- Email: [email protected]
Can Human Mesenchymal Stem Cells Reduce the Inflammatory Changes Associated with Disc Degeneration?
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NASS 31st Annual Meeting Proceedings / The Spine Journal 16 (2016) S113–S250
statistically greater gains in the amniotic tissue graft group from the 6-week postoperative time point through the 2-year time point. None of the subjects in the amniotic tissue graft group sustained a recurrent herniation at the same surgical level while 3 patients in the control group sustained a recurrent herniation at the same surgical level. CONCLUSIONS: The data from this study demonstrates statistically superior clinical outcomes following lumbar microdiscectomy as measured by ODI and SF-12 (PCS) and a lower rate of recurrent herniation with the use of an amniotic tissue graft. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2016.07.181
159. Cell-Based Annular Repair Using Cross-Linked High Density Collagen Gels in a Rodent Disc Model Brenton Pennicooke, MD1, Yu Moriguchi, MD2, Brandon Borde3; 1New York Presbyterian Hospital/Weill Cornell Medical Center, New York, NY, USA; 2Department of Orthopaedics, Osaka University Graduate School of Medicine, Suita, Japan; 3Cornell University, Ithaca, NY, USA BACKGROUND CONTEXT: Despite alleviating associated neurological symptoms, discectomy of herniated intervertebral discs (IVDs) fails to repair the underlying degenerative process. Persistent annular defect post discectomy is associated with increased risk of reherniation, progressive IVD degeneration, and chronic low back pain. To date, there is no established method in the repair of annular defects. We recently demonstrated that riboflavin crosslinked highdensity collagen gels (HDC) can facilitate annular repair in vivo. Collagen gels are commonly used in regenerative therapy due to their affinity to integrate with surrounding tissue. Previous studies have shown that fibrochondrocytes, component cells of annulus fibrosus (AF), seeded in the collagen gels are able to produce a matrix which is similar to the native AF and remodel the composite into a more biocompatible and mechanically-stable material [Mueller 1999, Bowles 2011]. The use of cell-based gels to repair annular defects in early to mid stage disc degeneration can potentially reduce the rate of reherniation and limit further degeneration, yielding favorable clinical outcome. PURPOSE: In this study, we assessed the in vivo efficacy of AF fibrochondrocytes seeded in the riboflavin crosslinked HDC gel in enhancing the reparative process at the site of annular defect and preventing further degenerative changes in a post-puncture rat-tail model. STUDY DESIGN/SETTING: In vivo, randomized, controlled study in the athymic rat intervertebral disc injury model. OUTCOME MEASURES: Quantitative analysis with a series of in vivo MRI were used to elucidate NP size and hydration based on T2-intensity, while X-rays were used to determine disc height index of treated segments. The viability of implanted fibrochondrocytes and the biological repair in the treated segment were assessed based on qualitative analysis of histology. METHODS: Forty athymic rats, punctured with an 18-gauge needle in the tail disc, were divided into 3 groups: untreated (n=6), injected with crosslinked HDC (n=16), and injected with fibrochondrocyte-laden crosslinked HDC (n=18). At 1, 2, and 5 weeks postoperatively, a series of in vivo images with X-ray and 7T MRI were conducted to determine the disc height index and nucleus pulposus (NP) size and hydration, respectively [Grunert P 2014]. Histological assessments were performed to evaluate the viability of implanted cells, degree of annular repair and secondary disc degeneration. The study was approved by and undertaken in accordance with guidelines outlined by the Hospital for Special Surgery Institutional Animal Care and Use Committee and New York State. For the analyses of continuous outcomes in disc height index, NP size, and NP hydration, we employed linear regression models with a generalized estimating equation and robust standard errors to estimate differences in mean changes from baseline controls (discectomy) across displaced and stable implantation groups. p values <.05 were considered statistically significant. RESULTS: The untreated discs showed substantial NP herniation at two weeks and NP absence with signs of degeneration by five weeks, resulting
