AOSpine Masters Symposium Abstracts

The Regenerative Effects of Notochordal Cell Matrix on Canine and Human Nucleus Pulposus Cells

Frances C. Bach¹, Stefan A. H. de Vries², Laura B. Creemers³, Björn P. Meij¹, Keita Ito^2,3, and Marianna A. Tryfonidou¹

¹Utrecht University, Utrecht, Netherlands ²Eindhoven University of Technology, Eindhoven, Netherlands ³University Medical Centre Utrecht, Utrecht, Netherlands

Introduction: Low back pain has been related to intervertebral disc (IVD) degeneration. Since dogs experience back pain and IVD degeneration with similar characteristics as humans, they are considered a suitable animal model for human IVD degeneration. Current treatments for IVD disease do not lead to repair, so there is urgent need for regenerative treatments resulting in functional IVD restoration. During IVD maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus (NP). NCs produce soluble factors that induce cell proliferation and healthy extracellular matrix (ECM) production, but identification of these specific factor(s) has proven difficult. A novel approach would be to directly use the matrix of NP tissue rich in NCs, the so-called notochordal cell matrix (NCM), which has already been shown to exert anabolic effects on bovine CLCs. Therefore, as a first step toward translation, the regenerative effects of NCM on canine and human CLCs derived from degenerated IVDs were determined. Materials and Methods: NCM was produced from healthy porcine NC-rich NP tissue (n = 6, pooled) by lyophilization and resuspension in basal culture medium (10 mg/mL). The effect of NCM was determined on canine (n = 6) and human (n = 6) CLCs derived from degenerated IVDs (Thompson grade III) cultured in micro-aggregates (35 000 cells) for 28 days under hypoxic conditions. Main read out parameters (determined by RT-qPCR, histology, DMMB assay, and ELISA) were the following: ECM anabolism/catabolism, cell proliferation, morphology, apoptosis, inflammation, and Smad signaling (phosphorylated Smad1 and Smad2). Results: Generally, the response to NCM of canine CLCs was more pronounced than the response to NCM of human CLCs. Although ACAN and COL2A1 gene expression was not upregulated at day 7, NCM significantly increased the glycosaminoglycan content of human and canine CLC micro-aggregates compared with controls at day 28. Furthermore, NCM induced mild collagen type I and abundant collagen type II deposition in human and canine CLC micro-aggregates. ECM catabolism and apoptosis marker gene expression was decreased by NCM treatment, whereas CCND1 gene expression (marker for proliferation) was increased in human and canine CLCs. Collagen type X deposition and (anti-)inflammatory and notochordal/nucleus pulposus marker gene expression were not considerably affected by NCM treatment, but VEGF gene expression was increased in human and canine CLCs. Last, NCM significantly induced rapid Smad1 (BMP) and Smad2 (TGF-β) signaling in canine CLCs, whereas it decreased Smad1 and Smad2 signaling in human CLCs. Conclusion: NCM induced an anabolic, anti-catabolic, anti-apoptotic, and proliferative effect on human and canine CLCs derived from degenerated IVDs, indicating that it holds promise as regenerative treatment for IVD disease. Increased Smad signaling can explain the regenerative effects of NCM on canine CLCs, but future studies should focus on determining the mechanism(s) of NCM-mediated regeneration in human CLCs. The current study shows that directly applying notochordal cell matrix of NP tissue could be a promising regenerative treatment for canine and human IVD disease, circumventing the (challenging) identification and application of bioactive NC-secreted substances. A preclinical in vivo (Beagle) study with NCM is ongoing at the moment (6-month follow-up).

Anabolic Effects of Bioactive Proteins and Extracellular Vesicles Derived From Notochordal Cell-Conditioned Medium

Frances C. Bach¹, Stefan A. H. de Vries², Frank M. Riemers¹, Janneke Boere¹, Ferdi W. M. van Heel¹, Marina van Doeselaar², Soenita Goerdayal¹, Peter G. J. Nikkels³, Karin Benz⁴, Laura B. Creemers³, Maarten F. M. Altelaar¹, Björn P. Meij¹, Keita Ito^2,3, and Marianna A. Tryfonidou¹

¹Utrecht University, Utrecht, Netherlands ²Eindhoven University of Technology, Eindhoven, Netherlands ³University Medical Centre Utrecht, Utrecht, Netherlands ⁴TETEC AG, Reutlingen, Germany

Introduction: During maturation and early degeneration of the IVD, notochordal cells (NCs) are gradually replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus (NP), suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. Materials and Methods: Mass spectrometry was performed on porcine, canine, and human NCCM, which was generated by culturing healthy porcine, canine, and human NC-rich NP tissue for 4 days in plain culture medium. As a first step to elucidate which bioactive substances are responsible for the effects of NCCM, porcine and canine NCCM were separated in a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles [EVs]) fraction by ultracentrifugation, and tested in vitro on bovine CLCs (4 porcine NCCM donors tested on 4 bovine CLC repeats) and canine CLCs (8 NCCM donors tested on a pool of 4 CLC donors), respectively. Main readout parameters were ECM production/degradation and cell proliferation (RT-qPCR, histology, DMMB assay). In a follow-up study, EVs and proteins derived from porcine NCCM (n = 7) were separated through qEV size exclusion columns and (separately) ultracentrifuged. Thereafter, to characterize EVs, the EV fraction was labelled with PKH67, floated in sucrose gradients, and quantitatively analyzed by high-resolution flow cytometry. The separate effects of the porcine NCCM-derived EVs and proteins were studied on micro-aggregates containing CLCs from degenerated canine IVDs (pool of 4 donors) in vitro. Results: Mass spectrometry of whole porcine, canine, and human NCCM identified 149, 170, and 217 proteins, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. In both bovine and canine CLCs, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P moderately enhanced GAG and collagen type II deposition by canine CLCs. Notably, the porcine NCCM-derived EVs (depleted of protein aggregates present in porcine NCCM-P) and porcine NCCM-derived proteins did not influence the DNA content, but they both increased the GAG content of the canine CLC micro-aggregates. Conclusion: Instead of a single regenerative NCCM candidate (growth factor), we found that porcine and canine NCCM exerted their anabolic effects mainly through soluble factors. However, also pelletable NCCM factors—containing both protein aggregates and EVs—showed moderate regenerative potential. Moreover, porcine NCCM contained a considerable amount of EVs, which (when applied without interfering protein aggregates that were present in NCCM-P) also exerted anabolic effects on canine CLCs. Altogether, this indicates that the bioactive factors within NCCM interact and may even negatively influence the anabolic effects exerted by specific bioactive factors. As such, the optimal combination of NC-secreted factors (proteins and EVs) needs to be determined to fully exploit the regenerative potential of NC-derived treatment strategies.

Optimization of 3D Printed Hydrogels With Primary Cells for Tissue Engineering

Ezgi Bakirci¹, Olivier Guenat², Andreas Hugi², Sufian S. Ahmad³, Sandro Kohl³, and Benjamin Gantenbein¹

¹Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland ²Artorg Center for Biomedical Engineering, Organs-on-Chip Technologies, University of Bern, Bern, Switzerland ³Department of Orthopedic Surgery and Traumatology, Insel Hospital, University of Bern, Bern, Switzerland

Background: Three-dimensional (3D) bioprinting, a flexible automated on demand platform for free form fabrication of complex living architectures, is a novel approach for design and engineering of human organs and tissues.¹ We here report on a tissue-engineered anterior cruciate ligament (ACL) scaffold fabricated using 3D bioprinting technology. The objective of this in vitro study was to optimize the printing parameters for maximum cell viability, determine cellular response, and characterize the mechanical properties of the materials. Here, human ACL cells were used as constituent cells and mixture of gelatin, fibrinogen, and hyaluronic acid were used as cell-laden hydrogel. Cell localization and proliferation were investigated to study cell functions and tissue formation process in 3D structure. Introduction: The anterior cruciate ligament is most commonly injured ligament of knee.² ACL does not heal due to poor healing potential and limited vascularization. Therefore, surgical intervention is usually used and several ACL reconstruction surgeries are performed such as autografts, allografts, and synthetic grafts. However, the long-term clinical outcome of these grafts was suboptimal due to poor abrasion resistance and limited integration between grafts and host tissues. These outcomes have prompted a growing interest in tissue engineering solutions for ACL reconstructions. Our approach is to demonstrate the potential of 3D bioprinting for ACL constructs. The aim of this study was to optimize in vitro 3D bioprinted scaffold design for ligament tissue engineering, focusing on material selection and printing parameters based on cellular response and mechanical properties of materials. Material and Methods: ACL scaffolds were produced using 3D Discovery RegenHU bioprinter equipped with 3 different print heads. The diameter of scaffolds is 3 mm and the height of scaffolds is 0.5 mm. Bioink solution includes gelatin (35 mg/mL), fibrinogen (25 mg/mL), and hyaluronic acid (3 mg/mL). Transglutaminase and thrombin were used as crosslinking agents. The bioink solution and the cross-linkers were printed from 2 different printing heads, a cell friendly ink-jet and a pressure driven nozzle, respectively. The bioink was mixed 500 μL of ACL cells (1 × 10⁶ to 4 × 10⁶ per mL) and the cell-laden ink was printed by 150 μL nozzles. As a control, the hydrogel was printed and ACL cells were seeded on the printed structures to test bioprinting process effect on cell viability. The viability of printed cells was examined using calcein-ethidium homodimer dyes under the LSM710 confocal microscopy for 4 different time points. Results: The printed constructs were cultured for 21 days. The viability of ACL cells was analyzed on day1, day 3, day 7, and day 14. High cell viability was observed for both the seeded and printed ACL cells group as seen Figure 1. After 7 days, seeding cells began to fill in the printed pattern. Discussion: In this work, we describe the development, optimization of a 3D bioprinted ACL scaffold, and determine an appropriate printing parameter with respect to cellular response and mechanical properties. To this end, this study examined the effect of printing process and the attachment and proliferation of primary human ACL cells. The results showed that the viability of cells was not affected by printing process. Future work will involve also the usage of primary intervertebral disc cells in order to test applications on microfluidic chip.

Figure 1.

Cell viability results of bioprinted and seeding cells.

Acknowledgments and Funding

This project was funded by the Gebert Rüf Stiftung project (# GRS-028/13). We thank Eva Roth for her valuable assistance in IVD isolation and the mechanical department for manufacturing the cross-incision tool. Financial support was obtained from the Swiss National Science Foundation (SNF PP00P2_163678/1) as well as from the Spine Society of Europe (Eurospine 2016_4).

References

1. Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat Biotechnol. 2014;32:773-785.

2. Gantenbein B, Gadhari N, Chan SC, Kohl S, Ahmad SS. Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair. World J Stem Cells. 2015;7:521-534.

Aneurysmal Bone Cyst of the Spine: An Alternative Treatment by Direct Injection of Concentrated Autologous Mesenchymal Stem Cells

G. Barbanti-Bròdano¹, M. Girolami¹, A. Cenacchi², A. Gasbarrini¹, S. Bandiera¹, S. Terzi¹, R. Ghermandi¹, and S. Boriani¹

¹Department of Oncological and Degenerative Spine Surgery, Istituto Ortopedico Rizzoli, Bologna, Italy

²Service of Immunohematology and Transfusional Medicine, Istituto Ortopedico Rizzoli, Bologna, Italy

Introduction: The nature of the aneurysmal bone cyst (ABC) is still controversial among benign tumor, often identifiable in the “aggressive” form (Enneking stage 3) or pseudotumoral lesion. It is well known for the very high risk of intraoperative bleeding, indicating a strongly unfavorable relationship between the surgical morbidity and the nature of the disease. Excellent results have been obtained in the treatment of ABC by repeated selective arterial embolization (SAE), which is considered the “gold standard.” Some alternatives have been proposed; recently, promising results have been achieved by the direct injection of mononuclear cells derived from bone marrow into the cyst. This kind of treatment takes advantage of the multipotent nature of adult mesenchymal stem cells (MSCs), which represent a promising source for bone engineering for their ability to differentiate into osteoblasts. This study presents the results of our initial experience in the treatment of vertebral ABC through the use of concentrated autologous MSCs. Patients and Methods: Two teenagers aged 15 years, male, and 14 years, female, came to our attention both with diagnosis of ABC in C2 vertebra, which was histologically confirmed. They were both neurologically intact; the girl complained of neck pain. The arteriography showed in both cases close relationships between the pathological ABC vascularization and the vertebral and cervical ascending arteries, making treatment by SAE unsuitable. Thus, it was decided to undertake the treatment by direct injection of MSCs preceded, in the same operative session, by harvesting from the iliac crest of 60 cc of bone marrow (by needle aspiration) and its separation with the use of concentration system Res-Q 60-BMC. In the second case, the treatment was repeated 2 times at an interval of 4 months. Results: The clinical and radiological follow-up is more than 24 months from the first treatment in both cases. In the first case, the presence of newly formed bone within the ABC appeared as a clear sign of recovery just a month after the first treatment and increased gradually, until the cyst appeared completely ossified 1 year after the treatment, with associated disappearance of the pain. In the second case, an initial sclerotic peripheral margin appeared after the second treatment and later ossification progressed, concurrently with the disappearance of the pain. Conclusion: SAE is considered effective in the treatment of ABC even if it is not without risks, mainly related to the frequent and repeated exposure to ionizing radiation. Furthermore, in a certain percentage of cases the procedure is not technically executable, especially for the presence of arteries afferent to the medullar vascularization. Based on the results obtained in the 2 cases described here, the injection of MSCs can be considered a valid alternative in the treatment of vertebral ABCs when embolization is not effective or unsuitable.

Biomaterials: A Good Alternative to Autologous Bone for Spine Fusion

Giovanni Barbanti Bròdano¹, Francesco Lolli¹, Gianluca Giavaresi¹, Milena Fini¹, Stefano Bandiera¹, Alessandro Gasbarrini¹, Silvia Terzi¹, Riccardo Ghermandi¹, Lisa Babbi¹, Marco Girolami¹, and Stefano Boriani¹

¹Department of Oncological and Degenerative Spine Surgery, Istituto Ortopedico Rizzoli, Bologna, Italy

Introduction: Spinal fusion is one of the most common surgical procedures in spine surgery, whose primary objective is the stabilization of the spine for the treatment of many degenerative, traumatic, and oncological diseases of the spine. Autologous bone is still considered the “gold standard” technique for spinal fusion. However, biomaterials that are potentially osteogenic, osteoinductive, and osteoconductive can be used to increase the process of spinal fusion. We proposed to evaluate 2 new bone substitutes as an alternative to autologous bone for spinal fusion, using an animal model of large size (adult sheep). Materials and Methods: A preclinical study was designed to compare the efficacy of SINTLife Putty and DBSINT biomaterials with conventional bone autograft in an ovine model of lumbar spine fusion. SINTlife is a biomaterial made from hydroxyapatite enriched with magnesium ions, resulting in a material very similar to natural bone. DBSINT is a paste composite bone, osteoinductive, pliable, and conformable, consisting of demineralized bone matrix carried by hydroxyapatite biomimetics. Eighteen adult female sheep were selected for 2-level spine surgical procedures. The animals were divided into 2 groups: in group A, one fusion level was treated with SINTLife Putty and the other level received cortical-cancellous bone autograft; in group B, one fusion level was treated with DBSINT and the other level received cortical-cancellous bone autograft. At the end of the experimental time, all the animals were euthanized. The spine segments were analyzed macroscopically, radiographically, microtomographically, histologically, and histomorphometrically. Results: The SINTLife Putty shows a perfect osteointegration in all the histological specimens. A high percentage of newly formed bone tissue is detected, with lots of trabeculae having structure and morphology similar to the preexisting bone. In all the specimens collected from DBSINT-treated animals the presence of hydroxyapatite alone is reported but not the demineralized bone matrix. The presence of newly formed bone tissue can be detected in all the specimens but newly formed bone shows very thin and irregular trabeculae next to the cartilage zone, while away from the border of ossification there are thicker trabeculae similar to the preexisting bone. Conclusion: The use of the experimental biomaterial SINTLife Putty in an ovine model of spine fusion leads to the development of newly formed bone tissue without qualitative and quantitative differences with the one formed with autologous bone. The experimental material DBSINT seems to lead to less deposition of newly formed bone with wider intertrabecular spaces. Following the results of preclinical studies, we evaluated SINTLife Putty for its safety in a postmarket surveillance analysis and we are starting now a clinical study aimed at evaluating the efficacy of this biomaterial as bone graft substitute in posterolateral vertebral arthrodesis.

