, there is no risk prediction score to predict such events in patients undergoing kyphosis surgery. PURPOSE: To develop a new preoperative MRI-based cord morphology classification (CMC) and risk prediction score for predicting IONM alerts in patients with kyphotic deformity. STUDY DESIGN: Retrospective analysis of prospectively collected data. PATIENT SAMPLE: About 114 patients undergoing surgical correction for kyphotic deformity. OUTCOME MEASURES: Intraoperative neuromonitoring alerts and postoperative neurological status using AIS grading. METHODS: Kyphotic deformity patients undergoing posterior spinal fusion were retrospectively reviewed. Based on the morphology of the spinal cord and surrounding CSF in MRI, there are 5 types of cord. Type 1 (normal cord): circular cord with surrounding visible CSF between the cord and the apex, Type 2 (flattened cord): cord with <50% distortion at the apex with obliteration of the anterior CSF; Type 3 (deformed cord): cord with >50% distortion at the apex with complete obliteration of the surrounding CSF; Type 4 (stretched cord): the cord is stretched and atrophied over the apex of the curve. Type 5 (translated cord): horizontal translation of the cord at the apex with buckling collapse of the vertebral column. Preoperative radiographs were used to measure the preoperative sagittal cobbs angle, sagittal deformity angular ratio (S-DAR), sagittal vertical axis (SVA), apex of the curve, and type of kyphosis. Clinical data like the duration of symptoms, clinical signs of myelopathy, neurological status (AIS grade), grade of myelopathy using the mJOA score, and type of osteotomy were documented. Multivariate logistic regression was used to determine the risk factors for IONM alerts and the risk prediction score was developed which was validated with new cohort of 30 patients. RESULTS: A total of 114 patients met the inclusion criteria. IONM alerts were documented in 33 patients (28.9%), with full recovery of the signal in 25 patients and a postoperative deficit in 8 patients. Rate of IONM alerts was significantly higher in Type 5 (66%), followed by Type 4 (50%), Type 3 (21.1%), Type 2 (11.1%), and Type 1 (11.1%) (p-value<.001). Based on multiple logistic regression, 7 factors, namely preoperative neurological status, mJOA score≤6, presence of signs of myelopathy, apex of the curve above T5, preoperative sagittal cobbs, S-DAR, and MRI-based CMC, were identified as risk predictors. The value for the risk factors varies from 0 to 4, and the maximum total risk score was 13. The cut-off value of 6 had good sensitivity (84.9%) and specificity (77.8%) indicating a high risk for IONM alerts. The AUC of the predictive model was 0.92, indicating excellent discriminative ability. CONCLUSION: We developed and validated a risk predictive score that identifies patients at risk of IONM alerts during kyphosis surgery. Identification of such high-risk patients (risk score≥6) helps in proper evaluation and preoperative counselling and helps in providing a proper evidence-based reference for treatment strategies.

Propensity matched outcome analysis following microdiscectomy versus interlaminar endoscopic discectomy for L5-S1 disc herniation.

Purpose: The main purpose of the study is to perform a propensity-matched functional outcome analysis following microdiscectomy (MD) versus interlaminar endoscopic lumbar discectomy (IELD) for L5-S1 disc herniation. Although many studies have compared endoscopic lumbar discectomy and microdiscectomy, few have compared the outcomes of microdiscectomy (MD) and interlaminar endoscopic discectomy (IELD) at the L5-S1 level. Methods: This is a propensity-matched analysis of 100 patients (50 MD patients, 50 IELD patients) based on baseline covariates with a minimum of one-year follow-up. Patient-reported outcome measures were obtained from EMR during follow-up visits. Back pain and sciatic pain were assessed by the Visual Analogue Scale (VAS-B and VAS-L). Functional outcome was assessed using Oswestry Disability Index (ODI) Score and 12-item Short Form Survey (SF-12) score. Data were obtained at baseline (pre-op) and at 0, 1, 3, and 12 months post-operatively. Results: Mean operative time was significantly lower (p < 0.001) in the IELD group (44 min) compared to the MD group (59 min). Mean VAS-B at the immediate and 1-month postoperative period was significantly (p < 0.001) lower in the IELD group (0.36, 0.24) when compared with the MD group (1.74, 1.16). There was no significant difference between IELD and MD groups with regard to improvement in sciatic pain (VAS-L). ODI scores at 1 month and 3 months post-operative period were significantly (p < 0.001) lower in the IELD group (30.1, 23.2) when compared with the MD group (41, 27.5). However, there was no significant difference between the two groups with regards to VAS-B, ODI, and SF-12 at 1-year follow-up. Conclusion: Our findings indicate that the IELD group achieved better immediate and early postoperative outcomes despite no significant difference at one-year follow-up.

