BMP-2 and TGF-β3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossification of the annulus fibrosus

Introduction: Different approaches for disc regeneration are currently under investigation. Beside gene therapy and tissue engineering techniques, the application of growth and differentiation factors own promising potential. Studies using reduced intervertebral disc models, such as cell or tissue fragment cultures, have limited validity and show controversial results depending on the employed experimental model. Therefore, the goal of the current study was to investigate the effect of BMP-2 and TGF-β3 on intervertebral disc degeneration using an in vitro full-organ disc/endplate culture system. Materials and methods: Intervertebral rabbit disc explants were cultured in the presence of 1μg/ml BMP-2 or TGF-β3 for 21days in DMEM/F12 media. Nucleus and annulus were analyzed for gene expression of collagen type I and II (Col I/II), aggrecan, collagenases (MMP-1/MMP-13) with RT-qPCR, histological changes with bone and proteoglycan-specific staining (von Kossa, toluidine blue) and differences in cellularity (DNA) and proteoglycan content (alcian blue binding assay). Results: The results demonstrate that disc proteoglycan concentration decreased with time in the TGF-β3 and BMP-2 groups. In the annulus fibrosus (AF), TGF-β3 and BMP-2 resulted in an up-regulation of Col I and type II, and of aggrecan gene expression. In contrast, MMP genes were inhibited. In the nucleus, the growth factors decreased gene expression of aggrecan and spontaneous Col I up-regulation was inhibited by TGF-β3, whereas expression of Col II was decreased with BMP-2. There was no effect on expression of MMP-1 and MMP-13 for most sampling points. However, TGF-β3 and BMP-2 induced ossification of the AF was demonstrated by histology. Conclusion: It can be concluded that both growth factors, at the tested concentrations, may not be suitable to regenerate the whole intervertebral disc organ but they are interesting candidates for being injected alone or in combination into a painful intervertebral disc to induce osseous fusion (spondylodesis

Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

There is a major controversy whether spinal trauma with vertebral endplate fractures can result in post-traumatic disc degeneration. Intervertebral discs, which are adjacent to burst endplates, are frequently removed and an intercorporal spondylodesis is performed. In any case, the biological effects within the discs following endplate factures are poorly elucidated to date. The aim of our investigations was therefore to establish a novel disc/endplate trauma culture model to reproducibly induce endplate fractures and investigate concurrent disc changes in vitro. This model is based on a full-organ disc/endplate culture system, which has been validated by the authors before. Intervertebral disc/endplate specimens were isolated from Burgundy rabbits and cultured in standard media (DMEM/F12, 10%FCS). Burst endplate fractures were induced in half of the specimens with a custom-made fracture device and subsequently cultured for 9days. The biological effects such as necrotic or apoptotic cell death and the expression of pro-apoptotic genes and other genes involved in organ degeneration, e.g. matrix metalloproteinases (MMPs) were analyzed. Cell damage was assessed by quantification of the lactate dehydrogenase (LDH) activity in the supernatant. The expression of genes involved in the cellular apoptotic pathway (caspase3) and the pro-apoptotic proteins FasL and TNF-α were monitored. The results demonstrate that LDH levels increased significantly post trauma compared to the control and remained elevated for 3days. Furthermore, a constant up-regulation of the caspase3 gene in both disc compartments was present. The pro-apoptotic proteins FasL and TNF-α were up regulated predominantly in the nucleus whereas the MMP-1 and -13 transcripts (collagenases) were increased in both disc structures. From this study we can conclude that endplate burst fractures result in both necrotic and apoptotic cell death in nucleus and annulus tissue. Moreover, FasL and TNF-α expression by nucleus cells may lead to continued apoptosis induced by Fas- and TNF-α receptor bearing cells. In addition TNF-α over-expression has potentially deleterious effects on disc metabolism such as over-expression of matrix proteinases. Taken together, the short term biological response of the disc following endplate fracture exhibits characteristics, which may initiate the degeneration of the orga

