P2. MiRNA-seq analysis of human vertebrae provides insight into the mechanism underlying GIOP

The Spine Journal 19 (2019) S158−S194

ePosters: Basic Science/Biologics P1. Improvement of motor function induced by skeletal muscle contraction in spinal cord injury rats Norito Hayashi, MD1, Naoyuki Himi, PhD1, Emi Nakamura-Maruyama, PhD1, Naohiko Okabe, PhD, DVM2, Issei Sakamoto, MD1, Toru Hasegawa, MD, PhD1, Osamu Miyamoto, MD, PhD1; 1 Kawasaki Medical School Hospital, Matushima Kurashiki, Okayama, Japan; 2 Second Department of Physiology, Kawasaki Medical School, Kurashiki City, Okayama, Japan BACKGROUND CONTEXT: Previous reports have shown that treadmill training after spinal cord injury (SCI) contributes to functional recovery and is associated with increased levels of neurotrophic factors in the spinal cord in a rat model. However, the precise mechanism underlying the above observation has not been elucidated. Brain-derived neurotrophic factor (BDNF) is associated with motor function recovery due to its neuroprotective and axon sprouting effects. Promoting BDNF expression in spinal cord lesions by strategies such as physical training and cell transplantation has been reported to be effective in treating SCI in animals. Myokines are muscle-derived bioactive molecules associated with muscle contraction. Although BDNF is a myokine and considered a potential mediator of neuroplasticity following exercise, its contribution to motor function recovery after SCI has not yet been described in detail. PURPOSE: To investigate the role of muscle contraction in motor function recovery after SCI, with a focus on BDNF. STUDY DESIGN/SETTING: Eight-week-old Sprague-Dawley male rats were used to establish the SCI model. Percutaneous electrical muscle stimulation (10 mA, 2 Hz, 10 min) was applied to both limbs of the rats immediately after SCI. The stimulation was performed once per day for 4 weeks. The sham, SCI (SCI), and SCI with electrical muscle stimulation (SCIapt-ent-b 002B apt-ent-e + apt-ent-uES) groups were compared. OUTCOME MEASURES: The effect of electrical muscle stimulation was assessed by the motor function recovery. Histological assessments were done by measuring the cavity volume, the amount of axon sprouting, the number of apoptotic cells, and the amount of BDNF. METHODS: Spinal injury was induced by dropping a 20 g rod with a 2 mm apex diameter from a height of 25 mm onto the spine of an anesthetized rat at the T9 level. Motor function was assessed using the Basso-Beattie-Bresnahan Locomotor Scale (BBB score), the inclined plane test, and the Rotarod test. One week after the injury, transferase dUTP nick-end labeling (TUNEL)-positive cells were counted at the injury epicenter, and the level of BDNF was measured in both the spinal cord and the lower limb muscle. Four weeks after injury, the cavity volume of the epicenter and the level of phosphorylated growth-associated protein 43 (pGAP43) in the spinal cord were measured. RESULTS: A significantly improved BBB score and inclined plane test results were observed in the SCIapt-ent-b 002B apt-ent-e + apt-ent-uES group when compared to the SCI group, 4 weeks post-SCI. We also observed a decrease in the cavity volume and an increase in pGAP43 levels in the SCIapt-ent-b 002B apt-ent-e + apt-ent-uES group. Electrical muscle stimulation decreased the numbers of TUNEL-positive cells in the epicenter and increased the levels of BDNF in the spinal cord and lower limb muscles 1 week post-SCI. CONCLUSIONS: Electrical muscle stimulation improved motor function and increased BDNF levels in both the muscles and the spinal cords of rats

subjected to SCI. Muscle contraction-induced BDNF expression might be involved in motor recovery during rehabilitation. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. https://doi.org/10.1016/j.spinee.2019.05.425