"Are we barking up the wrong tree? Too much emphasis on Cutibacterium acnes and ignoring other pathogens"- a study based on next-generation sequencing of normal and diseased discs.

BACKGROUND: The majority of literature on bacterial flora in the disc stands disadvantaged in utilizing traditional culture methods and targeting a single bacterium, Cutibacterium acnes. PURPOSE: Our objective was to document the diversity in the bacterial flora between normal and degenerated discs for shortlisting potential pathogens using next-generation genomic tools. STUDY DESIGN: Experimental case-control study. METHODS: Researchers employed 16S metagenome sequencing to profile bacterial diversity in magnetic resonance imaging normal healthy discs from brain-dead organ voluntary donors (n=20) and 40 degenerated disc samples harvested during surgery (Modic [MC]=20 and non-Modic [NMC]=20). The V3-V4 region was amplified using universal bacterial primers 341F and 806R, and the libraries were sequenced using Illumina NovoSeq 6000 platform. Statistical significance was set at bacteria with a minimum of 100 operational taxonomic unit (OTU) and present in at least 70% of the samples. The quality check-filtered reads were processed using the QIIME-2 pipeline. The OTU clustering and taxonomic classification were carried out for the merged reads using the Greengenes/SILVA reference database. Validation was done by identification of bacterial metabolites in samples using the liquid chromatography-mass spectrometry approach. RESULTS: Abundant bacteria differing widely in diversity, as evidenced by Alfa and Beta diversity analysis, were present in all control and degenerative samples. The number of bacterial genera was 27 (14-gram-positive: 13-gram-negative) in the control group, 23 (10-gram-positive: 11-gram-negative) in the Modic group, and 16 (11-gram-positive: 5-gram-negative) in the non-Modic group. In the Modic group, gram-negative bacteria OTUs were found to be predominant (more than 50% of the total bacteria identified), whereas in control and non-Modic groups the OTUs of gram-positive bacteria were predominant. Species-level analysis revealed an abundance of opportunistic gram-negative pathogens like Pseudomonas aeruginosa, Sphingomonos paucibacillus, and Ochrobactrum quorumnocens in the discs with Modic changes, more than in non-Modic discs. The presence of bacterial metabolites and quorum-sensing molecules like N-decanoyl-L-homoserine lactone, 6-hydroxynicotinic acid, 2-aminoacetophenone, 4-hydroxy-3-polyprenylbenzoate, PE (16:1(9Z)/18:0) and phthalic acid validated the colonization and cell-cell communication of bacteria in disc ruling out contamination theory. Cutibacterium acnes was not the predominant bacteria in any of the three groups of discs and in fact was in the 16th position in the order of abundance in the control discs (0.72%), seventh position in the Modic discs (1.41%), and 12th position (0.53%) in the non-Modic discs. CONCLUSION: This study identified a predominance of gram-negative bacteria in degenerated discs and highlights that Cutibacterium acnes may not be the only degeneration-causing bacteria. This may be attributed to the environment, diet, and lifestyle habits of the sample population. Though the study does not reveal the exact pathogen, it may pave the way for future studies on the subject. CLINICAL SIGNIFICANCE: These findings invite further investigation into causal relationships of bacterial profile with disc degeneration phenotypes as well as phenotype-driven clinical treatment protocols.

Breakage of a Pituitary Rongeur Tip: An Unforeseen Complication of Lumbar Microdiscectomy: A Case Report and Review of Literature.

