Novel Biomarkers of Health and Degeneration in Human Intervertebral Discs: In-depth Proteomic Analysis of Collagen Framework of Fetal, Healthy, Scoliotic, Degenerate, and Herniated Discs.

STUDY DESIGN: Profiling proteins expressed in the nucleus pulposus (NP) of intervertebral discs (IVDs) in five different biological states. PURPOSE: To evaluate the molecular complexity of the collagen (COL) framework and its role in the health and disease of human IVDs. OVERVIEW OF LITERATURE: Changes in COL composition have been linked to degenerative disk disease (DDD). Despite the fact that humans have 28 different types of COLs, most of the literature focuses solely on COL-1 and COL-2. This study used high-end proteomic technology to examine the entire COL composition of the human IVD across fetal (developmental-FD), normal (healthy-ND), scoliotic (early degeneration-SD), herniated (degenerate-DH), and degenerated (DD) disk phenotypes. METHODS: Forty NP tissues were snap-frozen in liquid nitrogen (-196°C) immediately before being subjected to proteomic and bioinformatic analyses from five different disk phenotypes (eight each). RESULTS: Tandem mass spectrometric analysis revealed a total of 1,050 proteins in FDs, 1,809 in ND, 1,487 in SD, 1,859 in DH, and 1,538 in the DD group. Of 28 major collagens reported in the human body, this study identified 24 different collagens with 34 subtypes in NP. Fibril-forming collagens (COL-1, 2, and 11A1) and fibril-associated collagens with interrupted triple helices (COL-9A1, 12A1, and 14A1) were abundantly expressed in FDs, representing their role in the development of NP. Multiplexin (COL-15), a hybrid proteoglycan-collagen molecule, was discovered only in FDs. Degeneration was associated with COL2A1 downregulation and COL-10A1 upregulation. CONCLUSIONS: COL10 was discovered to be a new biomarker for disk degeneration. Besides COL-1 and 2, other important COLs (6, 9, 11, 12, 14, 15) with anabolic potential and abundant expression in the fetal phenotype could be investigated for tissue engineering and novel DDD therapy.

Bacteria in human lumbar discs - subclinical infection or contamination? Metabolomic evidence for colonization, multiplication, and cell-cell cross-talk of bacteria.

BACKGROUND CONTEXT: The accumulating evidence associating sub-clinical infection with disc degeneration (DD) and the controversy of contamination versus infection mandates a further understanding of the microbial activity in the disc and host-microbiome interaction. PURPOSE: To utilize a novel approach of metabolomics to probe the presence of bacterial metabolites involved in colonization, survival, and replication in human lumbar intervertebral discs (LIVD). STUDY DESIGN: An observational case-control study. PATIENT SAMPLE: Nucleus pulposus from the LIVD of three brain-dead voluntary organ donors (MRI normal and classified as controls) and of three patients undergoing surgery for disc degeneration (DD) (cases) were utilized. METHODS: Untargeted metabolite profiling was carried out in six discs (3-controls and 3-cases) after extraction using methanol: acetonitrile: water (2:2:1) solvent system and acquired through HPLC-MS/MS platform using C18 reversed-phase column. From the total IVD metabolome, microbial metabolites were filtered by mapping against HMDB, ChEBI, SigMol, Siderophore database, ecdmb database, and PaMet databases. The biological functions of the metabolites were then studied by MSEA pipeline from Metaboanalyst, and the enrichment ratio, p-value, and Variably Importance Projection scores of the metabolites were calculated. Degeneration responsive changes in the abundance of the microbial metabolites were calculated based on the peak intensities between the control and cases. RESULTS: Mass spectrometry identified a total of 17601 and 15003 metabolites, respectively, in the control and degenerated discs. Preliminary mapping of the above metabolites against HMDB indicated the multiple sources, and of these, 64 metabolites were of microbial origin, accounting for 1.6% of the total IVD metabolome. Principle Component Analysis and Orthogonal Partial Least Square-Discriminant Analysis (OPLS-DA) showed distinct clustered patterns between control and disc degene`ration, indicating a strong variation in concentration, peak, and spectral values of the 64 metabolites between controls and cases. After the exclusion of metabolites that were also associated with humans, drugs, and food, 39 metabolites specific to bacteria were isolated. Nine were primary metabolites related to bacterial growth and survival, and the remaining 30 were secondary metabolites related to different environmental stress response activities. The three significant pathways (p<.001) which were predominant in the bacterial metabolites were autoinducer-2 biosynthesis, peptidoglycan biosynthesis, and chorismate pathway. In addition, a significant fold change of >1.0 was found for nine metabolites which included (S)-14-Methyilhexadecanoic acid related to P. acnes, 9-OxoODE, and 13-OxoODE related to gut flora, vibriobactin - a siderophore, tuberculosinol and iso-tuberculosinol, virulence factors of M. tuberculosis. There was also upregulation of Autoinducer- 2, an important "Quorum sensing molecule" involved in bacterial cross-talk. CONCLUSION: We identified several bacterial-specific metabolites participating in bacterial growth, survival, and cross-talk pathways. These were found in both groups but up-regulated in degenerated discs. The presence of Quorum sensing molecules and cell-cell interactions provides firm proof of colonization and growth. These findings indicate that the bacterial presence may not be mere contamination but could be colonization with a possible role in infection-mediated inflammation in DD. CLINICAL SIGNIFICANCE: Proof of subclinical infection as an initiator of DD and documentation of exact germ and drug sensitivity will change the way millions of patients with non-specific low back pain (NSLBP) are treated across the world.