Restorative Neurostimulation to Induce Episodic Contractions of the Lumbar Multifidus Leads to Clinical Improvements in Patients With Disabling Refractory Nonspecific Chronic Low Back Pain: Results of a Prospective International Multicenter Trial

Kris De Smedt¹, Kristiaan Deckers¹, Bruce Mitchell², David Vivian², Marc Russo³, Sam Eldabe⁴, Ashish Gulve⁴, Nick Harland⁴, Peter Georgius⁵, Jean-Pierre Van Buyten⁶, Iris Smet⁶, Mathew Green⁷, John Vieceli⁷, Ganesan Baranidharan⁸, Vivek Mehta⁹, Shankar Ramaswamy⁹, Richard Sullivan¹⁰, Robert Gassin¹⁰, James Rathmell¹¹, and Chris Gilligan¹¹

¹St. Augustinus Hospital, Antwerp, Belgium ²MetroPain Group, Melbourne, Victoria, Australia ³Hunter Pain Clinic, Newcastle, New South Wales, Australia ⁴The James Cook University Hospital, Middlesbrough, UK ⁵Georgius Practice, Noosa Heads, Queensland, Australia ⁶Algemeen Ziekenhuis Nikolaas, Sint-Niklaas, Belgium ⁷Ashford Community Hospital, Adelaide, South Australia, Australia ⁸Leeds Teaching Hospitals NHS Trust, Leeds, UK ⁹St. Bartholomew’s Hospital, London, UK ¹⁰Precision Neurosurgery, Melbourne, Victoria, Australia ¹¹Brigham and Women’s Healthcare, Harvard Medical School, Boston, MA, USA

Introduction: ReActiv8-A (clinicaltrials.gov/show/NCT01985230) is an international, multicenter, prospective, single-arm clinical trial to assess clinical performance of a novel implantable neurostimulation system for patients with disabling refractory nonspecific chronic low back pain (CLBP). The lumbar multifidus muscles, specifically the deep fascicles, play a key role in maintaining intersegmental functional stability. After an initial strain or sprain injury, lumbar multifidus function is often disrupted at the segmental level due to arthrogenic inhibition. Unless rehabilitated,¹ this can lead to self-sustained cycle of functional instability, re-injury and nociceptive joint pain, impaired motor control, and eventually atrophy. The implantable system bilaterally delivers electrical stimulation to the medial branch of the L2 dorsal ramus nerve to elicit episodic smooth contractions of the lumbar multifidus muscles. Restoration of impaired multifidus motor control and functional lumbar spine stability is the hypothesized mechanism of action that facilitates recovery. Key clinical outcome measures are presented for 90 days, 6 months, and 1 year. Materials and Methods: Fifty-three subjects (age 44 ± 10 years, pain duration 14 ± 11 years) were implanted at 9 centers in Europe and Australia. Selection criteria included disabling CLBP despite at least 90-days of medical management, no prior spine surgery, no identifiable spine pathology as a likely cause of CLBP, and no indications for spine surgery or spinal cord stimulation. Subjects were instructed to administer two 30-minute stimulation sessions per day for a minimum of 90 days. Outcome measures were ODI (Oswestry Disability Index) for disability, NRS (Numeric Rating Scale) for pain, EQ-5D for quality of life (QoL), and treatment satisfaction. Results: Utilizing matched data, significant improvements (P < .0001) were observed in disability, pain, and QoL (mean ± SE) compared to baseline. ODI (44.9 ± 1.4 baseline) improved 30 ± 5% at 90 days (n = 52), 28 ± 6% at 6 months (n = 51), and 34 ± 6% at 1 year (n = 47); NRS (6.8 ± 0.2 baseline) improved 35 ± 5%, 32 ± 5%, and 33 ± 6%; and EQ-5D (0.434 ± 0.025 baseline) improved 37 ± 4%, 31 ± 6%, and 39 ± 6%, respectively. The percentage of subjects with a clinically important improvement in ODI² was 52%, 57%, and 60%; in NRS³ 63%, 61%, and 57%; in EQ-5D⁴ 88%, 82%, and 81%; and in at least one of these measures 94%, 86%, and 87%, respectively. Similarly, 89%, 84%, and 81% of subjects reported being satisfied or very satisfied with the treatment. There were no unanticipated adverse events (AEs) and no serious device, therapy, or procedure related AEs. From a patient perspective, treatment success is a combination of increased ability to perform activities, pain reduction, and perceived QoL. The treatment’s ability to provide clinically important improvements in most subjects in one or more of these key measures may explain the high treatment satisfaction observed. Conclusion: In this difficult to treat population, significant and clinically important improvements in disability, pain, and QoL were observed within 90 days and maintained through 1 year with no serious related AEs. Electrical stimulation to contract the lumbar multifidus appears to be a safe and effective treatment option for patients with disabling refractory CLBP.

References

1. Hides JA, Richardson CA, Jull GA. Multifidus muscle recovery is not automatic after resolution of acute, first-episode low back pain. Spine (Phila Pa 1976). 1996;21:2763-2769.

2. Ostelo RW, Deyo RA, Stratford P, et al. Interpreting change scores for pain and functional status in low back pain: towards international consensus regarding minimal important change. Spine (Phila Pa 1976). 2008;33:90-94.

3. Dworkin RH, Turk DC, Wyrwich KW, et al. Interpreting the clinical importance of treatment outcomes in chronic pain clinical trials: IMMPACT recommendations. J Pain. 2008;9:105-121.

4. Soer R, Reneman MF, Speijer BL, Coppes MH, Vroomen PC. Clinimetric properties of the EuroQol-5D in patients with chronic low back pain. Spine J. 2012;12:1035-1039.

Model of Disc Degeneration in Rat Tail Induced Through a Vascular Isolation of Endplate

Maximo-Alberto Díez-Ulloa¹, Hector Fernandez-Susavila², Juan-Pablo Pardo-Seco¹, Ramón Iglesias-Rey², Ana Estany-Gestal³, Tomas Sobrino-Moreiras², and Francisco Campos-Perez²

¹Orthopaedics, Universitary Hospitalary Complex, Santiago de Compostela, Spain ²Neurology, Clnical Neurosciences Research Laboratory, IDIS, Universitary Hospitalary Complex, Santiago de Compostela, Spain ³Epidemiological Unit, IDIS, Universitary Hospitalary Complex, Santiago de Compostela, Spain

Purpose: Back pain is one of the major health problems in developed countries. The degenerative cascade of the spine begins with intervertebral disc degeneration, which is thought to happen due to an impairment in the blood supply through the vertebral endplates. The objective of this study was to develop a novel disc degeneration model in Sprague Dawley rat tail based on these premises, thus reproducing the intervertebral disc degeneration process in the human being, in contrast to other proposed models (puncture, enzyme injection, aberrant loads, etc). Materials and Methods: Disc degeneration was induced by means of vascular isolation of vertebral endplates either from only one side or both, so lowering down the nutrient supply to the disc. Afterwards, a volumetric analysis of the disc was performed by MRI (magnetic resonance imaging) and then a histological study of en bloc disc sections. There were 37 rats in 2 arms: (1) histological (n = 17, one died), with Van Gieson staining and classification after Nisimura-Mochida criteria and also collagen VI staining, as a marker of tissue oxidative stress, animals were euthanized at 2, 4, 6, and 8 months (4 at each); and (2) imaging (n = 20, 6 discarded for wound soft tissue slough), with MRI 9 Tesla protocol and then volumetric study of all animals at 24 hours, 1 week, and then 2, 3, 4, 6, and 8 weeks. Results: There was a progressive degeneration reflected in a highly significant reduction of disc volumen (P < .001) with both sides isolated; also, but less remarkable, between discs isolated from one sided isolation and those from both sides and barely meaningful between controls and those isolated from one side. Besides, there was an increasingly higher Nisimura-Mochida rating score with follow-up and also an increasing deposition of collagen VI in isolated disc compared to controls, with higher values for both sided isolation than in one sided. So, isolation from both sides caused a progressive degeneration of the disc than isolation from one side and no isolation (control), as rated by volumetric reduction, tissue structural changes (Nisimura-Mochida), and collagen VI deposition. Conclusion: The model described here represents a novel and translational tool that reproduces the intervertebral disc degeneration in a similar way to that taking place in human beings.

Affordable Surgical Navigation Based on Finite Element Simulation and 3D Printing Technology

Péter E. Éltes^1,2, Márton Bartos³, Damien Lacroix⁴, Varga Péter Pál¹, and Áron Lazáry¹

¹National Center for Spinal Disorders, Budapest, Hungary ²School of PhD Studies, Semmelweis University, Budapest, Hungary ³Do3D Innovations Ltd, Budapest, Hungary ⁴Department of Mechanical Engineering, INSIGNEO Institute for In Silico Medicine, The University of Sheffield, Sheffield, UK

Introduction: A revision surgery in case of a lumbosacral nonunion can be challenging especially if an implant-related failure is complicating the clinical situation. In the S1 segment the convergent bicortical screw trajectory provides a superior anchoring compared to any other directories, but the proper insertion of the new screws in a revision surgery can be impossible without surgical navigation. In this article, we present a case who has been surgically managed by the application of a computed tomography (CT)-based 3D reconstruction method combined with finite element analysis (FEA), computer assisted design (CAD), and 3D printing technology. Materials and Methods: A step-by-step approach was developed and performed to manage the clinical problem. (1) Quantitative CT-based patient-specific FE model of the sacrum was created. (2) CAD model of the pedicle screw was inserted in the sacrum model in a bicortical convergent and a monocortical divergent position. (3) Static FEAs (Abaqus, Dassault Systemes) were performed using 500 N tensile load applied to the screw head. (4) A template with the 2 screw guiding structures designed and created for the sacrum using 3D design and SLA 3D printing technologies. (5) The surgery was performed using the patient- and condition-specific template. Results: Based on the FEA results the modified bicortical convergent screw had better stability resulting in optimal von Mises stress distribution and less displacement compared to the monocortical divergent placement. The template design concept was shown to be accurate based on the CT scan and virtual model comparison. Intraoperatively the template fitted on the bone surface and screw insertion was completed successfully. Postoperative CT scans confirmed that the inserted pedicle screw reached the virtually planned position. Conclusion: The intraoperative pedicle screw navigation provided by a patient-specific screw-guiding template allows the surgeon to insert the screw into its optimal position considering the local bone material property and the challenging geometrical situation. Its advantages compared to the conventional surgical navigation techniques are the relatively low cost, minimized intraoperative X-ray exposure, and the possibility for the consideration of the patient-specific biomechanics. This new patient- and condition-specific approach can be widely used in revision spine surgeries or in challenging primary cases after its further clinical validations.

Comparing Two Annulus Fibrosus Injury Models in Loaded Bovine Organ Culture

Daniela A. Frauchiger¹, Lorin M. Benneker², and Benjamin Gantenbein¹

¹Institute for Surgical Technology & Biomechanics, University of Bern, Bern, Switzerland ²Department for Orthopaedic Surgery and Traumatology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Introduction: Models to mimic intervertebral disc (IVD) injury are mainly divided into 2 groups: (1) big biopsy or box cut injuries¹ or (2) smaller injuries caused by needle puncture or blade stabs.² These models have in common, that they do not take into consideration the structure of the annulus fibrosus (AF). Hence, we investigate the possibility to use a new design that will focus on affecting IVD performance while loaded physiologically. Materials and Methods: A custom-designed cross-incision tool was manufactured from stainless steel to produce a 7 mm deep and 2 mm wide cross-incision. Precision is given by an inner rod and outer shaft that allows for precise 90° turns (Figure 1). Fresh coccygeal bovine IVDs were isolated³ and either injured with a 2 mm biopsy punch or cross-incision tool. Control was a healthy undamaged IVD. Discs were suspected to 2 weeks of culture in HG-DMEM with 5% FCS under either (1) no load, (2) static load (0.2 MPa for 8 h/d), or (3) complex load (0.2 MPa and 0 ± 2° of torsion at 0.2 Hz for 8 h/d). Discs were investigated for mitochondrial activity (resazurin salt assay), glycosaminoglycan (dimethylmethylen blue), and DNA (Hoechst) content, gene expression of IVD-relevant genes, and histology (Safranin-O/Fast Green, Picosirirus red, H&E). Mitochondrial activity in cross-incision injury model seems lower than for 2 mm punch injury relative to healthy control discs (Figure 2A). DNA and GAG content do not show a significant difference between the 2injury models tested. Under complex load a recovery of DNA can be observed compared to no load or static load. Furthermore, no significant difference between the tissue harvesting point near the injury (injured) or the opposite side (intact) could be found (Figure 2B and C). Conclusion: It seems as if loading especially complex loading has a beneficial effect on DNA and GAG content.⁴ The 2 tested injury models mainly differed in disc height after 14 days of culture. Under complex loading this could be explained by the disruption of the aligned AF sheets. There, cross-incision might cause a bigger structural damage than a relatively large circular biopsy combined with torsion. Moreover, the cross-incision tool allowed for a highly reproducible injury due to its fixed dimensions.

Figure 1.

Custom designed cross-incision tool consisting of outer shaft.

Figure 2.

(A) Mitochondrial activity, (B) GAG content, and (C) DNA content of IVDs under different loading conditions. Mean ± SEM, N = 5.

References

1. Likhitpanichkul M, Dreischarf M, Illien-Junger S, et al. Fibrin-genipin adhesive hydrogel for annulus fibrosus repair: performance evaluation with large animal organ culture, in situ biomechanics, and in vivo degradation tests. Eur Cell Mater. 2014;28:25-37.

2. Michalek AJ, Iatridis JC. Height and torsional stiffness are most sensitive to annular injury in large animal intervertebral discs. Spine J. 2012;12:425-432.

3. Chan SCW, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp. 2012;(60):3490.

4. Gawri R, Moir J, Ouellet J, et al. Physiological loading can restore the proteoglycan content in a model of early IVD degeneration. PLoS One. 2014;9:e101233.