CASE: A 46-year-old man with left leg radiculopathy due to a left L4-5 disc extrusion had a lumbar microdiscectomy that was complicated by the pituitary rongeur tip breaking in the L4-5 disc space. The rongeur tip was successfully retrieved by widening the entry access without damaging the adjoining facet and utilizing a blunt nerve hook and probe dissector. CONCLUSION: Breakage of the pituitary rongeur tip is an unforeseen complication of lumbar microdiscectomy. Surgeons should be aware of this potential complication, ideally confirm the rongeur is intact prior to wound coverage, and understand the risks versus benefits of attempting to retrieve a broken rongeur tip.

C7 distal fixation anchor and its influence on sagittal profile in posterior cervical fusion; a retrospective analysis of 44 cases.

Study design: Retrospective. Purpose: In multilevel posterior cervical fusion, whether to stop distal fixation at C7 or T1, remains a matter of debate. We aimed to assess clinical feasibility of C7 as distal fixation point and sought to compare complication rates and radiological outcome between lateral mass screws and pedicle screws at C7. Overview of literature: Current literature remains inconclusive regarding need for thoracic extension of instrumentation in multilevel posterior cervical fusion. Methods: We did a retrospective review of 44 consecutive patients who underwent posterior instrumented cervical decompression and fusion for degenerative cervical myelopathy with C7 as distal fixation point, and a minimum follow-up period of two years. We had two groups of patients based on C7 instrumentation. Group 1: Lateral mass screw fixation. Group 2: Pedicle screw fixation. Primary outcome: Post-operative clinico-radiological evaluation of whole study population Secondary outcome: Comparison of complication rates and radiological outcome between groups 1 and 2. Results: Mean age was 58.06 ± 14.4 years with average follow-up duration of 35.4 ± 4.5 months. There were 18 patients in Group 1 and 26 patients in Group 2. Mean pre-operative mJOA score was 10.51 and post-operative mJOA score was 15.74 with mean recovery rate (RR) 69.82%, of which 30 patients (70.23%) had good recovery and 14 patients (29.77%) had fair recovery at final follow up. The two groups didn't show any significant difference in complication rates and outcome. Conclusion: C7 as distal fixation anchor is safe and effective in maintaining cervical sagittal balance following multilevel posterior cervical fusion. C7 lateral mass screws are found to be equally efficacious as pedicle screws in preventing worsening of sagittal profile.

Lumbar Disc Herniation and Preoperative Modic Changes: A Prospective Analysis of the Clinical Outcomes After Microdiscectomy.

STUDY DESIGN: Prospective comparative cohort study. OBJECTIVES: The study aims to elucidate the relationship between Modic endplate changes and clinical outcomes after a lumbar microdiscectomy. METHODS: Consecutive patients undergoing microdiscectomy for lumbar disc herniation (LDH) were prospectively studied. Pre-operative clinical and radiological parameters were recorded. The pain was assessed by Numeric pain rating scale (NPRS), and functional assessment by Oswestry Disability Index (ODI). Minimal clinically important difference (MCID) in outcome was calculated for both the groups. Complications related to surgery were studied. Follow-up was done at 6 weeks, 3 months, 6 months and 1 year. Mac Nab criteria were used to assess patient satisfaction at 1 year. RESULTS: Out of 309 patients, 86 had Modic changes, and 223 had no Modic changes. Both groups had similar back pain (p-value: 0.07) and functional scores (p-value: 0.85) pre-operatively. Postoperatively patients with Modic changes had poorer back pain and ODI scores in the third month, sixth month and 1 year (p-value: 0.001). However, MCID between the groups were not significant (p-value: 0.18 for back pain and 0.58 for ODI scores). Mac Nab criteria at 1 year were worse in Modic patients (p-value: 0.001). No difference was noted among Modic types in the pre-operative and postoperative pain and functional outcomes. Four patients in Modic group (4.7%) and one patient in the non-Modic group (0.5%) developed postoperative discitis (p-value: 0.009). CONCLUSIONS: Preoperative Modic changes in lumbar disc herniation is associated with less favorable back pain, functional scores and patient satisfaction in patients undergoing microdiscectomy.

Influence of endplate avulsion and Modic changes on the inflammation profile of herniated discs: a proteomic and bioinformatic approach.