Comparative biomechanical investigation of a modular dynamic lumbar stabilization system and the Dynesys system

The goal of non-fusion stabilization is to reduce the mobility of the spine segment to less than that of the intact spine specimen, while retaining some residual motion. Several in vitro studies have been conducted on a dynamic system currently available for clinical use (Dynesys®). Under pure moment loading, a dependency of the biomechanical performance on spacer length has been demonstrated; this variability in implant properties is removed with a modular concept incorporating a discrete flexible element. An in vitro study was performed to compare the kinematic and stabilizing properties of a modular dynamic lumbar stabilization system with those of Dynesys, under the influence of an axial preload. Six human cadaver spine specimens (L1-S1) were tested in a spine loading apparatus. Flexibility measurements were performed by applying pure bending moments of 8Nm, about each of the three principal anatomical axes, with a simultaneously applied axial preload of 400N. Specimens were tested intact, and following creation of a defect at L3-L4, with the Dynesys implant, with the modular implant and, after removal of the hardware, the injury state. Segmental range of motion (ROM) was reduced for flexion-extension and lateral bending with both implants. Motion in flexion was reduced to less than 20% of the intact level, in extension to approximately 40% and in lateral bending a motion reduction to less than 40% was measured. In torsion, the total ROM was not significantly different from that of the intact level. The expectations for a flexible posterior stabilizing implant are not fulfilled. The assumption that a device which is particularly compliant in bending allows substantial intersegmental motion cannot be fully supported when one considers that such devices are placed at a location far removed from the natural rotation center of the intervertebral join

Paradoxien der Geschichte. Anmerkungen zu den Fotografien von Gerhard Gäbler

PURPOSE: Surgical treatment of early-onset scoliosis (EOS) requires a balance between maintained curve correction and the capacity for spinal and thoracic growth. Spinal fusion creates irreversible conditions that prevent the implementation of further treatment methods. Our hypothesis was that non-fused anchors in growth guidance show a comparable outcome as the technique described in the literature, which involves spondylodesis of the anchoring segments. METHODS: This retrospective study analysed 148 surgeries in 22 EOS patients (11 female, 11 male) over a 15-year period. Patients underwent surgery with non-fused anchors and growth guidance techniques. Scoliosis, kyphosis, growth and anchoring segments were measured. For the latter, a new measuring technique was developed. Complications were recorded and classified. RESULTS: The mean Cobb angle reduced from 73.5 ± 24.4° to 28.4 ± 16.2° (60.2 ± 22.9%, p < 0.001) at the last follow-up. Spinal growth T1-S1 and T1-T12 were 41.1 ± 23.3 mm and 24.9 ± 16.6 mm (p < 0.001), respectively. Growth at the cranial and caudal anchoring segment was 1.5 mm/segment/year and 1.9 mm/segment/year, respectively. A total of 63 complications were documented in 20 patients, with 40 requiring unplanned revision surgery. Definitive spondylodesis was performed in three patients. CONCLUSION: Patients demonstrated a significant spinal growth including the anchoring segments. A comparable correction in Cobb angle and the type of complications was noted, although the rate of device-related complications was higher. No permanent impairment was reported. The rate of device-related complications is acceptable and outweighed by the significant degree of growth preservation and more flexible and individualised treatment strategy for patients with EOS. These slides can be retrieved under Electronic Supplementary Material

Anatomically and mechanically conforming patient-specific spinal fusion cages designed by full-scale topology optimization

