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Abstract Book 2021 MAY 31 – JUNE 1: COMMITTEE MEETINGS JUNE 2‐4: SCIENTIFIC PROGRAM ABSTRACT BOOK Oral presentations at the ISSLS Virtual Annual Meeting, May 31‐June 4, 2021 O01 Human Lumbar Discs are not sterile! - Defining the Normal Intervertebral Disc Microbiome Shanmuganathan Rajasekaran1, Soundararajan Dilip Chand Raja1, Tangavel Chitraa2, Nayagam Sharon Miracle2, Muthurajan Raveendran3, Matchado Monica Steffi2, K S Vijayanand1 1. Ganga hospital, Coimbatore, TAMIL NADU, India 2. Department of Proteomics, Ganga Research Centre, Coimbatore, Tamil Nadu, India 3. Department of Plant Biology , Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India Introduction: Traditionally, Central Nervous System, spinal cord, eye, fetus and Intervertebral Disc were considered to be to be sterile and immune privileged. With the advent of 16SrRNA sequencing, it has been proved that each of the above have a microbiome which plays an essential role in maintaining homeostasis. With increasing reports of sub-clinical infection as an etiology of disc degeneration and Modic changes, it remains unknown, whether lumbar discs are truly sterile. The primary aim of the study was to investigate the presence of Disc microbiome and its microbial composition. Materials and Methods: In order to obtain true normal controls, intervertebral discs were collected from MRI normal brain-dead voluntary organ donors with no history of back pain. The study was performed after the approval of IRB. The discs were dissected in surgical sterile conditions and were snap frozen immediately to -160⁰C and the processed samples were subjected to total DNA extraction using QIAamp® DNA Mini Kit and enrichment using NEBNext® Microbiome DNA Enrichment Kit (Cat # E612S/L; New England BioLabs, Ipswich, MA). The purified DNA was amplified using v1-V9 specific primers and the sequencing was performed using Illumina MiSeq platform. Raw data was analysed using Bioinformatic tools such as Greengenes database and kraken2. Proteomic analysis was also performed to confirm bacterial presence. Results: All the eight normal disc samples had bacteria. 42.75% OTUs were classified at the Kingdom level, 44% at Phylum level, 22.62% at Genus level and 5.5% at Species level. A total of 355 bacterial species were identified in normal discs which were distributed under five major phyla- Proteobacteria, Parcubacteria, Firmicutes, Cyanobacteria and Actinobacteria. 21 abundant genera including Pseudomonas, Acinetobacter, Anoxybacillus, Sphingomonas, Bordetella, and Brevundimonas were identified. At species level, the five most abundant bacteria identified were Anoxybacillus kestanbolensis, Acinetobacter lwoffii, Sphingomonas yabuuchiae, Stenotrophomonas acidaminiphila and Pseudomonas veronii. The much- discussed Propionibacterium acnes ranked ninth in abundance in the normal discs. Bacterial presence was confirmed through proteomics data by identification of bacteria specific proteins and vital enzymes. Conclusion: This is the first metagenome study in world literature to document the entire microbiome of MRI normal disc. The results of this study prove that lumbar discs are not sterile structures as traditionally thought. This brings out the question of what spectrum of organisms could invoke sub-clinical infection and disc degeneration. In addition, the presence of Propionibacterium acnes in normal disc samples raises a doubt in its etiopatholgical role in disc degeneration. It would be interesting to analyze the microbiome of degenerated discs to analyze whether a differential microbial composition is associated with disc degeneration. 2 Oral presentations at the ISSLS Virtual Annual Meeting, May 31‐June 4, 2021 O02 Kyphosis and early disc degeneration induced by paraspinal muscle insufficiency in female TSC1mKO mice Wing Moon Raymond Lam1, Wenhai Zhuo1, Way Cherng Chen2, Elisa Mariae Combie3, Kimberly Anne Tan4, Simon Cool5, Chloe Xiaoyun Chan4, Shih-Yin Tsai3, Hwee Weng Dennis Hey1 1. Department of Orthopaedic Surgery, YLL School of Medicine, National University of Singapore, Singapore 2. Bruker Centre, SBIC, A*Star, Singapore 3. Physiology, YLL School of Medicine, National University of Singapore, Singapore 4. Orthopaedic Surgery, National University Health System, Singapore 5. Institute of Medical Biology:: Agency for Science, Technology and Research, Singapore INTRODUCTION: The progression of degenerative thoracolumbar kyphosis is known to be associated with intervertebral disc degeneration and sarcopenia. However, the cause-effect relationship remains unclear as human cohort studies are confounded by various comorbidities. TSC1 inhibitor muscle knockout (TSC1mKO) mice has constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) leads to chronic muscle loss and kyphosis in