PURPOSE: The aim of this observational radiographic and proteomic study is to explore the influence of both Modic change (MC) and endplate avulsion (EPA) on the inflammation profile of herniated discs using a proteomic and bioinformatics approach. METHODS: Fifteen nucleus pulposus (NP) harvested from surgery underwent LC-MS/MC analysis, the proteome was subsequently scanned for inflammatory pathways using a bioinformatics approach. All proteins that were identified in inflammatory pathways and Gene Ontology and present in > 7 samples were integrated in a multiple regression analysis with MC and EPA as predictors. Significant proteins were imputed in an interaction and pathway analysis. RESULTS: Compared to annulus fibrosus tear (AFT), six proteins were significantly altered in EPA: catalase, Fibrinogen beta chain, protein disulfide-isomerase, pigment epithelium-derived factor, osteoprotegerin and lower expression of antithrombin-III, all of which corresponded to an upregulation of pathways involved in coagulation and detoxification of reactive oxygen species (ROS). Moreover, the presence of MC resulted in a significant alteration of nine proteins compared to patients without MC. Patients with MC showed a significantly higher expression of clusterin and lumican, and lower expression of catalase, complement factor B, Fibrinogen beta chain, protein disulfide-isomerase, periostin, Alpha-1-antitrypsin and pigment epithelium-derived factor. Together these altered protein expressions resulted in a downregulation of pathways involved in detoxification of ROS, complement system and immune system. Results were verified by Immunohistochemistry with CD68 cell counts. CONCLUSION: Both EPA and MC status significantly influence disc inflammation. The beneficial inflammatory signature of EPA illustrates that endplate pathology does not necessarily have to worsen the outcome, but the pathological inflammatory state is dependent on the presence of MC.

Can Scoliotic Discs Be Controls for Molecular Studies in Intervertebral Disc Research? Insights From Proteomics.

STUDY DESIGN: Proteomic analysis of human intervertebral discs. OBJECTIVES: To compare the characters of scoliotic discs and discs from magnetic resonance imaging (MRI)-normal voluntary organ donors controls used in disc research employing proteomics and establish "true controls" that can be utilized for future intervertebral disc (IVD) research. METHODS: Eight MRI-normal discs from 8 brain-dead voluntary organ donors (ND) and 8 scoliotic discs (SD) from 3 patients who underwent anterior surgery for adolescent idiopathic scoliosis were subjected to tandem mass spectrometry, and further analysis was performed. RESULTS: Mass spectrometry identified a total of 235 proteins in ND and 438 proteins in the SD group. Proteins involved in extracellular matrix integrity (Versican, keratins KRT6A, KRT14, KRT5, and KRT 13A1, A-kinase anchor protein 13, coagulation factor XIII A chain, proteoglycan 4) and proteins involved in transcription and DNA repair (Von Willebrand factor A domain-containing 3B, eukaryotic initiation factor 2B, histone H4, leukocyte cell-derived chemotaxin 2) were found to be downregulated in SD. Inflammatory proteins (C3, C1S), and oxidative stress response proteins (peroxiredoxin-2,6, catalase, myeloperoxidase, apolipoprotein E) were found to be upregulated in SD. These changes were reflected at the pathway level also. CONCLUSION: Findings of our study confirm that scoliotic discs have an abundance of inflammatory, oxidative stress response proteins, which are either absent or downregulated in the ND group indicating that scoliotic discs are not pathologically inert. Furthermore, this study has established MRI-normal discs from voluntary organ donors as the "true" control for molecular studies in IVD research.

Anatomical changes in vertebra in dystrophic scoliosis due to neurofibromatosis and its implications on surgical safety.

STUDY DESIGN: Detailed radiological analysis by multimodality imaging. OBJECTIVE: To document anatomical changes jeopardizing instrumentation safety in Neurofibromatosis deformity correction surgeries. MATERIALS AND METHODS: The apical and 3 adjacent vertebral segments above and below amounting to 70 segments in 10 NF scoliosis were studied by radiographs, CT and MRI. The changes in lamina, pedicle and vertebral body that could jeopardize pedicle screw and sublaminar wire placement were documented and changes were appropriately classified. RESULTS: Extensive anatomical changes were noted. These changes were more severe at the apex and independent of the curve severity. Both laminae were normal in only 36 (Type 1), rest had either gross asymmetry in length and shape (Type 2; 21) or also in sloping (Type 3; 13). Of the 140 pedicles, normal pedicles were found only in 48 (Type 1); while they were divergent (Type 2; 4) or abnormally elongated with only thinning (Type 3a; 26); or with sclerosis (3b; 34); or very curved and wavy (3c; 23) and even fractured or indistinct (Type 4; 5). It was notable that 92 of the 140 pedicles were unsuitable for pedicle screws. A unique phenomenon of body drift was identified in 29 segments which could jeopardize screw placement and rib dislocation into the canal was found in 18 segments. CONCLUSION: Gross anatomical changes jeopardizing both sublaminar wire strength and trajectory of pedicle screws were common in NF and independent of curve severity. Therefore, detailed preoperative assessment and planning by a 3D CT are essential.