Nucleus Pulposus Progenitor Cells: Isolation and Injection Into Degenerative Disc

Daniela A. Frauchiger¹, Adel Tekari², Lorin M. Benneker³, Daisuke Sakai⁴, Sibylle Grad⁵, Jivko Stoyanov⁶, Alessandro Bertolo⁶, and Benjamin Gantenbein¹

¹Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland ²Institute of Textile Machinery and High Performance Material Technology, TU Dresden, Dresden, Germany ³Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland ⁴Department for Orthopaedic Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan ⁵AO Research Institute, Davos, Switzerland ⁶Swiss Paraplegic Centre, Nottwil, Switzerland

Introduction: Recently, a nucleus pulpous (NP) subpopulation positive for angiopoietin-1 receptor (Tie2) was described as NP progenitor cells (NPPC), which possesses multipotency.^1,2 Therefore, NPPC might be a promising source of cells for intervertebral disc (IVD) regeneration. However, culture parameters for NPPC maintenance in vitro are yet unknown as is the optimal cell number required for successful restoration of mildly degenerated discs. Hence, we investigated different isolation methods for Tie2 cells and observed their survival when injected into IVDs and their regenerative potential. Materials and Methods: Fresh bovine IVDs were isolated as described previously.³ Injection of ∼100 μL of 60 U/mL papain solution followed by 8 days of incubation at 37°C was used to induce degeneration in IVDs.⁴ In the meantime, on day 7, NP cells were isolated from allogeneic tissue using a sequential 2-step digestion protocol with 0.19% pronase and 64.5 U/mL collagenase type 2. On day 8, Tie2+ cells and Tie2− cells were sorted using antibody against Tie2 by fluorescence-activated cell sorting (FACS, Facsaria, BD, N = 9), magnetic-activated cell sorting (MACS, Miltenyi Biotec, N = 4), or pluriBeads (pluriSelect Life Science, N = 2). Subsequently, cells were labelled with DiL (red fluorescence dye, Thermo-Scientific). 20 000 Tie2+ and 200 000 Tie2− cells (control) were either mixed with fibrin hydrogel (Tisseel, Baxter) or PBS (phosphate-buffered saline) and injected into the cavity of the degenerative group and nondegenerated control discs. On day 15, after 7 days of culture in free swelling conditions organ cultures were stopped and NP was separated from the annulus fibrosus (AF) tissue. Outcome measurements were cell viability (CV) by confocal laser scanning microscopy (cLSM) and semi-automated cell counting.⁵ All cells were stained with green fluorescence calcein-AM staining and DAPI, to distinguish living and dead exogenous and native cells. Furthermore, glycosaminoglycan (GAG) and DNA ratio was determined. FACS (5.033 ± 4.041) showed a higher percentage of Tie2+ cells than MACS (34) or pluriSelect (0.3376 ± 0.1568; Figure 1A). On injection, 3D cLSM stacks showed a cluster and spheroid-like arrangement of the NPPC in the NP region in contrast to the autochthonous IVD cells. NPPC survival was considerably reduced when embedded in fibrin hydrogel whereas CV in PBS was high with NPPC clustered in the native IVD environment. CV of exogenous Tie2+ cells in PBS (n = 3) was estimated to be 71 ± 29.2% and 64.5 ± 46.12% for the AF (n = 2), respectively in the NP region of nondegenerated discs. Tie2- cell’s CV was considerably lower in PBS (∼13% for NP, n = 1). In the degenerative discs with fibrin hydrogel, CV of Tie2+ in NP (n = 3) dropped from ∼90 down to ∼33% (Figure 1B). Conclusion: Despite being a cumbersome procedure, FACS showed highest Tie2+ cell yield probably due to the fine settings of gates. Sorted Tie2+ cells showed differentiation into osteo- and adipogenesis using all 3 methods. Low CV of injected cells was possibly due to problems with nutrition diffusion through fibrin hydrogel or the particular IVD degeneration model. Future studies should focus on NPPC phenotype maintenance in culture and identification of a carrier for NPPC injection.

Acknowledgment

This project was supported by funds from the Swiss National Science Foundation (Project # 310030_153411), the Gebert Rüf Foundation (Project # GRS-X028/13), and the Lindenhof Project (# 16-05F). We thank Eva Roth for her help in IVD isolation and biochemical assays. Microscopy was performed by the Microscopy Imaging Center (MIC) and the FACS work was conducted at the FACS Lab core facility of the University of Bern.

References

1. Sakai D, Nakamura Y, Nakai T, et al. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc. Nat Commun. 2012;3:1264.

2. Tekari A, Chan SC, Sakai D, Grad S, Gantenbein B. Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells. Stem Cell Res Ther. 2016;7:75.

3. Chan SC, Gantenbein-Ritter B. Preparation of intact bovine tail intervertebral discs for organ culture. J Vis Exp. 2012;(60).3490.

4. Chan SC, Bürki A, Bonél HM, Benneker LM, Gantenbein-Ritter B. Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy. Spine J. 2013;13:273-283.

5. Gantenbein-Ritter B, Potier E, Zeiter S, van der Werf M, Sprecher CM, Ito K. Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds. Tissue Eng Part C Methods. 2008;14:353-358.

Figure 1.

(A) Percentage Tie2+ cell yield using FACS, MACS and pluriSelect. (B) Cell viability of injected Tie2+ and Tie2− cells either in PBS or fibrin hydrogel.

Proliferation and Differentiation on Engineered Silk Scaffolds: From MSC Toward NP-Like Cells

Daniela A. Frauchiger¹, Silvan Heeb¹, Michael Wöltje², Lorin M Benneker³, and Benjamin Gantenbein¹

¹Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland ²Institute of Textile Machinery and High Performance Material Technology, TU Dresden, Dresden, Germany ³Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Introduction: An increasing number of people suffer from low back pain caused by intervertebral disc (IVD). Yet no treatment option is available that is capable of restoring the biological function of the IVD. In this study, we aimed to investigate the feasibility of using a silk scaffold from engineered Bombyx mori silk containing growth and differentiation factor 6 (GDF6) for IVD repair and assess the potential of GDF6 thriving human mesenchymal stem cells (MSC) toward a “disc-like” phenotype.¹ Materials and Methods: MSC and annulus fibrosus (AF) of each of 5 donors were used to test cell cytocompatibility and differentiation potential of functionalized silk composites. The silk was manufactured from B mori larvae infected with baculovirus to produce silk protein functionalized with either GDF6 or transforming growth factor β3 (TGFβ3) (Spintec Engineering GmbH).² A total of 120 000 cells were seeded per 5 × 5 mm² scaffold and cultured for 21 days in high glucose DMEM with 2% fetal calf serum. Silk without growth factors (cSilk), cSilk with exogenous GDF6 (exGDF6, 100 ng/mL), and cSilk with exogenous TGFβ3 (exTGFβ3, 10 ng/mL, both Peprotech) were used as controls. On days 1, 7, 14, and 21 mitochondrial activity, glycosaminoglycan (GAG), and DNA content were determined. Furthermore, live/dead stain was performed using confocal laser scanning microscopy. Additionally, relative gene expression of IVD marker genes was assessed on day 21 relative to day 0. All silk scaffolds revealed a good cell cytocompatibility regarding mitochondrial activity, AF data not shown (Figure 1). For MSC a drop in DNA content from days 21 to 14 was observed (Figure 1B). The cell cytocompatibility of silk scaffolds could be confirmed by live/dead stain where cells adhered to the scaffold and proliferated along silk fibers. qPCR showed an aggrecan/collagen type 2 ratio that indicated differentiation toward nucleus pulposus-like cells for MSC on GDF6 silk³ (Figure 2). The control with exogenous TGFβ1 lead to a decrease in ACAN/COL2 ratio by stimulating COL2 expression more strongly than ACAN. Conclusion: All tested engineered silk scaffolds showed equal biocompatibility regarding mitochondrial activity and DNA and GAG content. Scaffolds containing GDF6 induced differentiation of MSC toward an NP-like phenotype. Hence, the combination of silk and GDF6 might be part of a future inside-out treatment option that might be able to restore the biological function of the disc.

Figure 1.

(A) Mitochondrial activity, (B) DNA content, (C) GAG content of MSCs. All 2-way ANOVA, Bonferroni’s multiple comparison test ± SEM of n = 5 donors; values are means ± SEM, P values: ***<.0006, **<.0095.

Figure 2.

Relative Gene expression of aggrecan to collagen2 of MSC (top) and AF cells (bottom). One sample t test with a hypothetical value of 1.0 was used. P value: ***≤0.0005, *<.048.

Acknowledgments

This project was funded by the Gebert Rüf Stiftung (Project # GRS-028/13). Microscopy was performed on equipment supported by the Microscopy Imaging Center (MIC), University of Bern, Switzerland. We thank Eva Roth for assistance in cell isolation and biochemical assays.

References

1. Clarke LE, McConnell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA. Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition, and micromechanical properties of nucleus pulposus constructs. Arthritis Res Ther. 2014;16:R67.

2. Wöltje M, Böbel M, Rheinnecker M, et al. Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus. Appl Microbiol Biotechnol. 2014;98:4571-4580.

3. Thorpe AA, Binch AL, Creemers LB, Sammon C, Le Maitre CL. Nucleus pulposus phenotypic markers to determine stem cell differentiation: fact or fiction? Oncotarget. 2016;7:2189-2200.

Genipin-Enhanced Fibrin Hydrogel Combined With Engineered Silk Composite for Intervertebral Disc Repair

Daniela A. Frauchiger¹, Adel Tekari², Michael Wöltje², Lorin M. Benneker³, and Benjamin Gantenbein¹

¹Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland ²Institute of Textile Machinery and High Performance Material Technology, TU Dresden, Dresden, Germany ³Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Introduction: Low back pain caused by the intervertebral disc (IVD) is an increasing problem without satisfactory treatment option. Hence, we aim for an “inside-out” approach for repairing herniated IVDs or injuries of the outer annulus fibrosus (AF) by using a fibrin hydrogel in combination with engineered silk. For doing so, we investigated in vitro and ex vivo organ culture the feasibility of a genipin cross-linked fibrin in combination with an engineered silk composite. Materials and Methods: Fresh coccygeal bovine IVDs were isolated under aseptic conditions and a 2 mm biopsy punch was used to induce a circular injury. The injury was filled with genipin-enhanced human-based fibrin hydrogel (Tisseel, Baxter).^1,2 In order to seal the AF, a GMP-compliant silk membrane-fleece composite was placed on the hydrogel filled cavity (Figure 1A). Immediately after repair, IVDs were subjected to in vitro organ culture for 14 days using either one of the 3 loading regimes: (1) complex: 0.2 MPa compression and 0 ± 2° torsion at 0.2 Hz for 8 h/day; (2) static diurnal: 0.2 MPa compression for 8 h/day; (c) no loading. To achieve complex loading a custom-built 2 degree of freedom bioreactor was used.³ Measured end-point parameters were disc inspection for seal failure (herniation), disc height, metabolic activity (resazurin sodium salt assay), DNA, glycosaminoglycan (GAG), and collagen (hydroxyproline) contents. Additionally, histology was performed to visualize injury and integration of hydrogel and silk. Furthermore, cytocompatibility of genipin was tested in vitro with low-passage human mesenchymal stem cells (MSC, n = 6) and donor-matched (n = 3) human AF and nucleus pulposus (NP) cells. Culture medium with increasing genipin concentrations and DMSO (dimethyl sulfoxide) only as control was cultured with 120 000 cells/well (48-well plate) for 14 days, media was exchanged every 2 to 3 days. Mitochondrial activity (resazurin salt) was investigated on days 1, 4, 7, and 14. Results: No herniation of repaired discs could be observed throughout 14 days of culture. The genipin-enhanced fibrin hydrogel was capable of holding the silk composite in place (Figure 1B). Repair of discs did not lead to a restoration of disc height. Nevertheless, metabolic activity, DNA/GAG content, and disc height of repaired discs did not differ significantly from the injured IVDs. Except higher DNA content under static loading for the repaired discs compared to the injured IVDs (P ≤ .004). In vitro experiments on the cellular level showed barely any cell activity for the cells treated with genipin. Genipin showed a high cytotoxicity in vitro on cells tested by reducing the mitochondrial activity to zero (Figure 2). Conclusion: Genipin-enhanced fibrin hydrogel in combination with silk composite was able to form a durable AF seal that withstood instant loading. Nevertheless, our in vitro cytocompatibility test on primary MSC, AF, and NP cells suggests a strong cytotoxic effect of genipin. Future challenge is now to identify a less toxic but still mechanically load bearing hydrogel for AF repair.

Figure 1.

(A) Drawing of healthy disc, injury induced by biopsy punch and repaired disc with hydrogel and silk composite. (B) Macroscopic pictures of IVDs after 14 days of no loading culture. Blue discoloration of repaired disc arises from genipin.

Figure 2.

Mitochondrial activity measured for MSC exposed to media containing different concentrations of genipin dissolved in DMSO, DMSO only, and control medium (n = 6).

Acknowledgments

This project was funded by the Gebert Rüf Stiftung (Project # GRS-028/13). We thank Eva Roth for her valuable assistance in cell isolation, histology, and biochemical assays. We further thank Jochen Walser for assistance with the bioreactor.

References

1. Guterl CC, Torre OM, Purmessur D, et al. Characterization of mechanics and cytocompatibility of fibrin-genipin annulus fibrosus sealant with the addition of cell adhesion molecules. Tissue Eng Part A. 2014;20:2536-2545.

2. Likhitpanichkul M, Dreischarf M, Illien-Junger S, et al. Fibrin-genipin adhesive hydrogel for annulus fibrosus repair: performance evaluation with large animal organ culture, in situ biomechanics, and in vivo degradation tests. Eur Cell Mater. 2014;28:25-38.

3. Davies J. Replacing Animal Models: A Practical Guide to Creating and Using Biomimetic Alternatives. Chichester, England: John Wiley; 2012.

Percutaneous Cement Discoplasty, a Method for Restoring the Weight-Bearing Capacity of Lumbar Spine in Cases of Unstable Vacuum Discs

Gábor Jakab¹ and Péter Pál Varga¹

¹National Center for Spinal Disorders, Budapest, Hungary

Introduction: Intervertebral disc degeneration has been in the center of interest for decades due to its clinical and economical significance. It is observed that in advanced disc degeneration the disappearance of disc material leads to loss of disc height and often is associated with “vacuum” formation. This radiologic appearance may team up with pain, generated in vertical position of the patient. That means, in these cases the axial loading of the spine causes pain, loss of walking distance, and eventually bad quality of life. Materials and Methods: From September 2009 we started injecting bone cement percutaneously into empty lumbar disc spaces in individuals, whose pain generates locally or is referred to buttocks and/or thigh posteriorly or have some sensory deficit due to foraminal up/down stenosis at these levels. We operated more than 300 patients and 600 levels with this method, most of them are elderly with comorbidities and present with multiple musculoskeletal disorders. Regarding patient selection the presence of both suitable disc morphology and relevant complaint of patient is mandatory. As in all unstable pathologies the well-trained stabilizing musculature is important. Gross central or lateral recess stenosis may sacrifice good result or increase the impact of cement outflow; therefore, open decompression is to be considered at the same time. Sagittal misbalance makes the candidate less suitable for this procedure, as it may cause or maintain similar complaints. During the procedure we use general anesthesia and image intensifier, operate only in 1 or 2 discs simultaneously with one cementing, and use the same bone cement as for vertebroplasty. The approach is that of discography through Kambin’s safe triangle and from one, preferably the more collapsed side, thus partially correcting degenerative scoliosis. Results: As patients undergoing this percutaneous procedures are elderly with comorbidities and may have accompanying musculoskeletal disorders, it is difficult to find objective data to measure the extent of improvement due to our operation on a longer run. However there are very few patients whom do not admit remarkable improvement after operation. Complications imply accidental outflow of bone cement (in about 20%, but clinically significant is only 7%, requiring reoperation in less than 4%), loosening of the injected bone cement (in about 3%, requiring reoperation in term of restabilizing the segment(s), preferable in percutaneous way). We had septic complication in only 2 cases, in that debridement and transpeducular fixation solved the problem. Conclusion: Patients presenting with thoracolumbar empty disc spaces in association with pain generating on upright position may benefit from a simple procedure, called percutaneous cement discoplasty, which restores the load-bearing capacity of the spine section. The procedure not only causes widening of spinal canal—predominantly the intervertebral foramina—but also stabilizes the moving segments, and thus eliminates pain raising from dynamic stenosis.