in 40% loss of disc height. Both HDC gel groups, cellular and acellular, had significant retention of disc height and NP voxel count over the course of five weeks. Average NP voxel counts of cellular gels were higher than those of acellular gels at all time points and statistical significance was achieved at 1 and 5 weeks. Only the cellular group restored NP hydration relative to that of the adjacent healthy control. Further histological assessments indicate that while HDC gels influence the sealing of the defect, the addition of cells generates abundant tissue growth and extracellular matrices at the site of the annular defect, accelerating the reparative process. Disruption of endplate was observed in the puncture group, but not in the treated segments. CONCLUSIONS: In vivo studies on the cell-based annular repair are few in number. Our preliminary findings suggest that fibrochondrocytes can potentially improve HDC gel-based annular repair. A long-term study with sufficient sample size are necessary to confirm these results. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2016.07.182
160. Can Human Mesenchymal Stem Cells Reduce the Inflammatory Changes Associated with Disc Degeneration? Gregory D. Schroeder, MD1, Dessislava Markova, PhD2, John D. Koerner, MD3, D. Greg Anderson, MD4, Panya Luksanapruksa, MD5, Paul W. Millhouse, MD, MBA4, Jeffrey A. Rihn, MD6, Alexander R. Vaccaro, MD, PhD4, Christopher K. Kepler, MD, MBA2; 1Rothman Institute Thomas Jefferson University, Philadelphia, PA, USA; 2Philadelphia, PA, USA; 3New Jersey Spinal Medicine & Surgery, Glen Rock, NJ, USA; 4Rothman Institute, Philadelphia, PA, USA; 5Department of Orthopedic Surgery, Faculty of Medicine, Siriyaj Hospital, Bangkoknoi, Bangkok, Thailand; 6Thomas Jefferson University Hospital, The Rothman Institute, Philadelphia, PA, USA BACKGROUND CONTEXT: No treatment currently exists to prevent or reverse intervertebral disc degeneration, however, early research suggests that human mesenchymal stem cells (hMSCs) may be able to mitigate this process. PURPOSE: To determine if hMSCs can reduce the inflammatory cascade associated with intervertebral disc degeneration in an in vitro disc degeneration model. STUDY DESIGN/SETTING: Laboratory study. PATIENT SAMPLE: Normal Human IVD tissue was obtained from the Cooperative Human Tissue Network. The donor had no documented clinical history of degenerative disc disease. OUTCOME MEASURES: Gene expression of inflammatory cytokines. METHODS: Three groups of nucleus pulposus (NP) cells were cultured for seven days: a control sample, NP cells treated with 10 pg/ml IL-1β and 100 pg/ml TNF-α, and NP cells that were co-cultured with human primary umbilical cord-derived mesenchymal stem cells (ATCC) and treated with 10 pg/ml IL-1β and 100 pg/ml TNF-α. Prior to co-culture, human primary umbilical cord-derived mesenchymal stem Cells were fluorescently labeled with Cell Trace Far Red (Life Technologies). All cultures including co-cultures were performed using an Alvetex Scaffold 6-well insert at 50:50 ratios (2.0x106 in total) and cells were treated with media containing 10 pg/ml IL-1β and 100 pg/ml TNF-α for seven days. Cell sorting (FACS Aria II high speed cell sorter with Diva 7 software) was used to separate NP cells from the hMSCs. RNA from NP cells was isolated using RNA extraction RNeasykit (Qiagen). Total RNA was reverse transcribed into complementary DNA with RT2First Strand Kit (Qiagen). The level of 84 different inflammatory cytokines was determined with RT2 Profiler PCR Array Human Cytokines & Chemokines (Qiagen). The expression levels of CXCL-1, CXCL-2, CXCL-5, IL-6, IL-8 and MMP-3 were verified by Real- time RT-PCR Analysis. Expression was normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) and control cells cultured alone (level expressed as 1).
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.