In Vertebral Hemangiomas with Neurological Deficit, Is a Less Extensive Approach Adequate.

STUDY DESIGN: This was a retrospective study. PURPOSE: To analyze the surgical and neurological outcomes following surgical decompression in patients with aggressive vertebral hemangioma (AVH) presenting with neurological deficit and to determine whether a less extensive approach is appropriate. OVERVIEW OF LITERATURE: AVHs are a rare subset of benign vascular tumors frequently presenting with neurological deficit because of spinal cord compression. Though the results of surgical management have improved over time, there is a lack of consensus on the ideal management in this group of patients. METHODS: Twenty-one patients who underwent surgery for AVH between 2009 and 2018 were analyzed. Demographic and clinical details of patients were retrieved from hospital information system. Imaging information (i.e., radiography, computed tomography, magnetic resonance imaging) of all patients was accessed and analyzed in picture archiving and communication system. Tumor staging was performed using Enneking and Weinstein-Boriani-Biagini classifications and Spinal Instability Neoplastic Score. At followup, neurological and radiological evaluations were performed. RESULTS: Twenty-one patients (13 [61.9%] females and 8 [38.1%] males) were included with a mean age of 44.29 years (range, 14-72 years). All patients in the study had neurological deficit. Back pain was present in 80.9% of patients. Mean duration of symptoms was 4.6 months (range, 1 day to 10 months). Most common lesion location was thoracic spine (n=12), followed by thoracolumbar (D11- L2; n=7) and lumbar (n=2) regions. Ten patients had multiple level lesions. All patients underwent preoperative embolization. Nine patients underwent intralesional spondylectomy with reconstruction; another nine patients underwent stabilization, decompression, and vertebroplasty; three patients underwent decompression and stabilization. Neurology improved in all patients, and only one case of recurrence was noted in a mean follow-up of 55.78±25 months (range, 24-96 months). CONCLUSIONS: In AVH, good clinical and neurological outcomes with low recurrence rates can be achieved using less extensive procedures, such as posterior instrumented decompression with vertebroplasty and intralesional tumor resection.

Comparative metagenomic analysis of human intervertebral disc nucleus pulposus and cartilaginous end plates.