Nasal Chondrocytes Are Potential Autologous Cell-Transplant Candidates for Treating Degenerative Disc Disease

Max H.-P. Gay^1,2, Arne Mehrkens², Andrea Barbero¹, Ivan Martin¹, and Stefan Schären²

¹Tissue Engineering, Department of Biomedicine, University Hospital Basel, Basel, Switzerland ²Department of Spinal Surgery, University Hospital Basel, Basel, Switzerland

Introduction: Intervertebral disc (IVD) degeneration is one of the main causes for chronic back pain. Injection of autologous stem cells is still an experimental treatment for disc degeneration showing limited success so far. This is attributed to a low survival rate of the injected cells due to the harsh environment within the disc, which is hypoxic, acidic, low in nutrients, and possibly inflamed in a degenerative state. Studies performed in animal models have reported that juvenile chondrocytes display a better cell survival and production of extracellular matrix than stem cells, possibly due to chondrocytes being more accustomed to an avascular environment. Recently, it has been shown that adult human nasal septum chondrocytes (ie, cells that can be easily available in an autologous setting, under minimally invasive conditions) have an increased rate of proliferation and synthesis of proteoglycan (GAG) and collagen in contrast to articular chondrocytes (ACs). This study was aimed at assessing whether human nasal chondrocytes (NCs) could be an opportune cell source for autologous cell transplantation therapy in the treatment of IVD degeneration. Methods: Human bone marrow stromal cells (MSCs), ACs, and NCs were isolated from biopsies and expanded in cell culture for 2 passages. Thereafter, cells were cultured in either normoxic or hypoxic (2% O₂) conditions for 4 weeks in 3D pellet culture. The chondrogenic media with either regular (4.5 g/L) or low (1g/L) glucose levels were complemented either with or without the growth factor TGFβ1. The cell survival and the capability to form IVD-like tissue were evaluated using histological and biochemical analysis. Results: Quantification of DNA shows that the cell number of MSC decreased by more than 25% in the absence of TGFβ1 and was barely retained in the presence of the growth factor independent of environmental condition. ACs behaved similar to MSCs with the exception that the cell number increased in hypoxic conditions with the addition of TGFβ1, but independent of the glucose concentration. Surprisingly, the number of NC slightly increased (>12%) in all conditions without TGFβ1. Furthermore, the supplement of TGFβ1 increased the cell number by at least 48% in any of environmental conditions. Histological Safranin O staining and biochemical analysis showed for all 3 cell sources that TGFβ1 was necessary for an adequate production of GAG. The reduction of glucose decreased the level of GAG in pellets formed by MSCs in both hypoxia and normoxia. In contrast, GAG production of ACs was unaffected by changes in glucose concentration; however, hypoxic conditions influenced ACs to synthesize more GAG. Interestingly, NCs do not favor hypoxic conditions for GAG production; nonetheless, in combination with decreased glucose levels, they show a trend to produce the most GAG (35 pgr) compared to ACs and MSCs. Discussion: Our findings indicate that compared to MSCs and ACs, NCs are more prone to survive and synthesize cartilaginous extracellular matrix in vitro in conditions resembling those of the IVD (ie, low oxygen and low glucose concentration) and are therefore an excellent candidate for a cell-based therapy for degenerative disc disease.

Analysis of Osteoarthritis-Specific Structural Changes of Subchondral Bone in Degenerative Lumbar Facet Joints

Cordula Netzer¹, Pascal Distel¹, Hans Deyhle², Uwe Wolfram³, Stefan Schären¹, and Jeroen Geurts¹

¹Spine Surgery, University Hospital Basel, Basel, Switzerland ²Biomaterials Science Center, Department of Biomedical Engineering, University of Basel, Allschwil, Switzerland ³School of Engineering & Physical Sciences, Mechanical, Process & Energy Engineering, Heriot-Watt University, Edinburgh, UK

Introduction: Facet joint osteoarthritis (FJOA) is a prominent clinical hallmark of degenerative spine disorders. During disease progression, cartilage and subchondral bone tissues undergo increased turnover and remodeling. The structural changes to the subchondral tissue of FJOA have not been studied thus far. In this study, we performed a micro computed tomography (µCT) study of the subchondral cortical plate (SCP) and trabecular bone (STB) in FJOA and determined osteoarthritis-specific alterations. Materials and Methods: Twenty-four patients (11 male, 13 female, median age 65) scheduled for decompression and stabilization surgery for degenerative spinal stenosis were included in this study. FJOA specimens were harvested during surgery and analyzed by µCT. Bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) were evaluated using CT Analyzer. Lumbar facet joints without chondropathy from cadaveric specimens (9 male, 6 female, median age 57) served as healthy controls. Age-, gender-, and disease-specific effects were identified by ANOVA (P < .05) and significant differences confirmed by Bonferroni`s posttest. Association between age and structural parameters was determined using correlation analysis. Results: Cortical and trabecular bone structural parameters of FJOA were similar between males and females. Compared to healthy controls, FJOA specimens demonstrated significantly greater trabecular Tb.N (1.97 ± 0.11 vs 1.24 ± 0.04 mm⁻¹) and decrease of Tb.Sp (0.44 ± 0.03 vs 0.69 ± 0.03 mm). Conversely, subchondral cortical plate thickness (0.62 ± 0.08 vs 1.60 ± 0.08 mm) and porosity (22.9 ± 1.9% vs 31.5 ± 2.1%) were significantly less compared to healthy specimens. Tb.Th was equal between patients and controls. Age was positively correlated with Tb.N (r = 0.48, P = .02) and negatively correlated with Tb.Sp (r = −0.44, P = .03) and cortical plate thickness (r = −0.52, P = .04) in FJOA. Cortical plate thickness did not correlate with trabecular bone parameters in healthy and osteoarthritic facet joints. Conclusion: FJOA bone remodeling is characterized by thinning of the SCP and an increase in the number of subchondral trabeculae. Remodeling of cortical and trabecular bone might occur in an uncoupled fashion. Targeting elevated subchondral bone remodeling might slow progression of lumbar FJOA.

Comparison of In Vitro Osteogenic Potential of Iliac Crest and Degenerative Facet Joint Bone Autografts for Intervertebral Fusion in Lumbar Spinal Stenosis

Jeroen Geurts^1,2, Daniela Ramp², Stefan Schären^1,2, and Cordula Netzer^1,2

¹Department of Spine Surgery, University Hospital Basel, Basel, Switzerland ²Department of Biomedical Engineering, University of Basel, Allschwil, Switzerland

Introduction: The promotion of spinal fusion using bone autografts is largely mediated by the osteoinductive potential of progenitors/mesenchymal stem cells (MSCs) that reside in the marrow spaces of cancellous bone. Iliac crest is the common autograft donor site, but its use presents an increased risk for donor-site pain, morbidity, and infection. Degenerative bone samples harvested during facetectomy might provide an alternative viable source of osteoinductive autografts. In this study, we conducted an intra-individual comparison of the osteogenic potential of isolated low passage MSC from both sources. Materials and Methods: Iliac crest and degenerative facet joints were harvested from 8 consecutive patients undergoing transforaminal lumbar interspinal fusion due to lumbar spinal stenosis. MSCs were isolated by collagenase digestion, selected by plastic adherence and minimally expanded for downstream assays. Clonogenic and osteogenic potential was evaluated by colony formation assays in control and osteogenic culture medium. Osteogenic properties, including alkaline phosphatase (ALP) induction, matrix mineralization, and type I collagen mRNA and protein expression were characterized using quantitative histochemical staining and reverse transcription PCR (polymerase chain reaction). Spontaneous adipogenesis was analyzed by adipocyte enumeration and gene expression analysis of adipogenic markers. Results: Average colony-forming efficiency in osteogenic medium was equal between iliac crest (38 ± 12%) and facet joint (36 ± 11%). Osteogenic potential at the clonal level was 55 ± 26% and 68 ± 17% for iliac crest and facet joint MSC, respectively. Clonogenic and osteogenic potential were significantly negatively associated with donor age. Osteogenic differentiation led to significant induction of ALP activity in iliac crest (6-fold) and facet joint (8-fold) MSC. Matrix mineralization quantified by Alizarin red staining was increased by osteogenic differentiation, yet similar between both MSC sources. Protein expression of type I collagen was enhanced during osteogenesis and significantly greater in iliac crest MSC. Correspondingly, COL1A2 mRNA expression was higher in osteogenically differentiated MSC from iliac crest. Adipocyte numbers showed significant differences between iliac crest (63 ±6 0) and facet joint (18 ± 15) MSC under osteogenic conditions. Negative (GREM1) and positive (FABP4) adipogenic markers were not differentially expressed between sources. Conclusion: MSCs from iliac crest and degenerative facet joints largely display similar clonogenic and osteogenic properties in vitro. Differences at the molecular level are not likely to impair the osteoinductive capacity of facet joint MSC. Bone autografts from facetectomy would be viable alternatives as bone autografts for intervertebral spinal fusion in lumbar spinal stenosis.

A Novel Microgravity-Augmented Model for Intervertebral Disc Aging

Christina Giger-Lange¹, Simon L. Wuest¹, Fabian Ille¹, Benjamin Gantenbein-Ritter², and Marcel Egli¹

¹Space Biology Group, Institute of Medical Engineering, Lucerne University of Applied Sciences and Arts, Lucerne, Switzerland ²Organ Mechanobiology Group, Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland

Introduction: Age-related lower back pain due to intervertebral disc (IVD) degeneration is developing into a considerable economic and societal burden. The slow scientific advances during the last 30 years in introducing potent cures and prevention strategies are mainly due to lacking human disc material and limitations of existing disease models. It has been known for years that astronauts experience disc degeneration due to mechanical unloading during space flight. We therefore introduce an accelerated disease model based on mechanical unloading by simulated microgravity. This model overcomes limitations of the classical models by not requiring the use of nonphysiological methods to induce disc degeneration. Materials and Methods: Bovine IVDs were isolated from fresh ox-tails. They were cultured in a newly designed bioreactor for IVD loading that fits into an incubator that is located on a random positioning machine (RPM). The RPM is commonly used to simulate microgravity through the principle of gravity vector averaging. Thus, the samples are continuously rotating around 2 perpendicular axes. This principle showed good correlation to space experiments on several mammalian cell types. To ensure physiological conditions for the IVDs, the reactor was equipped with a mechanical loading system that can induce different loading/unloading patterns such as static and cyclic compression as to ensure a good nutrient exchange. The samples were cultivated for 2 weeks under earth gravity and simulated microgravity. Subsequently IVDs were measured, dissected, and digested with papain. The tissue was then analyzed for metabolic activity, DNA content, and cell viability. Glycosaminoglycan (GAG) measurements were performed to quantify degeneration. Results: We developed a novel bioreactor system that can be operated under simulated microgravity conditions. Preliminary data suggest that cell viability and metabolic activity of cultured IVDs is comparable to IVDs cultured in a classical 1g state-of-the-art bioreactor system. Furthermore, mechanical unloading induced a decrease of GAG content in first experiments, suggesting that the discs had degenerated as expected. Conclusion: Our preliminary data indicate that the new bioreactor can be used as a novel model system for IVD degeneration. We will further validate our findings with more data and additional experiments (eg, Safranin-O staining). Moreover, we will compare our cultured IVDs with degenerated human IVDs. Following the validation, we are planning to test the effect of different mechanical loading patterns on disc degeneration in our model and to provide prevention and treatment strategies for intervertebral disc aging and degeneration.

MRI-Informed Biomimetic Design of Artificial Intervertebral Disc Scaffolds Using 3D Bioplotting

Ella Hodder^1,2,3,4, Derek Covill¹, Nick Dowell², Mark Best³, Mara Cercignani^2,5, and Lisa M. Harris^2,4

¹School of Computing, Engineering and Mathematics, Brighton and Sussex University, Brighton, UK ²Clinical Imaging Sciences Centre, Brighton and Sussex University, Brighton, UK ³School of Pharmacy and Biomolecular Sciences, Brighton and Sussex University, Brighton, UK ⁴Brighton and Sussex University Hospitals NHS Trust, Brighton, UK ⁵Brighton Centre for Regenerative Medicine, Brighton and Sussex University, Brighton, UK

Introduction: Intervertebral disc (IVD) replacements using artificial devices (AD) have typically been pursued through the engineering of polymeric and metallic materials. However, due to the properties of the materials used, current ADs are unable to comply with the compressive forces of the spine, becoming subject to wear and damaging debris release. A promising alternative to ADs lies in tissue engineering in the form of biologically active constructs. This work investigates the use of the novel material alginate methylcellulose (alg/MC) as a candidate for chondrocyte-embedded scaffolds using 3D bioplotting. With the intent to produce artificial disc alternatives, guided by quantitative medical imaging, capable of replicating the physiological properties of the IVD. Materials and Methods: Quantitative MRI: Ten healthy lumbar discs were scanned using a Siemens Avanto 1.5 T imager. In vivo quantification of magnetization transfer ratio (MTR) to determine collagen content was made following the Helms method.¹ MR spectroscopy to detect proteoglycan levels was performed using acquisition conditions TE = 30 ms, TR = 1500 ms, with 256 averages. A 3D gradient echo (GRE) sequence was used to measure water content with T2* (TR =100 ms; Figure 1). 3D bioplotting: The alg/MC hydrogel mixture was prepared as first described by Hoppe et al.² The mixture was left to swell for 1.5 hours before the addition of bovine primary chondrocytes (BPCs) at a cell seeding density of 5 × 10⁶ per gram of material. BPCs were isolated from bovine metacarpal joints by collagenase digestion. The scaffolds were plotted using a tip diameter of 610 μm, print speed 10 mm/s, pressure 0.8 bar, and crosslinked in 100 mM CaCl₂. Samples were analyzed at days 7, 14, and 21 of culture (37°C; 5% CO₂), using gene expression analysis for collagen type I and II. Safranin-O staining was done for proteoglycans. Fluorescent microscopy for live/dead cell analysis with CMFDA-green and ethidium bromide. Results and Discussion: The T2* map (Figure 2) shows the characteristic water content distribution of discs. The mean T2* values for all participants was 49.1 ms, similar to values reported in literature.^3,4 MTR results accurately reflect known differences in collagen content between the AF (high) and NP (low). The MRS spectrum attained gave clear singlet peaks for proteoglycan at 2.04 ppm in all discs, comparable to published data.⁵ Cell survival was calculated at 80% to 85% (Figure 3) at days 1, 7, and 14, quantified directly from Live/Dead cell counts. The gene expression ratio of collagen type I to II in day 14 samples was 4:96, indicating the chondrocyte phenotype is supported. Safranin-O staining of proteoglycans demonstrated increasing content with longer culture time. In summation, this work promotes the feasibility of using T2*, MTR, and MRS to characterize the microstructure of discs, while demonstrating the use of 3D bioplotted alg/MC as a biocompatible and viable material for supporting cartilage tissue growth. Future work will aim to utilize these methods in producing scaffolds that more closely resemble the properties of native discs, as guided by qMRI measurements and physiochemical testing.

Figure 1.

T2* map of a disc in a healthy 29-year-old.

Figure 2.

3D Bioplotting of an alg/MC scaffold.

Figure 3.