NASS 31st Annual Meeting Proceedings / The Spine Journal 16 (2016) S113–S250 RESULTS: After treatment of NP cells with IL-1β and TNF-α, 15 cytokines were significantly elevated compared to the control group (IL-8, CXCL-1, CXCL-2, CSF-2, BMP-6, IL-1β, IL-11, BMP-2, IL-24, CCL-2, CCL-5, IL-16, MIF, CSF-1 and CSCL-10, (p<.05), and 11 cytokines had a nonsignificant increase: CXCL-5, IL-6, IL-1α,(p<.06); LIF, IL-7,(p<.08); CCL20, CSF-3, CCL-11, IL-1RN and IFN-2α). When the co-cultured NP cells were compared to the treated NP cells, a significant decrease in expression (p<.05) was seen in IL-24, IL-16, CSF-1 and CXCL-10, and a nonsignificant decrease in IL-8, CXCL-1, CXCL-2, CXCL-5, CSC-2, IL-6, CSF-3, IL-1β, CCL-11, BMP-2, IL-1α, LIF, CCL-2, CCL-7, CCL-5, IL-7, MIF, IL-1RN and IFN-2α was identified. Real-time RT-PCR analysis was used to verified the effect of co-culture NP cells with hMSCs on specific cytokines involved in intervertebral disc degeneration, and there was a significant decrease in gene expression of CXCL-5 (-2.66fold, p<.01; IL-8 (-2.30 fold, p=.048) and MMP-3 (-2.84 fold, p=.049); additionally, strong trends toward decreased gene expression of CXCL-1 (-1.89 fold p=.055), CXCL-2 (-1.27 fold, p=.053) and IL-6 (-1.28 fold p=.054) was identified. CONCLUSIONS: Production of inflammatory mediators and degradative enzymes in vivo leads to degenerative changes within the intervertebral disc. The results of this study suggests this process may be partially mitigated by co-culture of NP cells with hMSCs. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2016.07.183
161. Serum Phosphorylated Neurofilament Heavy Subunit (pNF-H) Levels in Patients with Cervical Compressive Myelopathy So Kato, MD1, Hirotaka Chikuda, MD, PhD2, Kentaro Hayakawa, MD, PhD3, Junichi Ohya, MD4, Katsushi Takeshita, MD, PhD5, Sakae Tanaka, MD, PhD2, Toru Ogata, MD, PhD6; 1Toronto Western Hospital, Toronto, ON, Canada; 2 University of Tokyo, Tokyo, Japan; 3NTT Medical Center Tokyo, Tokyo, Japan; 4Tokyo, Japan; 5Department of Orthopaedic Surgery, Jichi Medical University, Tochigi, Japan; 6National Rehabilitation Center, Saitama, Japan BACKGROUND CONTEXT: Phosphorylated form of the high-molecularweight neurofilament subunit (pNF-H) is becoming increasingly recognized as a biomarker for axonal injury; however, it remains unclear whether serum pNF-H is elevated in presence of chronic spinal cord compression. PURPOSE: The purpose of this study was to investigate serum levels of pNF-H in patients with cervical compressive myelopathy. STUDY DESIGN/SETTING: Prospective cohort study. Department of Orthopaedic Surgery, University of Tokyo Hospital, Japan. PATIENT SAMPLE: Twenty-six consecutive patients who underwent surgery for cervical compressive myelopathy were examined. OUTCOME MEASURES: Serum pNF-H levels by using enzyme-linked immunosorbent assay. METHODS: Peripheral blood samples were obtained preoperatively and one week after the surgery. Serum obtained from scoliosis patients without neurological symptoms were used as control. The history of recent aggravation of myelopathy was also investigated. RESULTS: A total of 26 myelopathy patients met the study criteria. Preoperative serum pNF-H was negative in 20 patients and moderately elevated in 6 patients (range: 115 – 458 pg/ml). Control samples (n=8) were all negative for pNF-H. Patients who were positive for pNF-H were more likely to have had recent aggravation of myelopathy compared with pNF-H negative patients (83% vs 25%, p=.02). Postoperative blood samples were available in 18 myelopathy patients. All patients who were positive for pNF-H before surgery remained positive after surgery. Two patients who became positive for pNF-H after surgery had transient neurologic deterioration associated with surgeries. CONCLUSIONS: In this pilot study, serum pNF-H level was negative in the majority of patients with cervical compressive myelopathy. Our results