Study design: The diversity of microflora inhabiting endplate (EP) and nucleus pulposus (NP) tissues of human intervertebral disc (IVD) was profiled through NGS-supported 16S rRNA amplicon sequencing. Sixteen EP and their corresponding NP were excised from the brain-dead voluntary organ donors with no clinical history of low back pain, and 12 herniated and 8 degenerated NP tissues isolated from the patients undergoing spinal surgery were subjected to study the alteration in the microbial diversity. Objectives: To understand in normal IVD, whether the colonization of bacteria to the NP is through the EP in discs with intact annulus fibrosus. To identify significantly differing microbial population(s) between normal and diseased IVD (NP). Background of the study: There is increasing evidence for subclinical infection by fastidious low, growing bacteria to be a cause of disc degeneration. Although the presence of bacteria in NP has been reported well in literature, the source of bacteria is not clearly proved as the disc is avascular in healthy condition. Documentation of similar bacterial populations in the EP and NP may add proof that bacterial inoculation of NP occurs via the EP. Materials and methods: Sixteen EP and their corresponding NP excised from brain-dead voluntary organ donors with no history of back pain and 20 diseased discs collected from patients undergoing microdiscectomy/fusion surgery were used for profiling microbiome through 16S rRNA amplicon sequencing using primers specific for V1-V9 hypervariable regions. Changes in bacterial diversity and abundance were analysed to identify the key microbial populations in normal IVD NP and EP tissues and those significantly altered in diseased IVD (NP). Results: NP and EP shared a similar spectrum of microbiome but with varying abundance. The five dominant phyla identified were Proteobacteria, Firmicutes, Actinobacteria, OD1, and Bacteroidetes. Proteobacteria was found to be the most abundant phyla in both NP (62%) and EP (53%) of the normal IVD. This was followed by Firmicutes (16%), Actinobacteriota (11%), OD1 (Parcubacteria) (7.6%), and Bacteroidetes (2%) in NP and Firmicutes (23.4%), OD1 (Parcubacteria) (17.6%), Actinobacteriota (2.8%), and Bacteroidetes (2.6%) in EP, respectively. Under diseased conditions, Proteobacteria (68%) was dominant when compared with other phyla. However, there was no significant difference in the abundance of Proteobacteria between the normal and diseased discs. Interestingly, the other dominant phyla such as Firmicutes (Normal-NP: 16.2%; Diseased-NP: 4.02%) and Actinobacteria (Normal-NP: 11%; Diseased-NP: 0.99%) showed a significant reduction in degenerated discs. To understand the key microbial populations that are significantly altered during disease, correlation analysis was performed among the three phyla, which revealed a negative correlation in the ratio of Actinobacteria + Firmicutes vs. Proteobacteria (p = 0.001) in DD. Conclusion: Results of our study clearly demonstrated a similar bacterial diversity but with varying abundance between the EP and NP, suggesting the existence of the endplate-nucleus pulposus axis in the normal IVD microbiome. Further, our results have indicated that the changes in the abundance of Actinobacteria + Firmicutes vs. Proteobacteria during DDD need further investigation.

Impact of Patient Counseling and Socioeconomic Factors on Initiation of Rehabilitation Program in Spinal Cord Injury Patients Presenting to a Tertiary Spine Unit in India.

STUDY DESIGN: Prospective case series. PURPOSE: This study aimed to investigate the impact of education, financial income, occupation, and patient counseling on the timing of enrolment in a spinal cord injury (SCI) rehabilitation program. OVERVIEW OF LITERATURE: A rehabilitation program following SCI is essential to improve functional outcomes. Socioeconomic factors can affect the timing of enrolment to a rehabilitation program. Literature on the effects of socioeconomic factors among patients with SCI in the Indian scenario is limited. METHODS: A prospective, consecutive analysis of patients with SCI was performed with 1-year follow-up. Assessment of the timing of enrolment to a rehabilitation program was performed using the modified Kuppuswamy socioeconomic scores (MKSS). Patients admitted to the SCI unit (group A), underwent intensive individual, group, and family counseling sessions to encourage early enrolment into a rehabilitation program. Patients presenting directly for rehabilitation (group B) were analyzed for comparison. RESULTS: A total of 153 patients were recruited. Group A was composed of 122 patients who started the rehabilitation program after a mean of 28 days, compared with a mean of 149 days for 31 patients in group B. In group A, 104 patients (85%; mean MKSS, 14.02) and 18 patients (15%; mean MKSS, 15.61) enrolled for rehabilitation <6 weeks and ≥6 weeks, respectively. In group B, 12 patients (39%; mean MKSS, 13.69) and 19 patients (61%; mean MKSS, 12.10) enrolled for rehabilitation <6 weeks and ≥6 weeks, respectively. The total MKSS and scores for education, income, and occupation did not show a significant difference between the two both groups (p>0.05). CONCLUSIONS: Early patient counseling in the acute care unit helps in the early enrolment of patients with poor socioeconomic demographic profile to a rehabilitation program.