Live/dead staining of BPC alg/MC scaffolds, day 7.

References

1. Helms G, Dathe H, Kallenberg K, Dechent P. High-resolution maps of magnetization transfer with inherent correction for RF inhomogeneity and T1 relaxation obtained from 3D FLASH MRI. Magn Reson Med. 2008;60:1396-1407.

2. Hoppe S, Quirbach S, Mamisch TC, Krause FG, Werlen S, Benneker LM. Axial T2 mapping in intervertebral discs: a new technique for assessment of intervertebral disc degeneration. Eur Radiol. 2012;22:2013-2019.

3. Schütz K, Placht AM, Paul B, Brüggemeier S, Gelinsky M, Lode A. Three-dimensional plotting of a cell-laden alginate/methylcellulose blend: towards biofabrication of tissue engineering constructs with clinically relevant dimensions. J Tissue Eng Regen Med. 2017;11:1574-1587.

4. Zuo J, Joseph GB, Li X, et al. In vivo intervertebral disc characterization using magnetic resonance spectroscopy and T1ρ imaging: association with discography and Oswestry Disability Index and Short Form-36 Health Survey. Spine (Phila Pa 1976). 2012;3:214-221.

5. Zuo J, Saadat E, Romero A, et al. Assessment of intervertebral disc degeneration with magnetic resonance single-voxel spectroscopy. Magn Reson Med. 2009;62:1140-1146.

Autologous Disc Chondrocyte Transplantation in Lumbar Spine Disc Degeneration Disease: A Prospective, Controlled, Randomized Study

Christian Hohaus^1,2, Florian Didrigkeit², and Hans Jörg Meisel²

¹Department of Neurosurgery, Städtisches Klinikum Dessau, Dessau, Germany ²Department of Neurosurgery, BG Klinikum Bergmannstrost, Halle, Germany

Introduction: Morphological restoration of degenerated intervertebral discs through the use of autologous disc chondrocyte transplantation (ADCT) represents a strategy that offers a potential to accentuate disc metabolism that will achieve mechanic function. This could be demonstrated in a preclinical animal study and was translated into the clinical prospective, controlled, randomized study. Materials and Methods: Seventy-eight patients with single-level intervertebral disc herniation with acute neurological symptoms or after conservative treatment failure were enrolled from January 2002 to November 2006 in a single institution. Operative procedure was done as a minimal invasive open sequestrectomy done by an experienced neurosurgeon. Cells are transplanted approximately 12 weeks following sequestrectomy to assure that the annulus has healed and will contain the cells. Therefore, a single puncture was used. Oswestry low back pain disability questionnaire (OPDQ), Prolo, and VAS (Visual Analog Scale) scores were used for the evaluation. Disc height and water content were assessed by MRI (magnetic resonance imaging). Demographic data, neurological status, and body mass index (BMI) were collected from the patients 6, 12, and 24 months postoperatively. Results: Thirty-six patients were randomized into the autologous disc chondrocyte transplantation group and 43 patients into the control group. The mean age of the patients was 31.4 years; the mean BMI was 23.9. Twenty-three patients from the ADCT group were transplanted in level L5/S1 and 12 patients at level L4/5. There were 7 dropout patients for private reasons. After 24 months the patients of the treatment group scored an average of 16.4 in the Oswestry questionnaire and 20.4 on the VAS, while the controls got 21.1 in the Oswestry and 22.6 on the VAS. Regarding the Prolo scale, transplanted patients were able to reach a 4.7 and 4.5 in working situation and functional status in comparison to 4.6 and 4.4 in the nontreatment group. The differences between the 2 groups were much higher by comparison in changes in all scores after the operative treatment and in the 24-month follow-up. Reherniation rate was reduced by 52%. No neurological or inflammatory complications were reported. Conclusions: For once, we were able to prove that no harm was done with the transplantation that is at least as good as the standard procedure and that there is no common adverse effect. Furthermore, even though that there was no statistical difference between the 2 groups big enough to prove the benefit of the autologous disc chondrocyte transplantation, we were still able to show that there is a tendency toward that 24 months after transplantation. Indeed, having such small differences may only show how good our standard procedures already are. Knowing the degeneration process in younger patients is going on over more than 2 years and we might just need to oversee the results over a longer period of time.

Three-Dimensional MRI Analysis of Paraspinal Muscle Degeneration

Sven Hoppe¹, Waldo Valenzuela², Daniela Maurer¹, Sufian S. Ahmad¹, and Lorin Benneker¹

¹Inselspital Bern, Department of Orthopaedic Surgery, Spine Unit, University of Bern, Bern, Switzerland ²Institute for Surgical Technology & Biomechanics, University of Bern, Bern, Switzerland

Introduction: Factors influencing paraspinal muscle degeneration are still not well understood. Fatty infiltration (FI) is known to be one main feature of the degeneration cascade. The aim of this study was to illustrate the cluster of paraspinal lumbar muscle degeneration on MRI. Material and Methods: Monosegmental, plurisegmental, and multisegmental lumbar muscles groups were segmented on T2-weighted MR sequences using a novel semiautomatic technique for quantitative muscle/fat discrimination. The degree of fatty infiltration of t3 predefined muscle groups was compared on a 3-dimensional basis, with regard to segment involvement, age, and disc degeneration. General linear models were utilized for statistical comparison. Results: N = 78 segments (age: 52.7; range: 16-87years) could be included. The average fatty infiltration of the monosegmental muscles (21.1 ± 14.5%) was significantly higher compared to the multisegmental (16.0 ± 8.8%) and plurisegmental (8.5 ± 8.0%) muscles. Muscles on the levels L4/5 (P = .09) and L5/S1 (P = .016) showed a significant higher amount of fat compared to the levels L2/3 and L3/4 if degeneration was present. Higher age (P = .039) and disc degeneration (P = .025) were covariables associated with increased fatty infiltration. Conclusion: The complexity of the problematic issue of lower back pain is likely to be beyond the horizon of a simple bony disease. Muscle affection that was shown to correlate to the degree of degeneration may truly play a more important role than yet perceived by the general spinal community and would necessitate increased attention in the future.

Musculoskeletal Modeling of Sarcopenia for Prediction of Associated Spinal Loads

Dominika Ignasiak¹, Waldo Valenzuela², Mauricio Reyes², Stephen J. Ferguson¹

¹Institute for Biomechanics, ETH Zurich, Zurich, Switzerland ²Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland

Introduction: Sarcopenia, the age-related progressive loss of muscle mass and function, is related to the increased risk of osteoporotic vertebral fractures.¹ Associations between trunk musculature and vertebral fractures have been described² but the underlying biomechanical relationships are not well understood. Therefore, the purpose of this study was to investigate the effect of different stages of muscle ageing and sarcopenia on spinal load distribution, combining musculoskeletal modeling approach with experimentally assessed spinal kinematics and previously reported trunk muscle properties. Material and Methods: A detailed AnyBody multi-body model of the thoracolumbar spine^3,4 was used to simulate a forward flexion maneuver, previously recorded in a group of healthy volunteers⁵ in order to predict associated thoracolumbar (T1T2-L5S1) segmental loads and muscle forces for simulated conditions of muscle deterioration (Figure 1). Muscle deterioration was represented in the model by a reduction of maximum possible muscle stress (ie, muscle strength) of erector spinae and multifidus muscles. Stages of simulated normal ageing from the 3rd to 6th decades of life were based on the level-dependent fatty infiltration affecting these muscles, estimated from previously reported MR imaging study.⁶ Levels of muscle cross-sectional reduction⁷ and fat infiltration,⁸ mentioned in the literature, were used to scale erector spinae and multifidus homogenously along the spine to reflect several severity degrees of sarcopenia (mild: 60% normal muscle strength; moderate: 48%; severe: 36%; very severe: 24%). Results: The maximum compressive loads associated with normal muscle ageing conditions were found to be <3% higher for the thoracic and <7% lower for the lumbar segments, compared to the default (unscaled muscles) condition. Simulations of severe sarcopenia stages predicted considerable increase of compressive (Figure 2) and shear forces in the upper thoracic spine, T1T2-T6T7. While the maximum compressive loads acting on the thoracolumbar junction were lower with increasing severity of sarcopenia, shear magnitudes were increased. These changes in loading magnitude and direction might contribute to the increased risk of vertebral fractures in severe sarcopenia stages. Furthermore, decreasing erector spinae and multifidus strength resulted in increased activity of all major trunk muscles. This might provide a biomechanical explanation for increased muscle fatigue and compromised balance in the individuals with sarcopenia. Conclusion: The influence of sarcopenia on spinal loads was investigated using a complex musculoskeletal model of the thoracolumbar spine, combined with realistic measurement-derived spinal kinematics and trunk muscle properties. The results contribute to our understanding of biomechanical links between sarcopenia and vertebral fractures and demonstrate the potential of this combined experimental and computational approach for future analysis of patient-specific cases. The limitations of the current study, that is, normal spine kinematics and simplified modeling of muscle deterioration, can be overcome by including kinematics characteristic for sarcopenia patients and level-dependent properties of multiple trunk muscle groups. Insight into corresponding spinal loading conditions could improve prevention and clinical management of sarcopenia.

Figure 1.

The concept of musculoskeletal modeling of the thoracolumbar spine combined with measurement-derived spine kinematics and paraspinal muscle properties obtained from previously reported imaging studies.

Figure 2.

Maximum segmental compressive forces estimated for simulated stages of sarcopenia affecting erector spinae and multifidus muscles, normalized to default (unscaled muscle strength condition).

References

1. Hida T, Shimakata H, Sakai Y, et al. Sarcopenia and sarcopenic leg as potential risk factors for acute osteoporotic vertebral fracture among older women. Eur Spine J. 2016;25:3424-3431.

2. Mokhtarzadeh H, Anderson DE. The role of trunk musculature in osteoporotic vertebral fractures: implications for prediction, prevention, and management. Curr Osteoporos Rep. 2016;14:67-76.

3. Ignasiak D, Dendorfer S, Ferguson, SJ. Thoracolumbar spine model with articulated ribcage for the prediction of dynamic spinal loading. J Biomech. 2016;49:959-966.

4. Ignasiak D, Ferguson SJ, Arjamand N. A rigid thorax assumption affects model loading predictions at the upper but not lower lumbar levels. J Biomech. 2016;49:3074-3078.

5. Ignasiak D, Rueger E, Ferguson SJ. Thoracic spine kinematics in the young and elderly. Paper presented at: 21st Congress of the European Society of Biomechanics; July 5-8, 2015; Prague, Czech Republic.

6. Crawford RJ, Filli L, Elliott JM, et al. Age- and level-dependence of fatty infiltration in lumbar paravertebral muscles of healthy volunteers. Am J Neuroradiol. 2016;37:742-748.

7. Lang T, Streeper T, Cawthon P, Baldwin K, Taaffe DR, Harris TB. Sarcopenia: etiology, clinical consequences, intervention, and assessment. Osteoporos Int. 2010;21:543-559.

8. Kang CH, Shin MJ, Kim SM, Lee SH, Lee CS. MRI of paraspinal muscles in lumbar degenerative kyphosis patients and control patients with chronic low back pain. Clin Radiol. 2007;62:479-486.

Acknowledgement

This study has been supported by a research grant from AOSpine International, Switzerland (Project CPP FFOB_OC_14).

Introducing SpinePRO: Digital Clinical Data Collection for Spine Tango

T. R. Jansen¹, H. Kohlhof¹, R. Bornemann¹, S. Hebel¹, S. Koob¹, D. C. Wirtz¹, R. Pflugmacher¹, and Y. Rommelspacher¹

¹Department of Orthopedics and Trauma Surgery, University Clinic Bonn, Germany

Introduction: Permanent innovations in spinal surgery demand quality assurance. For that reason Spine Tango was created.^1,2 Patient-related outcomes (PROs) are an essential tool to achieve Spine Tango’s goals. A systematic collection of PRO data can be time consuming in a hospital setting.³ That is why we created SpinePRO, an iOS App for spine patients and a web app for doctors. The objective of this study was to investigate the feasibility of a completely electronic data collection. Materials and Methods: We developed a tablet computer application using the open source framework ResearchKit.⁴ The app was used to inform and consent patients and collect a medical history and PRO data (COMI back or neck and EQ5D). An additional web application for doctors was developed using the open source framework AngualarJS.⁵ Results: A total of 700 patients consented to the study, 500 medical histories, 450 COMI for neck or back pain, and EQ-5D-3L were completed. Patients’ average age was 55 years. Most patients complained of low back pain (67%), followed by neck pain (17%) and upper back pain (11%). Pain duration was in 54% over 1 year, in 15% the pain was posttraumatic, and 94% had prior conservative treatment. Patients with neck pain had a median COMI score of 5.75, EQ5D score of 0.845, and EQ5D VAS of 55.5%; patients with upper back pain had a median COMI score of 6.66, EQ5D score of 0.531, and EQ5D VAS of 51.2%; patients with low back pain had a median COMI score of 7.5, EQ5D score of 0.79, and EQ5D VAS of 45.6%. Conclusions: We successfully developed SpinePRO, an iOS App for patients and a web app for doctors, as an accessory for the international spine registry Spine Tango. SpinePRO provides an effective and useful tool to collect a digital patient history and PRO data in health care routine. Data can be sent directly to the hospital information system and Spine Tango. Patient satisfaction was excellent.

References

1. Melloh M, Staub L, Aghayev E, et al. The International Spine Registry SPINE TANGO: status quo and first results. Eur Spine J. 2006;17:1201-1209.

2. Roeder C, Chavanne A, Mannion AF, Grob D, Aebi M. SSE Spine Tango—content, workflow, set-up. Eur Spine J. 2005;14:920-924.

3. Goz V, Lakomkin N. Reliability of SRS-22 and ODI by phone: a step toward making PROs more accessible. Spine J. 2016;16:1047-1048.

4. Apple Inc. ResearchKit. https://developer.apple.com/researchkit/. Accessed September 20, 2018.

5. AngularJS. Home page. https://angularjs.org/. Accessed September 20, 2018.

The Expression of Transient Receptor Potential (TRP) Channels in Intervertebral Disc Cells Is Regulated by Pro-Inflammatory Cytokines

Olga Krupkova¹, Joel Zvick¹, Jürgen Klasen², Stephen J. Ferguson¹, Alfredo Franco-Obregon³, Karin Wuertz-Kozak¹

¹ETH Zurich, Switzerland ²Prodorso, Zurich, Switzerland ³National University of Singapore, Singapore

Introduction: The transient receptor potential (TRP) channels are calcium permeable ion channels located in the plasma membrane. TRP channels are involved in various processes controlling mechanosensing, osmosensing, and cellular homeostasis.¹ Dysregulation in TRP function has been implicated in multiple diseases (eg, osteoarthritis, cardiovascular disease, muscular dystrophy), where it has been linked to inflammation and hyperalgesia.² As painful degenerative disc disease (DDD) is strongly correlated with mechanical, osmotic, and inflammatory stress, TRPs are likely dysregulated during the course of this disease. However, the role of TRPs in intervertebral disc (IVD) degeneration remains largely unexplored. Our study aims to uncover the involvement of TRP channels in inflammatory responses of IVD cells and assess the importance of these channels as a new therapeutic target in DDD. Materials and Methods: IVD cells were isolated from patients undergoing surgeries related to DDD (n = 5) and cultured adherently up to passage 3. IVD cells were stimulated with the pro-inflammatory cytokines IL-1β and TNF-α at 5 and 10 ng/mL for 18 hours. Gene expression of TRP channels in stimulated cells was analyzed by RT-qPCR and compared to an untreated control group. Target TRP channels were selected based on available literature. Statistical analysis was performed by ANOVA with Tukey post hoc test (P < .05). Results: The pro-inflammatory cytokines IL-1β and TNF-α significantly increased gene expression of TRP anykyrin 1 (TRPA1), with TNF-α having more profound effect. Both IL-1β and TNF-α significantly reduced the expression of TRP canonical 6 (TRPC6), but did not significantly influence the expression of other tested TRP channels (Figure 1). Conclusion: It has been shown that gene expression of TRP vallinoid 4 (TRPV4) in IVD cells can be regulated by tissue osmolality.³ Our data suggest that pro-inflammatory cytokines do not influence the expression of TRPV4, but that these cytokines regulate the expression of TRPA1 and TRPC6 in IVD cells. TRPA1 is considered a key player in acute and chronic pain and inflammation, with a possible role in the pathophysiology of many organ systems.⁴ Recently, TRPA1 expression was found in inflammatory OA chondrocytes, with TRPA1 involved in catabolic and inflammatory responses.⁵ TRPC6 is expressed in muscle cells and neurons where it regulates growth, function, and survival,¹ but the role of TRPC6 in inflammation of cartilaginous tissues remains unknown. Our own unpublished data indicates that TRPC6 is relevant in regulating cell cycle progression and senescence in IVD cells, but may possess broader, yet unknown function in IVD health and disease. Our data are currently being verified on the protein level by Western blotting. In the near future, the significance of our findings will be evaluated by TRP activity assays and using specific TRP blockers.