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suggest that elevated serum level of pNF-H is associated with acute worsening of myelopathy. Our results indicate that positive conversion of pNF-H after surgery is a marker of perioperative neural damage. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2016.07.184
162. Influence of Riluzole on Osteogenic Differentiation of Human Bone Marrow Stromal Cells and Osteoblasts Gregory D. Schroeder, MD1, Christopher K. Kepler, MD, MBA2, Sibylle Grad, PhD3, Mauro Alini, PhD4, Dessislava Markova, PhD2, John D. Koerner, MD5, Shanmuganathan Rajasekaran, MD, PhD, FACS6, Jens R. Chapman, MD7, Frank Kandziora, MD, PhD8, Klaus J. Schnake, MD9, Marcel F. Dvorak, MD, FRCSC10, F. Cumhur Oner, MD, PhD11, Alexander R. Vaccaro, MD, PhD12; 1 Rothman Institute Thomas Jefferson University, Philadelphia, PA, USA; 2 Philadelphia, PA, USA; 3AO Research Institute, Switzerland; 4AO Research Institute Davos, Davos Platz, Switzerland; 5New Jersey Spinal Medicine & Surgery, Glen Rock, NJ, USA; 6Ganga Hospital Orthopaedic Dept, Coimbatore, Tamilnadu, India; 7Swedish Neuroscience Institute, Seattle, WA, USA; 8Unfallklinik Frankfurt am Main Friedberger, Frankfurt, Germany; 9Center for Spinal Therapy, Schon Klinik Nurnberg Furth, Furth, Germany; 10Blusson Spinal Cord Centre, Vancouver, BC, Canada; 11University Medical Center Utrecht, Utrecht, Netherlands; 12 Rothman Institute, Philadelphia, PA, USA BACKGROUND CONTEXT: Preliminary studies suggest that riluzole, a sodium channel-blocking medication, may be neuroprotective in patients with an acute spinal cord injury (SCI). However, its effect on bone formation has never been studied. PURPOSE: To determine the effects of riluzole on osteogenic differentiation of human bone marrow derived mesenchymal stromal cells (MSCs) and human primary osteoblasts (OBs). STUDY DESIGN/SETTING: Laboratory setting. METHODS: Human bone marrow aspirate and human femoral heads were used to obtain MSCs and OBs. For cell viability testing MSCs and OBs were seeded in 96-well plates at 7,500 cells/cm2. After 24 h, the cells were treated with osteogenic medium containing different concentrations of riluzole (50 ng/mL; 150 ng/mL; 450 ng/mL). Control groups of MSCs and OBs were cultured without riluzole. After 2 and 7 days, cell viability was determined. For quantification of alkaline phosphatase (ALP) activity, MSCs and OBs were plated in 24-well plates (10,000 cells/cm2) and cultured for 2 days in basal medium. They were then treated with standard osteogenic differentiation medium. Control groups without riluzole, and experimental groups that were exposed to different concentrations of riluzole (50 ng/mL; 150 ng/mL; 450 ng/mL) were cultured. After 7, 14, 21 and 28 days ALP activity was measured. ALP activity was normalized to the DNA content of the respective sample. Osteogenic differentiation was also identified qualitatively by alizarin red to staining. For gene expression analysis, total RNA was extracted from controls and cells treated with 50 ng/mL or 450 ng/mL riluzole at days 7, 21 and 28 to determine the expression of osteogenic genes (type I collagen, alkaline phosphatase, osteocalcin, and RUNX2). Quantitative real-time PCR was performed, and expression levels of the genes of interest were normalized to a housekeeping gene, and data expressed as relative to corresponding untreated control. The experiments were performed in duplicate (PCR) or triplicate (ALP activity) for 3 MSC donors and 3 OB donors. RESULTS: Riluzole affected neither the cell viability of human MSCs nor osteoblasts after up to seven days. There was also no influence of riluzole supplementation on the proliferation of the MSCs or OBs. In contrast, ALP activity was increased by 30% in MSCs after 14 days of culture in medium containing 150 ng/mL of riluzole (p=.035 vs control); after 21 days of culture, ALP activity was up-regulated 2.5 times and 1.9 times in MSCs upon supplementation of 150 ng/mL (p<.001 vs control) and 450 ng/mL (p=.010 vs control) of riluzole, respectively. In osteoblasts, 2–2.5 times increased ALP activity
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.