P2. MiRNA-seq analysis of human vertebrae provides insight into the mechanism underlying GIOP Xiang Yu, PhD1, Hui Ren2, Jiang Xiaobing, PhD2, De Liang, MD3; 1 Guangzhou University of Chinese Medicine, Guangzhou, Baiyun/ Guangdong, China; 2 1st Affiliated Hospital of Guangzhou University of Chinese Medicine, Guang Zhou City, China; 3 Guangzhou, China BACKGROUND CONTEXT: High-throughput sequencing (HTS) was recently applied to detect microRNA (miRNA) regulation in age-related osteoporosis. However, miRNA regulation has not been reported in glucocorticoid-induced osteoporosis (GIOP) patients and the mechanism of GIOP remains elusive. PURPOSE: To comprehensively analyze the role of miRNA regulation in GIOP based on human vertebrae and to explore the molecular mechanism. STUDY DESIGN/SETTING: A high-throughput sequencing strategy was employed to identify miRNAs involved in GIOP. Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 26 vertebral samples were verified by qRT-PCR. Bioinformatics was utilized for target prediction, to investigate the regulation of miRNA-mRNA networks, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. PATIENT SAMPLE: Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 26 vertebral samples were verified by qRT-PCR. OUTCOME MEASURES: A high-throughput sequencing strategy was employed to identify miRNAs involved in GIOP. Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 26 vertebral samples were verified by qRT-PCR. Bioinformatics was utilized for target prediction, to investigate the regulation of miRNA-mRNA networks, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. METHODS: A high-throughput sequencing strategy was employed to identify miRNAs involved in GIOP. Twenty-six patients undergoing spinal surgery were included in this study. Six vertebral samples were selected for miRNA sequencing (miRNA-seq) analysis and 6 vertebral samples were verified by qRT-PCR. Bioinformatics was utilized for target prediction, to investigate the regulation of miRNA-mRNA networks, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. RESULTS: Six significantly up-regulated miRNAs (including one novel miRNA) and three significantly down-regulated miRNAs were verified via miRNA-seq and verified in the vertebrae of GIOP patients. Up-regulated miRNAs included hsa-miR-214-5p, hsa-miR-10b-5p, hsa-miR-21-5p, hsamiR-451a, hsamiR-186-5p, and hsa-miR-novel-chr3_49,413 while downregulated miRNAs included hsa-let-7f-5p, hsa-let-7a-5p, and hsa-miR27a-3p. Bioinformatics analysis revealed 5,983 and 23,463 predicted targets in the up-regulated and down-regulated miRNAs respectively, using

Refer to onsite annual meeting presentations and postmeeting proceedings for possible referenced figures and tables. Authors are responsible for accurately reporting disclosure and FDA device/drug status at time of abstract submission.

Proceedings of the 34th Annual Meeting of the North American Spine Society / The Spine Journal 19 (2019) S158−S194 the miRanda, miRBase and TargetScan databases. The target genes of these significantly altered miRNAs were enriched to 1,939 GO terms and 84 KEGG pathways. GO terms revealed that up-regulated targets were most enriched in actin filament-based processes (BP), anchoring junction (CC), and cytoskeletal protein binding (MF). Conversely, the down-regulated targets were mostly enriched in multicellular organismal development (BP), intracellular membrane-bounded organelles (CC), and protein binding (MF). Top-10 pathway analysis revealed that the differentially expressed miRNAs in GIOP were closely related to bone metabolismrelated pathways such as FoxO, PI3K-Akt, MAPK and Notch signaling pathway. CONCLUSIONS: These results suggest that significantly altered miRNAs may play an important role in GIOP by targeting mRNA and regulating biological processes and bone metabolism-related pathways such as MAPK, FoxO, PI3K-Akt and Notch signaling, which provides novel insight into the mechanism of GIOP and lays a good foundation for the prevention and treatment of GIOP. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. https://doi.org/10.1016/j.spinee.2019.05.426

P3. Perioperative immunonutrition in spine and total joint surgery Michael Shumaker, DO1, Bryan P. Hooks, DO1, Dhanunjay S. Boyalakuntla, PhD2, Matt S. Bishop, CRNP3; 1 Horizon Orthopedics, Hermitage, PA, US; 2 Canfield, OH, US; 3 TRMC, Butler, PA, US BACKGROUND CONTEXT: In the current era of adding value to health care, modifiable risk factors have become a major focus. Quality and outcomes are now measured, reported, and tied to reimbursement. Recent studies in orthopedic surgery and spine surgery have shown nutritional status to be one of the most significant risk factors for post-operative complications. Our purpose for this study is to evaluate if perioperative nutritional support can reduce post-operative complications and improve outcomes in orthopedic and spine surgery patients. PURPOSE: To evaluate if perioperative nutritional support with immunonutrition containing arginine, glutamine, omega 3 fatty acids, and nucleotides can reduce post-operative complications and improve outcomes in orthopedic and spine surgical patients. STUDY DESIGN/SETTING: For a 12-month period from November 2016 to October 2017 we prospectively collected data for inpatient total joint replacements and spine surgeries occurring at two hospitals. Surgeries were performed by one of two fellowship trained Orthopedic spine surgeons. PATIENT SAMPLE: A total of 125 patients were included. Patients were excluded from the study if they were receiving radiation, or chemotherapy, at the time of surgery or were non compliant with the nutritional protocol. Included patients were adults over the age of 18 undergoing elective spine or total joint replacement surgery OUTCOME MEASURES: Postoperative complications, readmissions, reoperations were recorded. Number needed to treat and absolute risk reduction were calculated to evaluate results. METHODS: Data were prospectively collected for inpatient total joint replacements and spine surgeries for 12 months beginning November 2016 and ending October 2017. All patients were followed a minimum of 90 days after surgery. The immunonutrition group was instructed to consume two 8 oz bottles of Ricochet Nutrition (Revive Medical, Brighton, MO USA) each day for 6 days before, and 6 days after, surgery. Outcome data was collected on reoperations, readmissions, and complications including: surgical site infections, Pneumonia, UTI, sepsis, and acute MI. RESULTS: A total of 125 patients (62 in immunonutrition group and 63 in the control group) were included and no difference was found between the two groups regarding surgery type, comorbidities, sample size, and demographics. The control group had a total of 8 patients with complications after surgery: 1 pneumonia, 3 UTIs, 1 incisional hernia, and 2 deep surgical site infections. There was a total of 3 reoperations and 4 readmissions in