Figure 1.

Gene expression of TRP channels in IVD cells treated with pro-inflammatory cytokines. IL-1β and TNF-α significantly induced the expression of TRPA1 and inhibited the expression of TRPC6. Graphs shows mean ± SEM (n = 5) evaluated by ANOVA with Tukey post hoc at P < .05, relative to untreated control.

References

1. Wu LJ, Sweet TB, Clapham DE. International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol Rev. 2010;62:381-404.

2. Kaneko Y, Szallasi A. Transient receptor potential (TRP) channels: a clinical perspective. Br J Pharmacol. 2014;171:2474-2507.

3. Walter BA, Purmessur D, Moon A, et al. Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells. Eur Cells Materials. 2016;32:123-136.

4. Nilius B, Appendino G, Owsianik G. The transient receptor potential channel TRPA1: from gene to pathophysiology. Pflugers Arch. 2012;464:425-458.

5. Nummenmaa E, Hamalainen M, Moilanen LJ, et al. Transient receptor potential ankyrin 1 (TRPA1) is functionally expressed in primary human osteoarthritic chondrocytes. Arthritis Res Ther. 2016;18:185.

Biological Disc Replacement Using Tissue-Engineered Intervertebral Discs Combined With a Resorbable Stabilization System: A Proof of Concept Study in an Ex Vivo Beagle Model

Gernot Lang^1,2, Jorge Mojica Santiago³, Rodrigo Navarro-Ramirez¹, Ibrahim Hussain¹, Roger Härtl¹, and Lawrence J. Bonassar^3,4

¹Weill Cornell Brain and Spine Center, Weill Cornell Medicine, New York-Presbyterian Hospital, New York, NY, USA ²Department of Orthopedic and Trauma Surgery, Freiburg University Medical Center, Freiburg, Germany ³Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA ⁴Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY, USA

Introduction: Recently, a tissue engineered (TE) intervertebral disc (IVD)-like construct has been evaluated in rat tail and beagle cervical spine models in vivo. While biological properties were encouraging, the biomechanical performance was inferior to native IVDs and implant displacement occurred occasionally. The purpose of this study was to assess the biomechanical responses of TE-IVDs combined with a resorbable stabilization system (BSS) to static compression in a canine ex vivo model. Materials and Methods: Cervical IVDs from beagles were obtained and separated into annulus fibrosus (AF) and nucleus pulposus (NP) tissues by macroscopic appearance. Then the tissues were digested in collagenase Type II at 37°C for 12 hours prior to being filtered and centrifuged. NP and AF cells were cultured individually to confluence. Alginate (3% weight/volume) was seeded with the cultured NP cells (2.5 × 10⁶ cells/mL) and injected into predesigned molds. Each of the molded NPs was placed in a well of a culture plate as a neutralized collagen gel solution (4 mg/mL seeded with 3 × 10⁶ cells/mL) was pipetted around the NP. All components were gelled at 37°C and TE-IVDs were cultured for 4 weeks. Whole cervical spinal motion segments from skeletally mature beagles were dissected and the mechanical compatibility was assessed for each motion segment at different time points: (1) intact, (2) after discectomy, (3) after implantation of the TE-IVD, and (4) after implantation of the TE-IVD and application of the BSS ventrally (Figure 1). Unconfined stress relaxation tests were performed up to 15% strain and their instantaneous and equilibrium modulus were calculated and reported normalized to the intact motion segment (Figure 1). One-way ANOVA was performed on all groups including the intact control segment and Tukey HSD was used to determine significant differences at P ≤ .05. Results: Intact motion segments revealed an equilibrium and instantaneous modulus of 174 ± 36 kPa and 1760 ± 430 kPa. Mechanical properties from motion segments after discectomy (Dx), TE-IVD implantation without BSS (IVD−), and TE-IVD implantation with BSS (IVD+) were all significantly lower than the intact motion segments. Results from all treatment groups were then normalized to their corresponding intact motion segment (N = 12). Dx motion segments resulted in 14 ± 6% and 13 ± 4% of the intact equilibrium and instantaneous modulus. Application of the BSS (IVD+) revealed a 2-fold increase in the equilibrium and instantaneous modulus (P ≤ .05) over the groups with TE-IVDs implantation only (IVD−). Conclusion: The present study demonstrates that a resorbable plate can be used in combination with a TE-IVD to help stabilize a canine cervical motion segment, while allowing load sharing to the TE-IVD. Implantation of the TE-IVD yielded relatively similar mechanical properties to those of the discectomized segments suggesting low magnitudes of loads are shared by the construct. The significant increase in mechanical properties of the motion segment with a plate suggests that the BSS increases the stability of TE-IVD construct and prevents implant displacement.

Figure 1.

(A) TE-IVD. (B) TE-IVD in situ. (C) TE-IVD with bioresorbable stabilization system. (D) Equilibrium modulus normalized to the intact motion segment. N = 12, Mean ± SD, *P < .05. (E) Instantaneous modulus normalized to the intact motion segment. N = 12, Mean ± SD, *P < .05.

Hypoplastic Vertebral Body and Recurrent Symptomatic Disc Herniation

Ralph Laeubli, MD¹ and Nicolas Fragnière, MD²

¹Head Spine Unit Hospitals FMI and Sportsclinic #1, Bern ²Hospitals FMI, Interlaken

Background Context: Pouriesa et al already demonstrated that difference of end plates anteroposterior diameter of lumbar vertebral bodies is a significant risk factor for occurrence of lumbar disc herniation. Operative activity in Spitäler FMI Interlaken in 2013 for cases of symptomatic lumbosacral disc herniation suggests that isolated lumbar or sacral vertebral body hypoplasy may play a major role in recurrence of symptomatic disc herniation. Purpose: To evaluate whether hypoplastic lumbar or sacral vertebral body is associated with increased risk of recurrence of disc herniation at its cranial level after surgical therapy. Design: Retrospective cohort study. Patient Sample: All cases of operated patients in 2013 in Spital FMI Interlaken because of symptomatic lumbar or lumbosacral disc herniation were studied. Patients with significant deformity or listhesis were excluded at once because of impairing measurement or possibly crossing etiology for radiculopathy. Also excluded were patients for whom MRI was not available. Among the remainders, 25 were men and 8 were women. Average age at the time of operation or re-operation was 50.4 years. Among this sample, 9 patients showed recurrence after former operation. Method: To assess vertebral body hypoplasy, cross section of 2 neighboring lumbar and sacral end plates lining symptomatic herniation was measured on section MRI pictures that were parallel to herniated disc, using a semi-manual method available on Phönix PACS program. A vertebral body was assessed as hypoplastic if its cranial end plate was measured greater than the caudal end plate of its cranial neighboring vertebral body with several measurements. Results: Among 33 cases of patients with radiculopathy due to documented lumbar or lumbosacral disc herniation, 20 were considered having vertebral hypoplasy according to our measurements, of which 7 were recurrent cases. In 2 cases of first occurrence of symptomatic disc herniation, a related hypoplastic vertebral body was assessed. Relative risk was calculated about 2.4. We also observed that exclusion of cases with poorly significant end plate difference makes the relative risk even raise. Conclusion: Results suggest that hypoplastic vertebral body represent a risk factor to recurrence of symptomatic proximal disc herniation. Nevertheless, this has to be confirmed among a much larger cohort of patients.

Comparison of Gene Expression of Discs From Diffuse Idiopathic Skeletal Hyperostosis (DISH) and Healthy (Trauma) Patient

Rahel D. May¹, Daniela A. Frauchiger¹, Lorin M. Benneker², and Benjamin Gantenbein¹

¹Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland ²Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Introduction: Diffuse idiopathic skeletal hyperostosis (DISH) is a common disease, affecting mostly middle-aged and elderly patients. DISH is characterized by formation of bone along the anterior spine. Often also extraspinal sites attached to the bones are affected by this disease. Abnormalities like osteophytes and following ossification anterior to the vertebral bodies and intervertebral discs (IVD) are frequently observed in DISH patients.¹ However, the nucleus pulposus remains unaffected from this disease. We have recently shown that the IVD cells are expressing BMP antagonists, which could lead to inhibition in bone formation.² The aim of this study is to compare the transcriptome of discs of the BMP pathway from DISH patients with discs from healthy donors. Here, we hypothesize that a possible reason of DISH could be a misconduct of the BMP signaling pathway. Methods: One fresh DISH IVD and one healthy trauma IVD (male 16 years) were obtained from 2 patients undergoing spinal surgery (proved by the Ethics Committee of the Canton of Bern). IVD cells were isolated from their native extracellular matrix by digestion of the tissue with pronase (1.9 mg/mL; Roche, Basel, Switzerland) for 1 hour and subsequent collagenase II digestion (129 U/mL; Worthington, London, UK). Cells were cultured up to passage 2 and then lysed in TRI reagent for total RNA extraction. RNA integrity and quality was controlled using Experion RNA electrophoresis prior qPCR (Bio-Rad). primePCR (TGFβ BMP Signaling Pathway Plus H96, 90 genes, Bio-Rad) was run on CFX96 (Bio-Rad). Results: The pairwise transcriptome comparison of a DISH and a healthy disc detected regulated BMP genes, such as upregulation of TGFβ1 and BMP2 in the DISH patient. Most interestingly, the BMP antagonist noggin (NOG) could be detected as downregulated in the DISH patient. Furthermore, there was a downregulation in nephroblastoma overexpressed (NOV) observed. NOV is involved in bone regeneration as a negative regulator by binding to BMP2. Finally, there was BMP-binding endothelial regulator protein (BMPER) downregulated, which is a negative factor of bone formation (Figure 1). Discussion: This is to our best knowledge the first study to shed light onto the phenotype of disc cells from patients suffering from DISH. Most interestingly, the disc cells showed an altered phenotype in BMP-related cytokines and their antagonists. This unexpected result considering the fact that the ossification happens in the neighboring ligaments and enthesis, leaving the inner part of the disc macroscopically unaffected. At the current stage, the nature of these transcript differences cannot be attributed to intraspinal level, age, or tissue-specific differences as more sampling is needed.

Figure 1.

TGFβ pathway transcriptome analysis. RNA transcripts that differed more than four times are indicated in re (up-regulated or in green (downregulated in DISH patient) relative to 16-years old trauma patient.

References

1. Resnick D, Guerra J Jr, Robinson CA, Vint VC. Association of diffuse idiopathic skeletal hyperostosis (DISH) and calcification and ossification of the posterior longitudinal ligament). AJR Am J Roentgenol. 1978;131:1049-1053.

2. Tekari A, May RD, Frauchiger DA, Chan SC, Benneker LM, Gantenbein B. The BMP2 variant L51P restores the osteogenic differentiation of human mesenchymal stromal cells in the presence of intervertebral disc cells. Eur Cell Mater. 2017;33:197-210.

The Influence of Primary Human Intervertebral Disc Cells on Primary Human Osteoblasts

Rahel D. May¹, Adel Tekari¹, Daniela A. Frauchiger¹, Lorin M. Benneker², Sandro Kohl², and Benjamin Gantenbein¹

¹Tissue and Organ Mechanobiology, Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland ²Department of Orthopaedic Surgery, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland

Introduction: For discogenic back pain the currently most popular strategy is lumbar interbody fusion (LIF), where the disc space is cleared and replaced with a cage (± bone substitute) to allow an intersomatic spondylodesis. Clinical observation showed that partial IVD tissue removal could lead to a failure of spinal ossification.¹ It was previously shown that human IVD cells inhibit ossification of human mesenchymal stem cells (MSCs).² However, whether this is a direct effect of IVD cells on osteoblasts (OB) is still unknown. We hypothesized that human primary OB co-cultured with IVD cells would show similar inhibitory effects in ossification as previously demonstrated for MSC. Methods: OB isolated from patients (N = 7) undergoing total knee replacement were seeded at a density of 10 000 cells/cm². IVD cells, as nucleus pulposus cells (NPC), annulus fibrosus cells (AFC), and cartilaginous endplate cells (CEPC), isolated from patients undergoing spinal surgery, were encapsulated in 1.2% alginate beads (∼30 µm in Ø at 4 M cells/mL, ∼75 000 cells/bead) and co-cultured via inserts (PET, high-density 0.4 µm pore size) with OB in the lower part as monolayer. Six, 9, and 12 NPC, AFC, or EPC beads, respectively, were investigated to test the dose response. The experimental groups were stimulated with osteogenic medium. Additionally, 3 controls were cultured: OB monolayer with osteogenic medium (± empty beads) and with control medium (α-MEM + 10% FCS). To quantify ossification, matrix mineralization of OB was visualized after 21 days by alizarin red (ALZR) staining and was quantified by absorbance. Furthermore, the ALP activity was measured at days 10 and 21. Results: After 21 days, the OB culture in osteogenic medium (positive control) (0.804 ± 0.189, mean ± SEM) and empty bead control (0.500 ± 0.053) showed a statistically significant higher mineralization (as shown with ALZR staining) compared to negative control (0.051 ± 0.01; P < .03). OB cultured with 9 NPC, AFC, and EPC beads showed a significant lower calcium deposition compared to the positive control (9 beads: NPC: 0.374 ± 0.085, AFC: 0.383 ± 0.070, CEPC: 0.402 ± 0.062, P < .03; Figure 1). The ALP activity showed a trend to be lower with increasing bead number in every cell type. However, no significant difference to the positive control was detected. Discussion: In this study, we could show a tendency that mineralization of primary OB could be inhibited by direct exposure to IVD cells. IVD cells seem to directly influence OB, by apparent inhibition of bone formation. Since empty beads seem to influence the mineralization of OB, it is possible that the alginate or the inserts played a role in matrix mineralization of the OB monolayer.

Figure 1.

Alizarin red (ALZR) staining and B. Quantification of ALZR staining of primary human osteoblasts, simulated with osteogenic medium and co-cultured with six or twelve, nucleus pulposus cell (NPC), annulus fibrosus cell (AFC) or cartilagenous endplate cell (CEPC) beads, respectively. Positive control groups were cultured with osteogenic medium (± insert and beads) and negative control group was cultured with αMEM only. Optical density of ALZR mean ± SEM. One-way ANOVA p-value *<0.03 ***<0.0001.