Part 1: profiling extra cellular matrix core proteome of human fetal nucleus pulposus in search for regenerative targets.

Intervertebral disc degeneration is accompanied by a loss of Extra-cellular matrix (ECM) due to an imbalance in anabolic and catabolic pathways. Identifying ECM proteins with anabolic and/or regenerative potential could be the key to developing regenerative therapies. Since human fetal discs grow and develop rapidly, studying these discs may provide valuable insights on proteins with regenerative potential. This study compares core matrisome of 9 fetal and 7 healthy adult (age 22-79) nucleus pulposus (NP), using a proteomic and bioinformatic approach. Of the 33 upregulated proteins in fetus NP's, 20 of which were involved in ECM assembly pathways: fibromodulin, biglycan, heparan sulfate proteoglycan 2, chondroitin sulfate proteoglycan 4, procollagen C-endopeptidase enhancer and Collagen-type 1a1, 1a2, 6a1, 6a3, 11a1, 11a2, 12a1, 14a1 and 15a1. Moreover, 10 of the upregulated proteins were involved in growth pathways 'PI3L-Akt signaling' and 'regulation of insulin like growth factor transport and uptake.' Thrombospondin 1,3 and 4, tenascin C, matrilin-3, and collagen- type 1a1, 1a2, 6a1, 6a3 and 9a1. Additionally, matrillin-2 and 'Collagen triple helix repeat containing 1' were identified as possible regenerative proteins due to their involvement in 'Regeneration' and 'tissue development' respectively. In conclusion, the consistency of human fetal NP's differs greatly from that of healthy adults. In view of these outcomes, the core matrisome of human fetal discs contains an abundant number of proteins that could potentially show regenerative properties, and their potential should be explored in future machinal experiments.

Proteomic Signatures of Healthy Intervertebral Discs From Organ Donors: A Comparison With Previous Studies on Discs From Scoliosis, Animals, and Trauma.

OBJECTIVE: To catalog and characterize the proteome of normal human intervertebral disc (IVD). METHODS: Nine magnetic resonance imaging (MRI) normal IVDs were harvested from 9 different brain dead yet alive voluntary organ donors and were subjected to electrospray ionization-liquid chromatography tandem mass spectrometry (ESI-LC-MS/MS) acquisition. RESULTS: A total of 1,116 proteins were identified. Functional enrichment analysis tool DAVID ver. 6.8 categorized: extracellular proteins (38%), intracellular (31%), protein-containing complex (13%), organelle (9%), membrane proteins (6%), supramolecular complex (2%), and 1% in the cell junction. Molecular function revealed: binding activity (42%), catalytic activity (31%), regulatory activity (14%), and structural activity (7%). Molecular transducer, transporter, and transcription regulator activity together contributed to 6%. A comparison of the proteins obtained from this study to others in the literature showed a wide variation in content with only 3% of bovine, 5% of murine, 54% of human scoliotic discs, and 10.2% of discs adjacent to lumbar burst fractures common to our study of organ donors. Between proteins reported in scoliosis and lumbar fracture patients, only 13.51% were common, further signifying the contrast amongst the various MRI normal IVD samples. CONCLUSION: The proteome of "healthy" human IVDs has been defined, and our results show that proteomic data on IVDs obtained from scoliosis, fracture patients, and cadavers lack normal physiological conditions and should not be used as biological controls despite normal MRI findings. This questions the validity of previous studies that have used such discs as controls for analyzing the pathomechanisms of disc degeneration.