Cage subsidence after instrumented lumbar spinal fusion surgery remains a significant cause of treatment failure, specifically for posterior or transforaminal lumbar interbody fusion. Recent advancements in computational techniques and additive manufacturing, have enabled the development of patient-specific implants and implant optimization to specific functional targets. This study aimed to introduce a novel full-scale topology optimization formulation that takes the structural response of the adjacent bone structures into account in the optimization process. The formulation includes maximum and minimum principal strain constraints that lower strain concentrations in the adjacent vertebrae. This optimization approach resulted in anatomically and mechanically conforming spinal fusion cages. Subsidence risk was quantified in a commercial finite element solver for off-the-shelf, anatomically conforming and the optimized cages, in two representative patients. We demonstrated that the anatomically and mechanically conforming cages reduced subsidence risk by 91% compared to an off-the-shelf implant with the same footprint for a patient with normal bone quality and 54% for a patient with osteopenia. Prototypes of the optimized cage were additively manufactured and mechanically tested to evaluate the manufacturability and integrity of the design and to validate the finite element model.Comment: 34 pages, 8 figure

Transoral Closed Reduction of Fixed Atlanto-Axial Rotatory-Subluxation (AARS) in Childhood and Adolescence

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In silico medical device testing of anatomically and mechanically conforming patient-specific spinal fusion cages designed by full-scale topology optimisation

A full-scale topology optimisation formulation has been developed to automate the design of cages used in instrumented transforaminal lumbar interbody fusion. The method incorporates the mechanical response of the adjacent bone structures in the optimisation process, yielding patient-specific spinal fusion cages that both anatomically and mechanically conform to the patient, effectively mitigating subsidence risk compared to generic, off-the-shelf cages and patient-specific devices. In this study, in silico medical device testing on a cohort of seven patients was performed to investigate the effectiveness of the anatomically and mechanically conforming devices using titanium and PEEK implant materials. A median reduction in the subsidence risk by 89% for titanium and 94% for PEEK implant materials was demonstrated compared to an off-the-shelf implant. A median reduction of 75% was achieved for a PEEK implant material compared to an anatomically conforming implant. A credibility assessment of the computational model used to predict the subsidence risk was provided according to the ASME V&amp;V40–2018 standard

Automatic Calculation of Cervical Spine Parameters Using Deep Learning: Development and Validation on an External Dataset

STUDY DESIGN Retrospective data analysis. OBJECTIVES This study aims to develop a deep learning model for the automatic calculation of some important spine parameters from lateral cervical radiographs. METHODS We collected two datasets from two different institutions. The first dataset of 1498 images was used to train and optimize the model to find the best hyperparameters while the second dataset of 79 images was used as an external validation set to evaluate the robustness and generalizability of our model. The performance of the model was assessed by calculating the median absolute errors between the model prediction and the ground truth for the following parameters: T1 slope, C7 slope, C2-C7 angle, C2-C6 angle, Sagittal Vertical Axis (SVA), C0-C2, Redlund-Johnell distance (RJD), the cranial tilting (CT) and the craniocervical angle (CCA). RESULTS Regarding the angles, we found median errors of 1.66° (SD 2.46°), 1.56° (1.95°), 2.46° (SD 2.55), 1.85° (SD 3.93°), 1.25° (SD 1.83°), .29° (SD .31°) and .67° (SD .77°) for T1 slope, C7 slope, C2-C7, C2-C6, C0-C2, CT, and CCA respectively. As concerns the distances, we found median errors of .55 mm (SD .47 mm) and .47 mm (.62 mm) for SVA and RJD respectively. CONCLUSIONS In this work, we developed a model that was able to accurately predict cervical spine parameters from lateral cervical radiographs. In particular, the performances on the external validation set demonstrate the robustness and the high degree of generalizability of our model on images acquired in a different institution

The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules – dexamethasone, triiodothyronine (T3) and insulin – on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences

Analysis of cytotoxic activity of naïve resident peritoneal cells against Listeria-infected hepatocytes