NASS 31st Annual Meeting Proceedings / The Spine Journal 16 (2016) S113–S250
statistically greater gains in the amniotic tissue graft group from the 6-week postoperative time point through the 2-year time point. None of the subjects in the amniotic tissue graft group sustained a recurrent herniation at the same surgical level while 3 patients in the control group sustained a recurrent herniation at the same surgical level. CONCLUSIONS: The data from this study demonstrates statistically superior clinical outcomes following lumbar microdiscectomy as measured by ODI and SF-12 (PCS) and a lower rate of recurrent herniation with the use of an amniotic tissue graft. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2016.07.181
159. Cell-Based Annular Repair Using Cross-Linked High Density Collagen Gels in a Rodent Disc Model Brenton Pennicooke, MD1, Yu Moriguchi, MD2, Brandon Borde3; 1New York Presbyterian Hospital/Weill Cornell Medical Center, New York, NY, USA; 2Department of Orthopaedics, Osaka University Graduate School of Medicine, Suita, Japan; 3Cornell University, Ithaca, NY, USA BACKGROUND CONTEXT: Despite alleviating associated neurological symptoms, discectomy of herniated intervertebral discs (IVDs) fails to repair the underlying degenerative process. Persistent annular defect post discectomy is associated with increased risk of reherniation, progressive IVD degeneration, and chronic low back pain. To date, there is no established method in the repair of annular defects. We recently demonstrated that riboflavin crosslinked highdensity collagen gels (HDC) can facilitate annular repair in vivo. Collagen gels are commonly used in regenerative therapy due to their affinity to integrate with surrounding tissue. Previous studies have shown that fibrochondrocytes, component cells of annulus fibrosus (AF), seeded in the collagen gels are able to produce a matrix which is similar to the native AF and remodel the composite into a more biocompatible and mechanically-stable material [Mueller 1999, Bowles 2011]. The use of cell-based gels to repair annular defects in early to mid stage disc degeneration can potentially reduce the rate of reherniation and limit further degeneration, yielding favorable clinical outcome. PURPOSE: In this study, we assessed the in vivo efficacy of AF fibrochondrocytes seeded in the riboflavin crosslinked HDC gel in enhancing the reparative process at the site of annular defect and preventing further degenerative changes in a post-puncture rat-tail model. STUDY DESIGN/SETTING: In vivo, randomized, controlled study in the athymic rat intervertebral disc injury model. OUTCOME MEASURES: Quantitative analysis with a series of in vivo MRI were used to elucidate NP size and hydration based on T2-intensity, while X-rays were used to determine disc height index of treated segments. The viability of implanted fibrochondrocytes and the biological repair in the treated segment were assessed based on qualitative analysis of histology. METHODS: Forty athymic rats, punctured with an 18-gauge needle in the tail disc, were divided into 3 groups: untreated (n=6), injected with crosslinked HDC (n=16), and injected with fibrochondrocyte-laden crosslinked HDC (n=18). At 1, 2, and 5 weeks postoperatively, a series of in vivo images with X-ray and 7T MRI were conducted to determine the disc height index and nucleus pulposus (NP) size and hydration, respectively [Grunert P 2014]. Histological assessments were performed to evaluate the viability of implanted cells, degree of annular repair and secondary disc degeneration. The study was approved by and undertaken in accordance with guidelines outlined by the Hospital for Special Surgery Institutional Animal Care and Use Committee and New York State. For the analyses of continuous outcomes in disc height index, NP size, and NP hydration, we employed linear regression models with a generalized estimating equation and robust standard errors to estimate differences in mean changes from baseline controls (discectomy) across displaced and stable implantation groups. p values <.05 were considered statistically significant. RESULTS: The untreated discs showed substantial NP herniation at two weeks and NP absence with signs of degeneration by five weeks, resulting