later stages of their life cycle [1]. The aims of this study are to understand the roles of paraspinal muscle insufficiency, intervertebral disc degeneration and trabecular bone architectural changes in the development of kyphosis in female TSC1mKO mice. METHODS: Twenty-four female mice were studied at 9 months (TSC1mKO, n = 3 and control, n= 7) and 12 months (TSC1mKO, n = 7 and control, n = 7), respectively. High resolution MicroCT was used to measure thoracolumbar kyphosis, disc height, and trabecular bone architecture. Myopathic changes in the paraspinal muscle were examined via H&E stain and Wheat Germ Agglutinin (WGA)/DAPI stain, and the extent of intervertebral disc degeneration was evaluated via FAST stain. RESULTS: The H&E stained paraspinal muscle sections from female TSC1mKO mice shows signs of myopathic alterations at 12 months, including the presence of central nuclei, triangular fibres and vacuole formation (Figure 1A). WGA/DAPI stain analysis showed greater heterogeneity in muscle fiber size distribution (Figure 1B) and increase central nucleus formation/fiber in TSC1mKO mice compared to controls (TSC1mKO 20.0 ± 5.0% vs control 3.1 ± 3.2%, p=0.034). These confirm the TSC1mKO does accelerate paraspinal muscle insufficiency. Development of thoracolumbar kyphosis is more significant in TSC1mKO mice than the control mice at 12 months (Figure 1C) (TSC1mKO 83.67 ± 17.18 vs control 52.23 ± 14.35°, p < 0.01). Comparing disc histological examination, mild NP cleft/fissure an early degeneration of the TSC1mKO mice disc is observed at 9 months before disc height loss is detectable on microCT (Figure 1D). At 12 months, AF clefts/fissure, more severe NP cleft/fissure and reduction of disc height are observed in TSC1mKO mice disc. The result matched with significantly greater reduction of disc height at L3/4 (TSC1mKO 166 ± 38µm vs control 235 ± 37 µm, p= 0.013) and L4/5 (TSC1mKO 164 ± 27 µm vs control 247 ± 59 µm, p= 0.027) in 12 months old TSC1mKO mice. Compared to the aging mice, vertebral bone density of lumbar spine is higher in 9 and 12 months old TSC1mKO mice (L4: 27.97 ± 9.17% vs 13.18 ± 5.57%, p=0.037) as there is increase trabecular thickness (113 ± 21 vs 81 ± 3µm, p= 0.0015) and number (2.45± 0.50 vs 1.25 ±0.59/mm, p=0.0046). This suggests that osteoporosis does not have an effect on the development of kyphosis. DISCUSSION: This study demonstrates that paraspinal muscle insufficiency can accelerate the development of thoracolumbar kyphosis and cause early degeneration of the intervertebral disc. It provides a strong rationale for further study on the treatment of paraspinal muscle insufficiency to reduce susceptibility to degenerative thoracolumbar kyphosis and degenerative disc disease. 1. Castets P, Lin S, Rion N, Di Fulvio S, Romanino K, Guridi M, Frank S, Tintignac LA, Sinnreich M, Rüegg MA. Sustained activation of mTORC1 in skeletal muscle inhibits constitutive and starvation-induced autophagy and causes a severe, late- onset myopathy. Cell Metab. 2013 May 7;17(5):731-44. 3 Oral presentations at the ISSLS Virtual Annual Meeting, May 31‐June 4, 2021 O03 Chromatin accessibility maps of human nucleus pulposus cells identify degeneration grade- specific epigenome signatures and gene regulatory networks Steven M. Presciutti1, 2, Nazir M. Khan1, 2, Justin Cotney3, Hicham Drissi1, 2 1. Emory University, Atlanta, GA, USA 2. Atlanta Veteran Affairs Medical Center, Decatur, GA, USA 3. University of Connecticut School of Medicine, Farmington, CT, USA INTRODUCTION: Intervertebral disc degeneration (IDD) is characterized by substantial clinical heterogeneity despite relatively few conserved genetic alterations. To provide a basis for studying epigenome dysregulation in the setting of IDD, we present unbiased genome-wide single-cell (sc) chromatin accessibility maps for both healthy and degenerated human nucleus pulposus (NP) cells measured by the scATAC-seq assay, a first in the field. METHODS: This study was approved by the ethics committees of Emory University (IRB# 00099028). Human NP cells were selected to reflect the clinical and biological heterogeneity of IDD, including those isolated from: (1) a Pfirrmann grade 1 disc (no degeneration) from an 18yo female, (2) a Pfirrmann grade 3 disc (mild/moderate degeneration) from a 35yo male, (3) a Pfirrmann grade 5 disc (severe degeneration) from a 66yo female. Additionally, notochordal cells from embryonic NP (ScienCell, CA) were included to provide an embryonic reference point (hereafter ‘NP-E’). scATAC was performed using the 10X Genomics Chromium platform and libraries were sequenced (paired-end 40bp reads, Illumina NextSeq500). Peaks were identified for each cluster using ArchR using default parameters (FDA cutoff = 0.01, Log2FC cutoff = 1) and a Markov
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