In vivo, in vitro and in silico investigations on disc nucleus replacements in the sheep model

Bandscheibenbedingte Rückenschmerzen stellen eine Gesundheitsstörung von herausragender Bedeutung dar. Innovative Therapiekonzepte sind darauf ausgerichtet, schmerzhaft degenerierte Bandscheiben in ihren natürlichen Strukturen zu regenerieren. Allein durch den chirurgischen Eingriff zur Anwendung dieser Therapiekonzepte wird jedoch die mechanische Kompetenz der Bandscheibe empfindlich gestört. Derzeit ist nicht bekannt, ob neue Nukleusersatzmaterialien für Tissue engineering Strategien an der Bandscheibe diesen Verlust kompensieren können. Daher war es das Ziel der Dissertation in einem kombinierten experimentellen Versuchsansatz aus In-vivo-, Ex-vivo-, In-vitro- und In-silico- Untersuchungen, neu entwickelte Hydrogele als Nukleusersatz im Tiermodell Schaf zu untersuchen und das Schaf als Tiermodell im Bereich der Bandscheibenforschung näher zu charakterisieren. Um ein physiologisches Lastprotokoll für die In-vitro-Untersuchungen zu etablieren, wurde an drei Schafen der intradiskale Druck (IDP) über je 24 Stunden gemessen. Der gesamte Datenpool des ersten Schafes wurde in eine Aktivitäts- und Erholungsphase unterteilt und ex vivo aus den IDP-Durchschnittswerten beider Phasen die entsprechenden axialen Kompressionskräfte abgeleitet. In vitro wurde ein Kriech-Relaxations-Test an 36 ovinen lumbalen Bewegungssegmenten durchgeführt. Die Segmente wurden drei Belastungszyklen ausgesetzt, die jeweils aus einer 15-minütigen Belastungsphase (130 N) und einer 30-minütigen Erholungsphase (58 N) bestanden. IDP-Verlauf und Höhenverlust der Segmente wurden in sechs verschiedenen Versuchsgruppen untersucht: (i) INTAKT; (ii) DEF-AN: Eine schräge Anulusinzision. Der Defekt wurde durch Naht und Cyanoacrylatkleber verschlossen. (iii) DEF-NUKn+k: Nukleusgewebe wurde entfernt und anschließend reimplantiert. Der Anulusverschluss erfolgte wie in DEF-AN. (iv) DEF-NUKp: Entsprechend dem Vorgehen in Testgruppe DEF-NUKn+k wurde der Nukleus entfernt und reimplantiert. Um eine Volumenverdrängung reimplantierten Gewebes in den inneren Anulusdefekt zu vermeiden, erfolgte der Verschluss mittels eines Plugs. Abschließend wurden zwei Hydrogele als Nukleusersatz untersucht: (v) DDAHA und (vi) iGG-MA. Zur besseren Interpretation der In-vitro-Ergebnisse wurden Finite-Elemente-Analysen an einem Bandscheibenmodell durchgeführt. In vivo lag der Bandscheibendruck beim Schaf nahezu konstant höher als beim Menschen. Niedrigste Druckwerte wurden intraoperativ mit ~0,5 MPa ermittelt. Höchste Druckwerte wurden für Aufstehen oder Drehen mit 3,6 bzw. 2,6 MPa gemessen und waren damit ungefähr zwei- bis viermal höher in der ovinen Bandscheibe. Die IDP-Mittelwerte der Aktivitäts- und Erholungsphasen des ersten Schafes lagen bei ~0,75 bzw. ~0,5 MPa, welche axialen Kompressionskräften von 130 bzw. 58 N entsprachen. Im Kriech-Relaxations-Test hatte ein isolierter Anulusdefekt (DEF-AN) keinen Einfluss auf Höhenverlust und IDP der Segmente. DEF-NUKn+k, DEF-NUKp, DDAHA und iGG-MA hingegen steigerten den Höhenverlust und verringerten signifikant den IDP im Vergleich zu INTAKT. Die Modellvorhersagen belegten erhebliche Auswirkungen eines reduzierten Wassergehalts, Kompressionsmoduls und osmotischen Potentials des reimplantierten Gewebes auf den Höhenverlust und IDP des Segmentes. Die Lastübertragung innerhalb der Bandscheibe veränderte sich hierdurch deutlich und ging mit einer erhöhten Belastung des Anulus einher. Die vergleichsweise hohen Bandscheibendrücke des Schafes stehen der weit verbreiteten Meinung gegenüber, dass aufgrund der horizontal ausgerichteten Wirbelsäule des Vierbeiners, intradiskale Lasten geringer sein müssten als beim Menschen. In Kenntnis der vorliegenden Untersuchungen sollte die Rechtfertigung bzw. der Ausschluss des Schafes als Modell im Bereich der Wirbelsäule nicht auf Unterschieden im Gang begründet werden, sondern auf mechanischen Überlegungen bzgl. künftiger Einsatzgebiete. Die In-vitro-Ergebnisse zeigen, dass der Erfolg von Hydrogelen als Nukleusersatz nicht nur vom Ersatzmaterial selbst abhängt, sondern auch von der Wiederherstellung zerstörter Bandscheibenstrukturen, wie der Grenzflächen zwischen Nukleus und Umgebung sowie dem gesetzten Anulusdefekt. Die vorliegende Dissertation konnte die Bedeutung iatrogen induzierter struktureller Schädigungen der Bandscheibe für Nukleusersatzstrategien herausarbeiten und stellt somit wesentliche Anforderungskriterien an das zukünftige Designkonzept von Hydrogelen als Nukleusersatz für Tissue engineering Strategien an der Bandscheibe. Hydrogele, die allein das mechanische