in 40% loss of disc height. Both HDC gel groups, cellular and acellular, had significant retention of disc height and NP voxel count over the course of five weeks. Average NP voxel counts of cellular gels were higher than those of acellular gels at all time points and statistical significance was achieved at 1 and 5 weeks. Only the cellular group restored NP hydration relative to that of the adjacent healthy control. Further histological assessments indicate that while HDC gels influence the sealing of the defect, the addition of cells generates abundant tissue growth and extracellular matrices at the site of the annular defect, accelerating the reparative process. Disruption of endplate was observed in the puncture group, but not in the treated segments. CONCLUSIONS: In vivo studies on the cell-based annular repair are few in number. Our preliminary findings suggest that fibrochondrocytes can potentially improve HDC gel-based annular repair. A long-term study with sufficient sample size are necessary to confirm these results. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2016.07.182
160. Can Human Mesenchymal Stem Cells Reduce the Inflammatory Changes Associated with Disc Degeneration? Gregory D. Schroeder, MD1, Dessislava Markova, PhD2, John D. Koerner, MD3, D. Greg Anderson, MD4, Panya Luksanapruksa, MD5, Paul W. Millhouse, MD, MBA4, Jeffrey A. Rihn, MD6, Alexander R. Vaccaro, MD, PhD4, Christopher K. Kepler, MD, MBA2; 1Rothman Institute Thomas Jefferson University, Philadelphia, PA, USA; 2Philadelphia, PA, USA; 3New Jersey Spinal Medicine & Surgery, Glen Rock, NJ, USA; 4Rothman Institute, Philadelphia, PA, USA; 5Department of Orthopedic Surgery, Faculty of Medicine, Siriyaj Hospital, Bangkoknoi, Bangkok, Thailand; 6Thomas Jefferson University Hospital, The Rothman Institute, Philadelphia, PA, USA BACKGROUND CONTEXT: No treatment currently exists to prevent or reverse intervertebral disc degeneration, however, early research suggests that human mesenchymal stem cells (hMSCs) may be able to mitigate this process. PURPOSE: To determine if hMSCs can reduce the inflammatory cascade associated with intervertebral disc degeneration in an in vitro disc degeneration model. STUDY DESIGN/SETTING: Laboratory study. PATIENT SAMPLE: Normal Human IVD tissue was obtained from the Cooperative Human Tissue Network. The donor had no documented clinical history of degenerative disc disease. OUTCOME MEASURES: Gene expression of inflammatory cytokines. METHODS: Three groups of nucleus pulposus (NP) cells were cultured for seven days: a control sample, NP cells treated with 10 pg/ml IL-1β and 100 pg/ml TNF-α, and NP cells that were co-cultured with human primary umbilical cord-derived mesenchymal stem cells (ATCC) and treated with 10 pg/ml IL-1β and 100 pg/ml TNF-α. Prior to co-culture, human primary umbilical cord-derived mesenchymal stem Cells were fluorescently labeled with Cell Trace Far Red (Life Technologies). All cultures including co-cultures were performed using an Alvetex Scaffold 6-well insert at 50:50 ratios (2.0x106 in total) and cells were treated with media containing 10 pg/ml IL-1β and 100 pg/ml TNF-α for seven days. Cell sorting (FACS Aria II high speed cell sorter with Diva 7 software) was used to separate NP cells from the hMSCs. RNA from NP cells was isolated using RNA extraction RNeasykit (Qiagen). Total RNA was reverse transcribed into complementary DNA with RT2First Strand Kit (Qiagen). The level of 84 different inflammatory cytokines was determined with RT2 Profiler PCR Array Human Cytokines & Chemokines (Qiagen). The expression levels of CXCL-1, CXCL-2, CXCL-5, IL-6, IL-8 and MMP-3 were verified by Real- time RT-PCR Analysis. Expression was normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) and control cells cultured alone (level expressed as 1).
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.