References

1. Watkins R 4th, Watkins R 3rd, Hanna R. Non-union rate with stand-alone lateral lumbar interbody fusion. Medicine (Baltimore). 2014;93:e275.

2. Chan SCW, Tekari A, Benneker LM, Heini PF, Gantenbein B. Osteogenic differentiation of bone marrow stromal cells is hindered by the presence of intervertebral disc cells. Arthritis Res Ther. 2015;18:29.

Cell Transplantation in Lumbar Spine Disc Degeneration Disease

H. J. Meisel¹, C. Hohaus², Y. Minkus¹, and T. Ganey³

¹Department of Neurosurgery, BG Klinikum Bergmannstrost, Halle/Saale, Germany ²Department of Neurosurgery, Städtisches Klinikum Dessau, Dessau, Germany ³Atlanta Medical Center, Atlanta, GA, USA

Introduction: Low back pain is an extremely common symptom, affecting nearly three quarters of the population sometime in their life. Given that disc herniation is thought to be an extension of progressive disc degeneration that attends the normal aging process, seeking an effective therapy that staves off disc degeneration has been considered a logical attempt to reduce back pain. The most apparent cellular and biochemical changes attributable to degeneration include a decrease in cell density in the disc that is accompanied by a reduction in synthesis of cartilage-specific extracellular matrix components. With this in mind, one therapeutic strategy would be to replace, regenerate, or augment the intervertebral disc cell population, with a goal of correcting matrix insufficiencies and restoring normal segment biomechanics. Biological restoration through the use of autologous disc chondrocyte transplantation offers a potential to achieve functional integration of disc metabolism and mechanics. Methods: We designed an animal study using the dog as our model to investigate this hypothesis by transplantation of autologous disc-derived chondrocytes into degenerated intervertebral discs. As a result, we demonstrated that disc cells remained viable after transplantation; transplanted disc cells produced an extracellular matrix that contained components similar to normal intervertebral disc tissue; a statistically significant correlation between transplanting cells and retention of disc height could be displayed. Results: Following these results, the Euro Disc Randomized Trial was initiated to embrace a representative patient group with persistent symptoms that had not responded to conservative treatment where an indication for surgical treatment was given. In the interim analyses, we evaluated that patients who received autologous disc cell transplantation had greater pain reduction at 2 years compared with patients who did not receive cells following their discectomy surgery and discs in patients that received cells demonstrated a significant difference as a group in the fluid content of their treated disc when compared to control.^1,2 Discussion: Autologous disc-derived cell transplantation is technically feasible and biologically relevant to repairing disc damage and retarding disc degeneration. Adipose tissue provides an alternative source of regenerative cells with little donor site morbidity. These regenerative cells are able to differentiate into a nucleus pulposus-like phenotype when exposed to environmental factors similar to disc, and offer the inherent advantage of availability without the need for transporting, culturing, and expanding the cells. In an effort to develop a clinical option for cell placement and assess the response of the cells to the post-surgical milieu, adipose-derived cells were collected, concentrated, and transplanted under fluoroscopic guidance directly into a surgically damaged disc using our dog model. This study provides evidence that cells harvested from adipose tissue might offer a reliable source of regenerative potential capable of bio-restitution.

References

1. Ganey T, Libera J, Moos V, et al. Disc chondrocyte transplantation in a canine model: a treatment for degenerated or damaged intervertebral disc. Spine (Phila Pa 1976). 2003;28:2609-2620.

2. Ganey TM, Meisel HJ. A potential role for cell-based therapeutics in the treatment of intervertebral disc herniation. Eur Spine J. 2002;11(suppl 2):S206-S214.

Reduction of Pelvic Incidence and Patient-Reported Outcomes Following Surgical Correction and Stabilization of Lytic Spondylolisthesis

Michael Mokawem¹ and Rajesh Shah¹

¹Department of Trauma and Orthopaedic Surgery, Hull and East Yorkshire Hospitals NHS Trust, Hull, UK

Introduction: A correlation exists between an increased pelvic incidence and development of lytic spondylolisthesis. The increased pelvic incidence increases the sheer force at the affected level and contributes to instability during weight bearing. We present a case series of patients who underwent surgery for lytic spondylolisthesis and attempt to evaluate the effect of correcting the pelvic incidence on patient-reported outcomes. Materials and Methods: Single surgeon case series of 30 patients with lytic spondylolisthesis who had surgical correction over a 4-year period with a minimum follow-up of 6 months. Pelvic incidence and grade of spondylolisthesis were measured pre- and postoperatively by 2 observers. The Oswestry Disability Index was used to measure patient reported outcomes. Results: Male 16: female 14. Age range: 22-72 with mean of 46 years. Preoperative grade (Meyerding) of spondylolisthesis: 1 to 3 with mean of 1.7. Postoperative grade of spondylolisthesis: 0 to 2 with mean of 0.8. Average correction of 1 grade. Pelvic incidence preoperative: 40.5° to 112° with mean of 64.1°. Pelvic incidence postoperative: 38.9° to76.1° with mean of 58.5°. Therefore, pelvic incidence correction: ranged from no correction to 35.9° with mean correction of 5.6°. Oswestry Disability Index (ODI) preoperative: 28 to 78 with mean of 56. ODI postoperative: 0 to 32 with mean of 14. ODI improvement of 12 to 66 with mean 42. Conclusion: Our series demonstrates a marked improvement in these symptomatic patients. We believe that correcting the pelvic incidence is an important factor in achieving satisfactory outcomes, although in a series of this size it is difficult to draw specific conclusions as it is true that a number of patients achieved good patient-reported outcomes without a significant improvement in pelvic incidence. Pelvic incidence correction should be considered as a radiological outcome in this group of patients.

Bone Mineral Density of Vertebras at Instrumented Levels and Levels Above the Instrumentation Increases During the First Postoperative Year After the Lumbar Spine Fusion

Mikko A. Hakulinen^1,2, Liisa Pekkanen³, Kati Kyrölä³, Marko H. Neva⁴, and Arja Häkkinen^5,6

¹Department of Applied Physics, University of Eastern Finland, Kuopio, Finland ²Diagnostic Imaging Center, Kuopio University Hospital, Kuopio, Finland ³Department of Orthopaedics and Traumatology, Central Finland Central Hospital, Jyväskylä, Finland ⁴Department of Orthopaedics and Traumatology, Tampere University Hospital, Tampere, Finland ⁵Department of Physical Medicine and Rehabilitation, Central Finland Central Hospital, Jyväskylä, Finland ⁶Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland

Introduction: Bone mineral density (BMD) at adjacent levels has been reported to decrease after lumbar spine fusion (LSF). However, it has been shown to increase again in 10-years follow-up. Aim of the Study: To evaluate the change in vertebral body BMD during 1-year follow-up after LSF in both instrumented levels and levels above the instrumentation. Materials and Methods: Altogether 36 patients (mean age 59 years, 75% females) undergoing 1- or 2-level instrumented LSF were enrolled to the prospective study. BMD was assessed by using dual-energy X-ray (DXA) from proximal femurs and both instrumented vertebras and vertebras above during the first week after LSF and again 1 year later. In addition, before the surgery and at the 1-year follow-up the patients filled a questionnaire including Oswestry Disability Index (ODI), back and leg pain Visual Analog Scale (VAS, 100 mm), and leisure time physical activity (LPA). Results: In 1-year follow-up, BMD of the instrumented vertebras increased significantly (P < .05). The highest increase (+0.107 g/cm², +11.8%) was found at L4 level. The chance at levels above the instrumentation were smaller (<6.2%), but still statistically significant (P < .05). In both proximal femurs, the changes were not significant during the follow-up (P > .05). Mean (95% confidence interval) disability (ODI) decreased by 23 (18 to 28; P < .001), and back and leg pain VAS decreased by 41 (32 to 51) mm and 45 (33 to 57) mm (both P < .001), respectively. LPA increased by 104 (215) min/week (P = .006). Conclusion: During the first postoperative year after LSF the BMD of both instrumented vertebras and vertebras above increases significantly. Improved pain and disability together with increased loading stress of lumbar spine may partly explain the bone remodeling, which is demonstrated by increase in BMD.

Ferromagnetic Dissection and Discectomy in Scoliosis Surgery: Comparison With Electrical and Mechanical Tools

Alessandro Ramieri¹, Giuseppe Costanzo¹, and Cloe Curri¹

¹Orthopaedics, Don Gnocchi Foundation ONLUS, Milan and Rome Sapienza University, Rome, Italy

Introduction: The ferromagnetic dissection (FMD), recently used in neurosurgery, is based on inductive high-frequency magnetic fields able to cut, ablate, and vaporize the tissue, obtaining a highly hemostatic effect. FMD in scoliosis surgery represents a technological innovation. Materials and Methods: We used the FMD for posterior approach of 20 scoliosis (group A). All these procedures involving the use of evoked potentials. In XLIF, the FMD was used for discectomy by foot control long probes. We recorded surgical time and blood loss, comparing the data with other 20 scoliosis (group B), prepared by monopolar/bipolar electrical devices. Postoperatively, we evaluated tenderness, swelling, and bruise of the paravertebral tissue. Results: In group A, the mean preparation time was 21 minutes (range 17-55), while in group B it was 47 minutes (range 27-78; P < .01). In group A, mean blood loss was 55 cc (range 30-80) and in group B 85 cc (50-150; P < .05). Tenderness/swelling showed no particular differences. Evoked potentials showed no abnormalities. In XLIF, cutting and ablation of the annulus were quick, more than with mechanical tools. Conclusion: In vivo studies established that FMD decreases damages to the paraspinal muscles, edema, or postoperative scar. Our original application to paravertebral muscles in scoliosis showed positive results in terms of surgical time and bleeding. The absence of electrical stimulation did not affected intraoperative monitoring. The postoperative tenderness was related to psychological factors rather than the dissection. Finally, incision and ablation of the annulus were more rapid compared to that performed with mechanical instruments. Actually, we are evaluating the effect of electrical dissection and FMD on paravertebral tissue by magnetic resonance imaging.

In Toto and Expanded Human Vertebral Bone Marrow Cells Cultured Under Normoxic and Hypoxic Conditions: A Novel Strategy for Spine Surgery

F. Salamanna¹, S. Cepollaro², G. Barbanti Brodano³, C. Griffoni³, A. Gasbarrini³, S. Bandiera³, S. Terzi³, R. Ghermandi³, S. Boriani³, and M. Fini¹

¹Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopaedic Institute, Bologna, Italy ²Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy ³Department of Oncological and Degenerative Spine Surgery, Rizzoli Orthopaedic Institute, Bologna, Italy

Introduction: Spine surgeons are embracing advanced biologic technologies in an attempt to help millions of people achieve a better outcome in spine surgery and cell-based therapies seem to offer a promising approach. Due to their properties and characteristics, human mesenchymal stem cells (MSCs) appear to have great therapeutic potential. Many different populations of MSCs have been described, and to understand whether they have equivalent biological properties is a critical issue for their therapeutic application. In the last few decades, a number of emerging strategies as well as tissue engineering with mesenchymal stem cells (MSCs) have been planned to enhance spinal fusion rate. Notwithstanding MSCs represent a stimulating and hopeful panorama, many demands regarding their use in spinal arthrodesis procedures remain unanswered. As further studies are needed to determine the best protocol for MSCs use in spinal arthrodesis procedure, we proposed to investigate the potential of vertebral MSCs (vMSCs), which can be easily harvested from vertebral pedicles during spinal surgical procedures. Materials and Methods: Human vertebral bone marrow was harvested from vertebral pedicles during spinal surgical procedures involving posterolateral arthrodesis. Vertebral bone marrow in toto and expanded human vertebral bone marrow cells, cultured under normoxic and hypoxic conditions, were analyzed to evaluate multidifferentiation potential, cellular proliferation, and gene expression for markers of osteoblast differentiation and homeobox genes of HOX and TALE subfamilies. Results: Under hypoxic condition in toto human vertebral bone marrow can be maintained in culture for a greater number of steps with respect to concentrated vMSCs, and it also generates mature cells of all mesenchymal lineages with greater efficiency, when induced into osteogenic, adipogenic, and chondrogenic differentiation. In addition, analyses of homeobox genes of HOX and TALE subfamilies showed that in toto human vertebral bone marrow cultured under hypoxic condition displayed distinct and specific levels of expression for HOX and TALE signatures. Conclusion: The in vitro ability and capability of in toto human vertebral bone marrow cultured under hypoxic condition is very interesting in light of a clinical application for bone fusion in spine surgery. For this purpose, a clinical experimental trial will be designed after a complete in vitro characterization of vMSCs.

In Situ Photopolymerized Composite Hydrogels for Implants: Application to a Nucleus Pulposus Replacement

Andreas Schmocker^1,2, Azadeh Khoushabi^1,3, Daniela A. Frauchiger⁴, Benjamin Gantenbein⁴, Constantin Schizas⁵, Christophe Moser², Pierre-Etienne Bourban³, Dominique P. Pioletti¹

¹Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland ²Institute of Microengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland ³Institute of Materials, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland ⁴Institute for Surgical Technology and Biomechanics, University of Bern, Bern, Switzerland ⁵Neuro-orthopedic Spine Unit, Clinic Cecil, Lausanne, Switzerland

Introduction: The nucleus pulposus (NP) is the core of the intervertebral disc (IVD). NP replacements have been subject to highly controversial discussions over the last 40 years. Their utility seems to be eminent to treat herniated disc or degenerated disc disease (DDD). But in practice not a single implant or tissue replacement was able to withstand the loads within an IVD. Hence, spinal fusion remains the main treatment for DDD. The existing NP replacements suffer from extrusion and subsidence. Material and Methods: A composite hydrogel was designed by combining polyethylene glycol-dimethacrylate (PEGDA) with Nano Fibril Cellulose (NFC) fibers. To inject the PEGDA-NFC hydrogel and activate it by light illumination in situ, a minimally invasive surgical device was developed. To validate the approach, the NP of bovine IVDs was removed and hydrogel samples were placed within the degenerated IVD disc model. After surgery the IVDs were cyclically loaded in a bioreactor over more than 0.5 million cycles and the change of organ height was compared between healthy, degenerated, repaired, and cyclically loaded states. Results: The developed hydrogel could be tuned according to native material properties and was able to reach similar material properties in terms of swelling pressure, extrusion, and confined compression strength. It was successfully implanted and photopolymerized within the IVD model and was able to resist cyclic loading over 0.5 million cycles. The postoperative disc high increase was 33.4% and was significant between degenerated/repaired state (P < .0025) and between degenerated and loaded state (P < .025). Histology of the samples showed a continuous interface between native tissue and implanted hydrogel. Discussion: The minimally invasive implantation by in situ photopolymerization is a unique feature to control the surgery and provide optimal tissue integration. The restauration of disc height over 0.5 million loading cycles suggests that the orthopedic implant could also be functional in humans.

Bitten by Translation: Are Veterinary Patient Dogs Good Models for Intervertebral Disc Repair Using Cell Therapy?