Verhalten des Nukleus imitieren, können ansonsten bei der Wiederherstellung der Mechanik des Gesamtsegmentes versagen.Discogenic low back pain represents a major health disorder in the musculoskeletal field. Innovative therapeutic approaches aim to regenerate the painfully degenerated disc by restoring its original structure. Surgical procedures like nucleotomy, however, which are necessary for the application of such therapeutic concepts, however, perturb the mechanical competence of the disc. It has not been fully clarified whether biomaterials for nucleus replacement are able to compensate for this. Therefore, a combined experimental approach of in vivo, ex vivo, in vitro and in silico studies was carried out to evaluate the efficiency of newly developed hydrogels for nucleus replacement in an ovine disc model and to characterize the sheep as an animal model in intervertebral disc research. To establish a physiological loading protocol for the in vitro studies, intradiscal pressure (IDP) was measured in three sheep over 24 hours. The total data set of the first sheep was divided into an activity phase and a recovery phase, and the resulting average pressures of both phases were calculated. Subsequently, the corresponding axial forces were derived ex vivo. In vitro, a creep and recovery test was performed on 36 ovine lumbar motion segments. Specimens were subjected to three loading cycles, each consisting of a loading period of 15 minutes at 130 N and a recovery period of 30 minutes at 58 N. IDP and segment height loss were investigated in six different test groups: (i) INTACT; (ii) DEF-ANN: A small oblique incision in the annulus. The defect was closed by suturing and with cyanoacrylate glue. (iii) DEF-NUCs+g: Nucleus tissue was removed and subsequently re-implanted. The annulus defect was closed as in DEF-ANN. (iv) DEF-NUCp: As in DEF-NUCs+g, the nucleus tissue was removed and re-implanted. To avoid squeezing of nucleus tissue into the inner annulus defect, sealant was applied using a plug. Finally, two hydrogels were investigated as nucleus replacements: (v) DDAHA and (vi) iGG-MA. To better interpret ambiguous results obtained in vitro, finite element analyses were conducted on a disc model. In vivo, ovine IDPs were almost consistently higher than the human. The lowest IDPs were measured intra-operatively with ~0.5 MPa. The highest IDPs were found for standing up or turning around, where IDPs were with 3.6 MPa and 2.6 MPa, respectively, approximately two to four times higher within the ovine disc in comparison to humans. In the creep and recovery test, an isolated annulus incision (DEF-ANN) did not affect segmental height-loss or fluid pressurization. DEF-NUCs+g, DEF-NUCp, DDAHA and iGG-MA increased the height loss and decreased the fluid pressurization compared with INTACT. Model predictions demonstrated substantial effects of reductions in replaced nucleus water content, bulk modulus and osmotic potential on disc height loss and pressure similar to the experimental measurements. For these events in the model, the compression load transfer in the disc was markedly altered by substantially increasing the load on the annulus when compared with the nucleus. The finding of comparably high ovine IDPs in vivo conflicts with the widespread belief that, due to the horizontally aligned spine of quadrupeds, intradiscal loads should be less than in the upright positioned spine of humans. Given the sometimes multiple higher load amplitudes within the ovine disc combined with comparably low axial external forces, current results suggest that the justification of using sheep for spinal research questions should not be primarily based on differences in gait, but rather on mechanical considerations regarding the scientific field of application. In vitro results reveal that the success of hydrogels for nucleus replacement is not only dependent on the implant material itself but also on the restoration of the environment perturbed during surgery. The importance of the interface between the nucleus and its surrounding structures and the relevance of an appropriate annulus closure to avoid a displacement of implant material into the inner annulus defect are clearly indicated. By emphasizing the importance of surgically induced structural damages to the intervertebral disc, the present PhD thesis prescribes essential requirements for future design concepts for hydrogels as nucleus replacements for tissue engineering strategies of the intervertebral disc. Hydrogels that mimic the mechanical behavior of the native nucleus alone may otherwise fail in restoring the mechanical competence of the disc