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the control group. The immunon trition group had no reoperations, no readmissions, and no complications. CONCLUSIONS: Our study provides strong evidence that perioperative immunonutrition (containing Arginine, Glutamine, Omega 3 FAs and Nucleotides) reduces complications, reoperations and readmission for inpatient orthopedic and spine surgery patients.; Patient optimization prior to surgery should include optimizing nutritional status with immunonutrition. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. https://doi.org/10.1016/j.spinee.2019.05.427

P4. Autologous blood coagulum as a native carrier for rhBMP6 induces new bone formation and posterolateral lumbar spine fusion in rabbits and sheep Munish C. Gupta, MD1, Lovorka Grgurevic, MD, PhD2, Marko Pecin, PhD3, Igor Erjavec, PhD4, Marija Lipar, PhD5, Jadranka Bubi c, DVM, PhD4, Drazen Maticic, PhD, MSc3, Drazen Vnuk, PhD3, Kuber T. Sampath, PhD6, Slobodan Vuki cevi c, MD, PhD4; 1 Washington University School of Medicine, St. Louis, MO, US; 2 Medical School University of Zagreb, Zagreb, Croatia; 3 School of Veterinary Medicine, Zagreb, Zagreb, Croatia; 4 School of Medicine, University of Zagreb, Zagreb, Croatia; 5 Faculty of Veterinary Medicine University of Zagreb, Zagreb, Croatia; 6 perForm Biologics Inc, Holliston, MA, US BACKGROUND CONTEXT: RhBMP2 soaked in bovine tendon collagen and synthetic ceramics composite as well as rhBMP7/OP1 lyophilized in bovine bone collagen particles with additive carboxyl-methyl cellulose have been evaluated for posterolateral lumbar fusion (PLF). We investigated rhBMP6 delivered in autologous blood coagulum (ABC), a native carrier (scaffold), with and without allograft bone particles (ALLO) to induce new bone formation and promote posterolateral lumbar spine fusion (PLF) in rabbit and sheep. PURPOSE: To evaluate the efficacy of RhBMP6 with autologous blood coagulum (ABC) alone or with ABC and allograft (ALLO) will induce a successful posterolateral spinal fusion in animal models. STUDY DESIGN/SETTING: Preclinical evaluation of spine fusion in animal models. PATIENT SAMPLE: Multiple experimental groups of rats, rabbits and sheep. OUTCOME MEASURES: Radiographic and histologic evaluation for bone formation and spinal fusion. METHODS: An autologous bone graft substitute (ABGS) was fabricated by dispersing rhBMP6 in ABC with and without ALLO and evaluated for bone induction in rat subcutaneous implants and to promote bone formation in posterolateral lumbar fusion in preclinical models. The cellular response of bone induction and the quantity and quality of newly formed bone were examined by histology, radiographs, micro-CT analyses at various time intervals. RESULTS: ABGS with and without ALLO induced new bone formation in rat implants. ALLO alone induced formation of multinucleated foreign body giant cells (FBGCs). However, ALLO plus ABC decreased the formation of FBGCs. The addition of rhBMP6 to ABC/ALLO, resulted in fewer or no FBGCs. In Rabbits, the groups with ABC alone or with ALLO did not fuse. The 50 micrograms rhBMP6 group only 2 out of 4 animals successfully fused. In the 100 micrograms, 200 micrograms and 400 micrograms rhBMP6 groups all animals successfully fused. In sheep, the groups with ABC or ABC with ALLO failed to fuse. In the rhBMP6 187.5 micro grams/ml group with ABC and instrumentation fusion was achieved in 83% (10/12 animals). In the 187.5 micro grams/ml group with ABC, ALLO and instrumentation, fusion was achieved in 93% (11/12 animals). Bone formation occurred uniformly within ABGS implants where the ALLO was replaced by the newly formed bone via creeping substitution. CONCLUSIONS: RhBMP6 with autologous blood coagulum (ABC) alone or with ABC and allograft (ALLO) induced a robust bone formation in posterolateral spinal fusion in preclinical models.

Refer to onsite annual meeting presentations and postmeeting proceedings for possible referenced figures and tables. Authors are responsible for accurately reporting disclosure and FDA device/drug status at time of abstract submission.