NASS 31st Annual Meeting Proceedings / The Spine Journal 16 (2016) S113–S250 RESULTS: After treatment of NP cells with IL-1β and TNF-α, 15 cytokines were significantly elevated compared to the control group (IL-8, CXCL-1, CXCL-2, CSF-2, BMP-6, IL-1β, IL-11, BMP-2, IL-24, CCL-2, CCL-5, IL-16, MIF, CSF-1 and CSCL-10, (p<.05), and 11 cytokines had a nonsignificant increase: CXCL-5, IL-6, IL-1α,(p<.06); LIF, IL-7,(p<.08); CCL20, CSF-3, CCL-11, IL-1RN and IFN-2α). When the co-cultured NP cells were compared to the treated NP cells, a significant decrease in expression (p<.05) was seen in IL-24, IL-16, CSF-1 and CXCL-10, and a nonsignificant decrease in IL-8, CXCL-1, CXCL-2, CXCL-5, CSC-2, IL-6, CSF-3, IL-1β, CCL-11, BMP-2, IL-1α, LIF, CCL-2, CCL-7, CCL-5, IL-7, MIF, IL-1RN and IFN-2α was identified. Real-time RT-PCR analysis was used to verified the effect of co-culture NP cells with hMSCs on specific cytokines involved in intervertebral disc degeneration, and there was a significant decrease in gene expression of CXCL-5 (-2.66fold, p<.01; IL-8 (-2.30 fold, p=.048) and MMP-3 (-2.84 fold, p=.049); additionally, strong trends toward decreased gene expression of CXCL-1 (-1.89 fold p=.055), CXCL-2 (-1.27 fold, p=.053) and IL-6 (-1.28 fold p=.054) was identified. CONCLUSIONS: Production of inflammatory mediators and degradative enzymes in vivo leads to degenerative changes within the intervertebral disc. The results of this study suggests this process may be partially mitigated by co-culture of NP cells with hMSCs. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2016.07.183
161. Serum Phosphorylated Neurofilament Heavy Subunit (pNF-H) Levels in Patients with Cervical Compressive Myelopathy So Kato, MD1, Hirotaka Chikuda, MD, PhD2, Kentaro Hayakawa, MD, PhD3, Junichi Ohya, MD4, Katsushi Takeshita, MD, PhD5, Sakae Tanaka, MD, PhD2, Toru Ogata, MD, PhD6; 1Toronto Western Hospital, Toronto, ON, Canada; 2 University of Tokyo, Tokyo, Japan; 3NTT Medical Center Tokyo, Tokyo, Japan; 4Tokyo, Japan; 5Department of Orthopaedic Surgery, Jichi Medical University, Tochigi, Japan; 6National Rehabilitation Center, Saitama, Japan BACKGROUND CONTEXT: Phosphorylated form of the high-molecularweight neurofilament subunit (pNF-H) is becoming increasingly recognized as a biomarker for axonal injury; however, it remains unclear whether serum pNF-H is elevated in presence of chronic spinal cord compression. PURPOSE: The purpose of this study was to investigate serum levels of pNF-H in patients with cervical compressive myelopathy. STUDY DESIGN/SETTING: Prospective cohort study. Department of Orthopaedic Surgery, University of Tokyo Hospital, Japan. PATIENT SAMPLE: Twenty-six consecutive patients who underwent surgery for cervical compressive myelopathy were examined. OUTCOME MEASURES: Serum pNF-H levels by using enzyme-linked immunosorbent assay. METHODS: Peripheral blood samples were obtained preoperatively and one week after the surgery. Serum obtained from scoliosis patients without neurological symptoms were used as control. The history of recent aggravation of myelopathy was also investigated. RESULTS: A total of 26 myelopathy patients met the study criteria. Preoperative serum pNF-H was negative in 20 patients and moderately elevated in 6 patients (range: 115 – 458 pg/ml). Control samples (n=8) were all negative for pNF-H. Patients who were positive for pNF-H were more likely to have had recent aggravation of myelopathy compared with pNF-H negative patients (83% vs 25%, p=.02). Postoperative blood samples were available in 18 myelopathy patients. All patients who were positive for pNF-H before surgery remained positive after surgery. Two patients who became positive for pNF-H after surgery had transient neurologic deterioration associated with surgeries. CONCLUSIONS: In this pilot study, serum pNF-H level was negative in the majority of patients with cervical compressive myelopathy. Our results