Investigation of different hydrogels for nucleus replacement : a biomechanical study

Hydrogels are considered promising for disc regeneration strategies. However, it is currently unknown whether the destruction of the natural interface between nucleus and surrounding structures caused by nucleotomy and an inadequate annulus closure diminishes the mechanical competence of the disc. To clarify these mechanisms and to evaluate whether hydrogels are able to restore the biomechanical behaviour of the disc a combined in vivo and in vitro and approach was used

In vivo biofunctional evaluation of hydrogels for disc regeneration

Purpose Regenerative strategies aim to restore the original biofunctionality of the intervertebral disc. Different biomaterials are available, which might support disc regeneration. In the present study, the prospects of success of two hydrogels functionalized with anti-angiogenic peptides and seeded with bone marrow derived mononuclear cells (BMC), respectively, were investigated in an ovine nucleotomy model. Methods In a one-step procedure iliac crest aspirates were harvested and, subsequently, separated BMC were seeded on hydrogels and implanted into the ovine disc. For the cell-seeded approach a hyaluronic acid-based hydrogel was used. The anti-angiogenic potential of newly developed VEGF-blockers was investigated on ionically crosslinked metacrylated gellan gum hydrogels. Untreated discs served as nucleotomy controls. 24 adult merino sheep were used. After 6 weeks histological, after 12 weeks histological and biomechanical analyses were conducted. Results Biomechanical tests revealed no differences between any of the implanted and nucleotomized discs. All implanted discs significantly degenerated compared to intact discs. In contrast, there was no marked difference between implanted and nucleotomized discs. In tendency, albeit not significant, degeneration score and disc height index deteriorated for all but not for the cell-seeded hydrogels from 6 to 12 weeks. Cell-seeded hydrogels slightly decelerated degeneration. Conclusions None of the hydrogel configurations was able to regenerate biofunctionality of the intervertebral disc. This might presumably be caused by hydrogel extrusion. Great importance should be given to the development of annulus sealants, which effectively exploit the potential of (cell-seeded) hydrogels for biological disc regeneration and restoration of intervertebral disc functioningThis work was supported by the EU-project Disc Regeneration (NMP3-LA-2008-213904). Technical assistance of Iris Baum and the whole animal surgery team of the Institute of Orthopaedic Research and Biomechanics, Ulm, are gratefully acknowledged. DDAHA hydrogels were kindly provided by Cristina Longinotti (DDAHA, Anika Therapeutics, Abano Therme, Italy)

Integrating multidimensional data analytics for precision diagnosis of chronic low back pain

Low back pain (LBP) is a leading cause of disability worldwide, with up to 25% of cases become chronic (cLBP). Whilst multi-factorial, the relative importance of contributors to cLBP remains unclear. We leveraged a comprehensive multi-dimensional data-set and machine learning-based variable importance selection to identify the most effective modalities for differentiating whether a person has cLBP. The dataset included questionnaire data, clinical and functional assessments, and spino-pelvic magnetic resonance imaging (MRI), encompassing a total of 144 parameters from 1,161 adults with (n = 512) and without cLBP (n = 649). Boruta and random forest were utilised for variable importance selection and cLBP classification respectively. A multimodal model including questionnaire, clinical, and MRI data was the most effective in differentiating people with and without cLBP. From this, the most robust variables (n = 9) were psychosocial factors, neck and hip mobility, as well as lower lumbar disc herniation and degeneration. This finding persisted in an unseen holdout dataset. Beyond demonstrating the importance of a multi-dimensional approach to cLBP, our findings will guide the development of targeted diagnostics and personalized treatment strategies for cLBP patients

Nur 100 Jahre alt? Historische Friedhöfe zwischen Ignoranz, Akzeptanz und Relevanz

Friedhöfe und Bestattungen des 18. bis 20. Jh. werden auch in der Schweiz zunehmend zum Aufgaben- und Forschungsgebiet der Historischen Archäologie. Das zeigen Ausgrabungen der letzten Jahre in den Kantonen Bern, Basel und Graubünden

Effects of nucleotomy on segmental flexibility: a numerical analysis

Nucleotomy, a common treatment for disc herniations, aims to relieve pressure on spinal structures. While effective in alleviating symptoms, this intervention can compromise spinal stability. However, previous in vivo studies in sheep have demonstrated conflicting results with significant long-term stiffening of the spine following nucleotomy, with occasional spontaneous fusion of the affected motion segment. The objective of this study was to investigate the mechanical regulation of tissue adaptation processes post-nucleotomy using computational modeling. A parametric finite element model of the L4–L5 ovine spinal motion segment, developed previously, was modified to simulate surgical procedures that have been performed in prior in vivo studies. An iterative approach was used to simulate post-surgical tissue healing and adaptation processes. Two loading scenarios were simulated: one with combined axial compression and flexion moments, and the other incorporating axial rotation. An initial decrease in stability, with stiffness reduced by up to 50% due to disc decompression and nucleus removal, was followed by a gradual increase in stiffness over time as a consequence of bone healing and remodeling, with the most pronounced stiffening – up to 350% of the intact state – observed in axial rotation. The findings align with previous in vivo observations, suggesting that spontaneous fusion and increased rigidity may be natural consequences of mechano-biological adaptation. The results of this study highlight that healing processes accompanied by adaptive bone remodeling are directed towards restoration of spinal stability after nucleotomy. These findings align with previous in vivo observations, suggesting that spontaneous fusion and increased rigidity may be a natural consequence of post-nucleotomy mechano-biological adaptation. On the other hand, the results indicate a critical role of an appropriate loading regime on the outcome of these processes