Alessandro Bertolo¹, Frank Steffen², and Jivko Stoyanov¹

¹Swiss Paraplegic Research, Nottwil, Switzerland ²Clinic for Small Animals, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

Introduction: It is a considerable challenge to bridge the gap between basic research in intervertebral disc (IVD) biology, tissue engineering with mesenchymal stem cells (MSCs), biomechanics of the IVD, and the translation of the recent advances in these fields into new clinical practices aimed at the repair/regeneration of degenerated IVD. First, the present laboratory research-based tissue engineering methodology is not appropriate for clinical implementation. Development of a new, simplified, and robust but safe workflow, which can be handled with medical instead of research hardware, is necessary. Once this is done, we need to choose a model to translate the in vitro work on disc regeneration to the next stage. This very important step toward clinical IVD regeneration application calls for use of animal models—preferably with larger animals. However, all animal models using induced disc degeneration have been criticized that they do not mirror the real complex disc degeneration situation. Here we will discuss advantages and disadvantages of using veterinary patients. Methodology: We compare our translational approach to validate promising in vitro results in veterinary patients (large dogs), which have degenerative disc disease (DDD), to existing large animal models. We also compare and discuss the challenges in sample processing, reproducing in vitro otherwise robust data obtained with human cells, and the differences in stem cell senescence between humans and dogs. Results: Differences between human and dog bone marrow sample handling and in vitro behavior of human and dog MSC are reviewed, as well as differences between dog breeds. The results from the pilot study—using only stem cells and following studies using microcarriers and stem cells—are compared. Preoperative and postoperative magnetic resonance imaging (MRI) data are compared to functional outcomes. Conclusions: Dogs with DDD are a valuable unused resource which offer the advantages of reduced animal experimentation and of being a realistic model, because of the right combination of age, genetic predisposition, and disc biochemistry. Diagnostics and outcome evaluation of veterinary patients (MRI of disc height and functional and neurological tests) as well as treatment (comparison of stem cell therapy to standard operative therapy) closely resembles that of the human condition and human clinical trials. Further advantages are the possibility for long-term patient follow-up and eventually to obtain autopsy material after the natural death of the animal. Major disadvantages of using veterinary patients are problems with recruitment and follow-up (communications with the owner), breed variability, postoperative animal treatment, and discrepancy between high functional recovery score and imaging DDD score.

Success Stories and Challenges of Translational Studies

M. A. Tryfonidou, DVM, dipl ECVS¹

¹Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, The Netherlands

Successful translation of regenerative treatment strategies for degenerative disc disease demands a multidisciplinary approach. Results of in vitro and in vivo studies should be interpreted with a clinical directive and experiments need to be designed with the translation from bench to bed in mind. In-depth understanding of underlying degenerative and regenerative processes in animal models is facilitated by cutting-edge biomolecular techniques of “nonhuman species” and strong implementation of the 3Rs (reduction, refinement, replacement). Innovative technological platforms, including injectable drug delivery systems, enable local delivery and controlled release of medication for the purpose of pain management and tissue regeneration. On the course of collaborative projects, biomaterials that enable controlled release of anti-inflammatory medication and growth factors have been validated in a preclinical platform, that is, the canine model. The canine species is considered to be a suitable model to study the process of disc degeneration: like humans, dogs suffer from spontaneous disc degeneration and disease. The degenerative process involves similar macroscopic, histopathological, radiological, and biochemical changes as humans. To this end, we employed the Beagle as an experimental animal followed by successful first-in-dogs studies in canine patients suffering from back pain. Lessons learned during these preclinical studies will be discussed focusing on the 3Rs, fine tuning of the treatment strategies respecting the physiology of the IVDs (including needle size, injection volume, loading dose), and the additive value of imaging modalities for longitudinal follow-up.

Disc Regeneration Using MSC Transplanted via the Endplate Route

Gianluca Vadalà¹, Fabrizio Russo¹, Alessia Valentini², Marco Bernardini², Giulia De Benedictis², Luca Denaro³, Rosaria Giordano⁴, and Vincenzo Denaro¹

¹Department of Orthopaedic Surgery, Università Campus Bio-Medico di Roma, Rome, Italy ²Department of Veterinary, Univerisità di Padova, Padua, Italy ³Department of Neurosurgery, Univerisità di Padova, Padua, Italy ⁴Cell Factory, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico, Milan, Italy

Introduction: Stem cell–based intervertebral disc (IVD) regeneration is quickly moving toward clinical applications.¹ However, many aspects need to be investigated to routinely translate this therapy to clinical applications, in particular, the most efficient way to deliver cell to the IVD. Cells are commonly delivered to the IVD through the annulus fibrosus (AF) injection. However, recent studies have shown serious drawbacks of this approach such as accelerated disc degeneration^2,3 and cells leakage.⁴ As an alternative we have described and tested a new surgical approach to the IVD via the endplate-pedicles (transpedicular approach). The purpose of the study was to test MSCs/hydrogel transplantation for IVD regeneration in a grade IV preclinical model of IDD on large size animals⁵ via the transpedicular approach⁶ with cell dose escalation. Methods and Methods: Adult sheep (n = 18) underwent bone marrow aspiration for autologous MSC isolation and expansion. MSC were suspended in autologous platelet-rich plasma (PRP) and conjugated with hyaluronic acid and batroxobin⁷ at the time of transplant (MSCs/hydrogel). Nucleotomy was performed via the transpedicular approach in 4 lumbar IVDs and that were injected with (1) hydrogel, (2) low doses of MSC/hydrogel, (3) high doses of MSC/hydrogel, and (4) no injection (CTRL). The endplate tunnel was sealed using a polyurethane scaffold. X-ray and MRI (magnetic resonance imaging) were performed at baseline and 1, 3, 6, and 12 months. Disc macro- and micromorphology were analyzed at each time point. Results: The MRI index showed a significant decrease in the untreated group, the disc injected with hydrogel and those injected with low MSC dose compared to healthy discs in all time points. The discs treated with high dose of MSC showed maintenance of the MRI index compared to the healthy disc. Morphologically, the grade of degeneration evaluated using them were in agreement with the grades observed at the MRI. Conclusions: An effective dose of autologous MSC (1 × 10⁷ cell/mL) delivered via the alternative transpedicular approach regenerates the NP in a preclinical model of grade IV IDD maintaining the AF intact. This preclinical study has high translational value as large animal model with the long fallow-up were used, MSCs were expanded in GMP facility simulating the clinical scenario, and the hydrogel were composed of clinically available materials. This approach represent a new valuable strategy to regenerate grade IV degenerated disc of the aging spine.

Acknowledgments

The support of the Italian Ministry of Instruction, University and Research Grant (PRIN-200938NT8Z), the Young Investigator Research Grant of the Italian Ministry of Health (GR-2010-2 318 448), and the BIOSPINA Award of the Italian Society of Spine Surgery (SICV&GIS) are gratefully acknowledged.

References

1. Orozco L, Soler R, Morera C, et al. Intervertebral disc repair by autologous mesenchymal bone marrow cells: a pilot study. Transplantation. 2011;92:822-828.

2. Carragee EJ, Don AS, Hurwitz EL, Cuellar JM, Carrino JA, Herzog R. 2009 ISSLS Prize Winner: Does discography cause accelerated progression of degeneration changes in the lumbar disc: a ten-year matched cohort study. Spine (Phila Pa 1976). 2009;34:2338-2345.

3. Korecki CL, Costi JJ, Iatridis JC. Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model. Spine (Phila Pa 1976). 2008;33:235-241.

4. Vadala G, Sowa G, Hubert M, Gilbertson LG, Denaro V, Kang JD. Mesenchymal stem cells injection in degenerated intervertebral disc: cell leakage may induce osteophyte formation. J Tissue Eng Regen Med. 2012;6:348-355.

5. Vadala G, Russo F, Pattappa G, et al. A Nucleotomy model with intact annulus fibrosus to test intervertebral disc regeneration strategies. Tissue Eng Part C Methods. 2015;21:1117-1124.

6. Vadala G, Russo F, Pattappa G, et al. The transpedicular approach as an alternative route for intervertebral disc regeneration. Spine (Phila Pa 1976). 2013;38:E319-E324.

7. Vadalà G, Fabrizio R, De Strobel F, et. al. Novel stepwise model of intervertebral disc degeneration with intact annulus fibrosus to test regeneration strategies. J Orthop Res. 2018;36(9):2460-2468.

CD146-Positive Mesenchymal Stem Cells Possess a Superior Migration Potential Toward Induced Degenerative Intervertebral Discs

Sebastian Wangler¹, Marianna Peroglio¹, Zhen Li¹, Ursula Menzel¹, Lorin M. Benneker², R. Geoff Richards¹, Mauro Alini¹, and Sibylle Grad¹

¹AO Research Institute Davos, Davos, Switzerland ²Inselspital, University of Bern, Bern, Switzerland

Introduction: Recent research is targeting at the homing ability of human mesenchymal stem cells (hMSCs) for regeneration of degenerative intervertebral discs (IVDs).¹ While a general homing process could be observed, the migrating hMSC subpopulation remains unknown. hMSC migration described in other musculoskeletal tissues suggests an involvement of CD146, also known as melanoma cell adhesion molecule MCAM.² Therefore, we hypothesized that CD146+ hMSCs play a role in cell homing toward the degenerative IVD. The aims of this study were (1) to quantify the migration of CD146+ versus CD146− hMSC populations toward conditioned medium from induced-degenerative IVDs; and (2) to evaluate the migration of CD146+ versus CD146− hMSC populations into induced-degenerative IVDs in organ culture. Materials and Methods: hMSCs were isolated from bone marrow aspirates obtained according to local ethical regulations (KEK 2016-00 141). Bovine caudal IVDs with endplates (n = 24, 6-8 months) were loaded in a whole organ culture bioreactor under induced-degenerative conditions (loading at 10 Hz for 4 h/day; low glucose DMEM) for 7 days.¹ Disc culture medium was replaced daily and collected as conditioned medium. Aim 1: hMSCs (n = 6, age 44 ± 21.9 years) were separated in CD146+ and CD146− populations through fluorescence activated cell sorting (FACS). A trans-well system consisting of top and bottom chambers separated by an 8 µm porous membrane was used for the migration assay. Migration assay was performed for each population separately (6 × 10⁴ cells/cm² in DMEM low glucose); disc conditioned medium was placed as a chemoattractant in the bottom chamber. After 16 hours, the number of migrated cells was counted by automated cell counter. Aim 2: CD146+ and CD146− sorted hMSCs were labeled with PKH26 and PKH67 fluorescent dyes, respectively. Each population was placed on a separate degenerative bovine IVD at a density of 1 × 10⁶ cells/disc (hMSCs: n = 3, age 38.3 ± 14 years; discs n = 6). After 5 days of migration, IVDs were fixed, cut in sagittal sections, and the number of migrated cells was counted by dual channel fluorescence microscopy. Paired t tests were used for statistical comparisons. Results: (1) The proportion of hMSCs migrating toward disc conditioned medium was significantly higher for CD146+ (22.5% migrated cells) compared to CD146− (15.7% migrated cells; P < .0001) sorted hMSCs. (2) The organ culture experiment confirmed that CD146+ hMSCs had a superior migration potential toward induced-degenerative disc tissue (179.6 ± 29.6 cells/cm²) when compared to the CD146− hMSCs (99.2 ± 4.3 cells/cm²; Figure 1). Conclusion: CD146-positive hMSCs showed a superior migration potential compared to CD146-negative hMSCs both in vitro and in whole organ culture. Further studies will assess if the enhanced expression of CD146 is associated with an increased regenerative potential of this subpopulation. If so, enrichment or induction of CD146 may represent a new strategy to improve cell recruitment and eventually regenerative capacity.

Figure 1.

Combined red and green fluorescent image of migrated MSCs in a sagittal section of bovine IVDs. Left: CD146− MSCs (blue); Right: CD146+ MSCs (red). The IVD region without endplates is marked with a yellow line. Scale bar = 1 mm.

References

1. Pattappa G, Peroglio M, Sakai D. CCL5/RANTES is a key chemoattractant released by degenerative intervertebral discs in organ culture. Eur Cell Mater. 2014;27:124-136.

2. Lee CH, Lee FY, Tarafder S, Kao K, Jun Y, Yang G, Mao JJ. Harnessing endogenous stem/progenitor cells for tendon regeneration. J Clin Invest. 2015;125(7):2690-2701.

Short- and Mid-term Effect of TNF-α Intradiscal Injection and Detrimental Dynamic Loading in Intervertebral Disc Organ Culture

Zhen Li¹, Gernot Lang^1,2, Yishan Liu^1,2, Janna Geries^1,2, Zhiyu Zhou¹, David Kubosch², Norbert Südkamp², Mauro Alini¹, and Sibylle Grad¹

¹AO Research Institute Davos, Davos, Switzerland ²Department of Orthopedics and Trauma Surgery, University Hospital Freiburg, Freiburg, Germany

Introduction: Establishment of a pro-inflammatory and degenerative intervertebral disc (IVD) organ culture model can be instrumental in deciphering the role of therapeutic agents in a relevant and controlled environment. The aim of the current study was to (1) establish a pro-inflammatory and degenerative IVD organ culture model, by combining TNF-α intradiscal injection, detrimental loading, and limited nutrition; (2) investigate the short- and mid-term disc cell phenotype changes with the simulated early disc degeneration model. Materials and Methods: Bovine caudal IVDs were divided into 3 groups as follows (n = 8-10/group):

Group	Days 1-4	Days 5-11
1	Phy	Phy
2	Deg + TNF-α	Deg
3	Deg + TNF-α	Phy

Phy: physiological loading (0.02-0.2 MPa; 0.2 Hz; 2 h/day) and high glucose (4.5 g/L) medium; Deg: degenerative loading (0.32-0.5 MPa; 5 Hz; 2 h/day) and low glucose (2 g/L) medium; TNF-α: TNF-α was injected into the nucleus pulposus (NP) tissue, 100 ng/IVD at day 1.

Conditioned medium was collected for nitric oxide (NO) and glycosaminoglycan (GAG) analysis. After 4 or 11 days, disc tissue was harvested and gene expression was analyzed using real-time RT-PCR. After 11 days, cell viability was evaluated by lactate dehydrogenase (LDH)/ethidium homodimer staining of disc cryo-sections. Graphpad software was used to perform the statistics. P < .05 was considered statistically significant. Results: Group 2 vs Group 1: At day 4, NO and GAG release from IVD was increased in Deg compared with Phy group. Gene expression level of type II collagen (COL2) was downregulated in NP tissue, while type I collagen (COL1) was downregulated in AF tissue, under Deg condition and TNF-α injection. TNF-α injection combined with Deg condition induced an upregulation of interleukin-6 (IL6) and matrix metalloproteinase 1 (MMP1) in NP tissue, and interleukin-1β (IL1B) in AF tissue. At day 11, cell viability was markedly lower in Group 2 compared to Group 1. Group 3, Day 11 vs Day 4: After another 7 days of culture under Phy condition, the IL6 and IL1B gene expression decreased, while the COL2, COL1, and MMP1 gene expression stayed at the same level compared with day 4. Conclusion: After 4 days culture under combined TNF-α injection and Deg condition, an upregulation of catabolic and inflammatory marker gene expression and downregulation of ECM gene expression was observed in disc tissue, together with enhanced GAG and NO release, as well as cell death, which indicate an accelerated inflammatory and degenerative effect. After another 7 days of culture under Phy condition, the inflammation vanished, while the degeneration was maintained. To conclude, an acute pro-inflammatory and sustained degenerative organ culture model was established by TNF-α intradiscal injection, detrimental loading, and limited nutrition to mimic the early course of DDD. This model will be of high interest for screening of therapeutic agents in further preclinical studies.

Acknowledgments

Funded by the Foundation for the Promotion of Alternate and Complementary Methods to Reduce Animal Testing (SET). Zhiyu Zhou was funded by China Scholarship Council and Sino-Swiss Science and Technology Cooperation.