Proteomic Signature of Nucleus Pulposus in Fetal Intervertebral Disc.

STUDY DESIGN: Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). PURPOSE: To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. OVERVIEW OF LITERATURE: Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. METHODS: IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. RESULTS: A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. CONCLUSIONS: This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.

Inflammaging determines health and disease in lumbar discs-evidence from differing proteomic signatures of healthy, aging, and degenerating discs.

BACKGROUND CONTEXT: The true understanding of aging and disc degeneration (DD) is still elusive. MRI has not helped our attempts to understand the health and disease status of the discs as it reflects mainly the end morphologic changes and not the changes at a molecular level. Understanding degeneration at a molecular level through proteomics might allow differentiation from normal aging and also aid in the development of biomarkers for early diagnosis and preventive therapies. PURPOSE: To utilize proteomics to understand the molecular basis of healthy, aging, and degenerating discs and conclusively differentiate normal aging and degeneration. STUDY DESIGN: Proteomic analysis of human intervertebral disc samples. METHODS: L4-L5 disc samples from three groups were acquired and subjected to proteomic analysis. Samples from individuals aged in the second, third, and fourth decades were used to represent young healthy discs (Group A). Those from MRI normal donors aged in the fifth, sixth, and seventh decades represented normal aging (Group B). Five degenerated discs obtained from patients at surgery represented degeneration (Group C). The entire proteome map and alteration in protein expressions were further analyzed using bioinformatics analysis. This was a self-funded project. RESULTS: There were 84 common proteins. Specific proteins numbered 225 in A, 315 in B, and 283 in C. By gene ontology biological process identification, Group A predominated with extracellular matrix organization, cytoskeletal structural and normal metabolic proteins. Group B differed in having additional basal expression of immune response, complement inhibitors, and senescence proteins. Group C was different, with upregulation of proteins associated with oxidative stress response, positive regulators of apoptosis, innate immune response, complement activation and defense response to gram positive bacteria indicating ongoing inflammaging. CONCLUSIONS: Our study documented diverse proteome signatures between the young, aging and degenerating discs. Inflammaging was the main differentiator between normal biological aging and DD. CLINICAL SIGNIFICANCE: Multiple inflammatory molecules unique to DD were identified, allowing the possibility of developing specific biomarkers for early diagnosis and thereby provide evidence-based metrics for preventive measures rather than surgical intervention and also to monitor progress of the disease.

Does Sarcopenia Increase the Risk for Fresh Vertebral Fragility Fractures?: A Case-Control Study.

STUDY DESIGN: Case-control study. PURPOSE: Sarcopenia is an age associated condition characterized by decrease in muscle mass, strength, and physical performance. We aimed to investigate whether sarcopenia increased the risk of vertebral fragility fractures among the elderly. OVERVIEW OF LITERATURE: Initial reports on sarcopenia suggest its contribution to the development of vertebral fragility fractures. However, recent studies showed contradictory findings. METHODS: Fifty-one consecutive patients with vertebral fragility fractures and matched controls without fractures were evaluated for sarcopenia, T-score, body mass index, and presence of preexisting vertebral fractures. Sarcopenia was diagnosed as total psoas cross-sectional area (TPA) 2 standard deviations below normative value from normal young adults and decreased handgrip strength (26 kg for men and 18 kg for women). Univariate and multivariate analyses were performed using the fresh fracture occurrence as the dependent variable. RESULTS: Sarcopenia was confirmed in 29.4% and 7.8% of cases and controls (p=0.005), respectively; 56.8% and 13.7% of cases and controls had previous vertebral fractures. Sarcopenia prevalence was greater among those with previous fractures (38% vs. 7.6%; odds ratio, 7.76; p<0.001). TPA was lower among the cases (1,278 mm2 vs. 1,569 mm2 , p=0.001) and those with previous fractures (1,168 mm2 vs. 1,563 mm2 , p<0.001). Handgrip strength was greater among those without previous fractures (19.6 kg vs. 16.3 kg, p=0.05). In multivariate analysis, sarcopenia was not identified as a significant predictor of fresh fractures whereas previous fractures and lower T-score were found to be significant. CONCLUSIONS: Sarcopenia is not an independent risk factor for fresh vertebral fragility fractures in the elderly.