Titel, Inhalt, Danksagung, Lebenslauf 1\. Einleitung 1 2\. Material und Methoden 10 3\. Ergebnisse 27 4\. Diskussion 45 5\. Zusammenfassung 61 6\. Literatur 63Für die Überwindung der Infektion mit dem intrazellulären Bakterium Listeria monocytogenes sind CD8+ zytotoxische T-Lymphozyten essentiell. Welche Zellpopulationen aber in der präimmunen Phase der Infektion das Wachstum der Erreger begrenzen, bevor spezifische T-Zellen vorhanden sind, ist bisher nicht ausreichend beantwortet. Einige Autoren schreiben Neutrophilen Granulozyten eine zytotoxische Aktivität gegenüber listerieninfizierten Hepatozyten zu. Diese Aussage stützt sich hauptsächlich auf histologischen Beobachtungen und den Einsatz von monoklonalen Antikörpern, die den CD18/11b-abhängigen Zellinflux in das infizierte Gewebe hemmen bzw. Neutrophile Granulozyten in vivo lysieren. Sie erlauben aber aufgrund der vielfältigen direkten und indirekten Auswirkungen dieser experimentellen Maßnahmen nicht die Schlussfolgerung, dass diese Zellpopulation direkt zytotoxisch für infizierte Wirtszellen ist. Tatsächlich zeigen in-vitro-Studien zur Zytotoxizität von Peritonealexsudatzellen gegen-über listerieninfizierten Hepatozyten, dass Granulozyten keine zytolytische Potenz besitzen. Die hierfür verantwortliche Zellpopulation ist aber bis dato unklar. Ziel der vorliegenden Arbeit war daher, die Grundlage für eine detaillierte Analyse der Zell-Zell-Interaktion in der präimmunen Phase der Listeriose zu schaffen, und die Effektormechanismen näher zu untersuchen. Hierfür wurde ein ex-vivo-in-vitro- Experimentalsystem etabliert, welches die quantitative Erfassung und Modulation der Zytotoxizität von naiven murinen Peritonealleukozyten (Balb/c) gegenüber listerieninfizierten Hepatozyten (ATCC TIB73) erlaubt. Die Ergebnisse der in dieser Arbeit dargelegten in-vitro-Untersuchungen legen nahe, dass (naive residente) Makrophagen zur Lyse listerieninfizierter Hepatozyten in der Lage sind. Ihre zytotoxische Aktivität war durch IFN-γ zu steigern, aber von TNF-α und Stickstoffmonoxid (NO) unabhängig. Diese Befunde zeichnen ein neues Bild über die Rolle von Makrophagen bei der Abwehr der Listeriose. So sind sie nicht nur durch Phagozytose und Abtötung extrazellulärer Listerien an der Überwindung der Infektion beteiligt, sondern auch an der präimmunen Abtötung permissiver Wirtszellen.For the resolution of an infection with the Gram-positive facultative intracellular bacterium Listeria monocytogenes CD8+ cytotoxic lymphocytes are essential. But which cell population in the preimmune phase of infection is limiting bacterial growth before specific T cells are generated is not entirely known. Some authors favour polymorphonuclear neutrophils (PMNs) being cytotoxic to Listeria-infected hepatocytes. But this finding is based mainly on histological observations and the use of monoclonal antibodies abrogating the CD18/11b dependant cellular influx in infected organs or antibodies that antagonize PMNs. These experiments do not allow the conclusion that this cell population is directly cytotoxic to Listeria-infected cells because of the vast direct and indirect consequences of those experimental means. And indeed in-vitro-experiments on the cytotoxic capacity of peritoneal cells show that PMNs are not able to lyse Listeria-infected hepatocytes. The responsible cell population is until now un-known. Aim of our experiments was to establish the basis for a detailed analysis of cell-cell interaction in the preimmune phase of listeriosis. Therefore an ex-vivo-in-vitro-experimental system was created allowing the quantitative measurement and modulation of cytotoxic effects of naïve murine peritoneal cells (BALB/c) against Listeria-infected hepatocytes (ATCC TIB 73). The results of the experiments show that naïve resident macrophages are able to lyse Listeria-infected hepatocytes. The cytotoxic capacity could be increased with IFN-γ but was independent of TNF-α and nitric oxide (NO). These findings suggest a new role for macrophages in the early phase of defence. Thus macrophages do not only phagocytose and kill extracellular bacteria they also help to resolute the infection by preimmune killing of permissive parenchymal host cells