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suggest that elevated serum level of pNF-H is associated with acute worsening of myelopathy. Our results indicate that positive conversion of pNF-H after surgery is a marker of perioperative neural damage. FDA DEVICE/DRUG STATUS: This abstract does not discuss or include any applicable devices or drugs. http://dx.doi.org/10.1016/j.spinee.2016.07.184
162. Influence of Riluzole on Osteogenic Differentiation of Human Bone Marrow Stromal Cells and Osteoblasts Gregory D. Schroeder, MD1, Christopher K. Kepler, MD, MBA2, Sibylle Grad, PhD3, Mauro Alini, PhD4, Dessislava Markova, PhD2, John D. Koerner, MD5, Shanmuganathan Rajasekaran, MD, PhD, FACS6, Jens R. Chapman, MD7, Frank Kandziora, MD, PhD8, Klaus J. Schnake, MD9, Marcel F. Dvorak, MD, FRCSC10, F. Cumhur Oner, MD, PhD11, Alexander R. Vaccaro, MD, PhD12; 1 Rothman Institute Thomas Jefferson University, Philadelphia, PA, USA; 2 Philadelphia, PA, USA; 3AO Research Institute, Switzerland; 4AO Research Institute Davos, Davos Platz, Switzerland; 5New Jersey Spinal Medicine & Surgery, Glen Rock, NJ, USA; 6Ganga Hospital Orthopaedic Dept, Coimbatore, Tamilnadu, India; 7Swedish Neuroscience Institute, Seattle, WA, USA; 8Unfallklinik Frankfurt am Main Friedberger, Frankfurt, Germany; 9Center for Spinal Therapy, Schon Klinik Nurnberg Furth, Furth, Germany; 10Blusson Spinal Cord Centre, Vancouver, BC, Canada; 11University Medical Center Utrecht, Utrecht, Netherlands; 12 Rothman Institute, Philadelphia, PA, USA BACKGROUND CONTEXT: Preliminary studies suggest that riluzole, a sodium channel-blocking medication, may be neuroprotective in patients with an acute spinal cord injury (SCI). However, its effect on bone formation has never been studied. PURPOSE: To determine the effects of riluzole on osteogenic differentiation of human bone marrow derived mesenchymal stromal cells (MSCs) and human primary osteoblasts (OBs). STUDY DESIGN/SETTING: Laboratory setting. METHODS: Human bone marrow aspirate and human femoral heads were used to obtain MSCs and OBs. For cell viability testing MSCs and OBs were seeded in 96-well plates at 7,500 cells/cm2. After 24 h, the cells were treated with osteogenic medium containing different concentrations of riluzole (50 ng/mL; 150 ng/mL; 450 ng/mL). Control groups of MSCs and OBs were cultured without riluzole. After 2 and 7 days, cell viability was determined. For quantification of alkaline phosphatase (ALP) activity, MSCs and OBs were plated in 24-well plates (10,000 cells/cm2) and cultured for 2 days in basal medium. They were then treated with standard osteogenic differentiation medium. Control groups without riluzole, and experimental groups that were exposed to different concentrations of riluzole (50 ng/mL; 150 ng/mL; 450 ng/mL) were cultured. After 7, 14, 21 and 28 days ALP activity was measured. ALP activity was normalized to the DNA content of the respective sample. Osteogenic differentiation was also identified qualitatively by alizarin red to staining. For gene expression analysis, total RNA was extracted from controls and cells treated with 50 ng/mL or 450 ng/mL riluzole at days 7, 21 and 28 to determine the expression of osteogenic genes (type I collagen, alkaline phosphatase, osteocalcin, and RUNX2). Quantitative real-time PCR was performed, and expression levels of the genes of interest were normalized to a housekeeping gene, and data expressed as relative to corresponding untreated control. The experiments were performed in duplicate (PCR) or triplicate (ALP activity) for 3 MSC donors and 3 OB donors. RESULTS: Riluzole affected neither the cell viability of human MSCs nor osteoblasts after up to seven days. There was also no influence of riluzole supplementation on the proliferation of the MSCs or OBs. In contrast, ALP activity was increased by 30% in MSCs after 14 days of culture in medium containing 150 ng/mL of riluzole (p=.035 vs control); after 21 days of culture, ALP activity was up-regulated 2.5 times and 1.9 times in MSCs upon supplementation of 150 ng/mL (p<.001 vs control) and 450 ng/mL (p=.010 vs control) of riluzole, respectively. In osteoblasts, 2–2.5 times increased ALP activity
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.