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)

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

The mode of interfragmentary movement affects bone formation and revascularization after callus distraction.

Callus distraction is sometimes associated with a delay in the maturation process and serious complications. It is believed that these complications are often caused by instability of the bone segment fixation. Typical fixation devices, such as ring-fixators, show significant deformations in all directions under external loading and muscle forces. This leads to axial compression and tension as well as shear movements in the healing area. Herein we investigated the hypothesis that the direction of interfragmentary movement after callus distraction affects the bone formation and revascularization during the maturation process. Two custom fixator systems were designed to apply a protocol of lateral callus distraction and subsequent cyclic stimulation of the regenerate tissue. One fixator system was used to apply either compressive or tensile stimulation while the other was used to apply shearing stimulation. The fixators were applied to the tibial surface of the right hind leg of sheep specimens. During lateral callus distraction, a titanium plate was elevated by 0.275 mm perpendicular to the long axis of the bone twice daily, resulting in a 5.5 mm gap at the end of the ten-day distraction phase. Following a seven-day consolidation phase, the regenerate in the gap between tibial cortex and titanium plate was stimulated once daily by cyclic movement for 120 cycles. The stimulation was applied for 18 days with amplitudes of 0.6 mm in compression (Group C) or tension (Group T), or a 1.0 mm shear amplitude (Group S). Seven weeks postoperatively the specimens were analyzed for quantity of bone formation, the presence of cartilage and fibrous tissue, and blood vessel density. There was a significantly higher blood vessel density (4.6 ± 1.6%) in Group C than in Group T (1.2 ± 0.4%) or Group S (1.0 ± 0.5%) (p < 0.01). The amount of bone was significantly higher in Group C (25.6% ± 13.0%) than in Group T (13.5 ± 4.9%) (p < 0.05). Group S showed a similar amount of bone (14.0 ± 10.7%) to Group T. The results show that bone formation and revascularization are dependent on the direction of interfragmentary movement and that the cyclic compression best stimulates the healing process

Pestgräber der Zweiten Pandemie aus Stans (NW) und Domat/Ems (GR): Archäologie, Anthropologie, Paläogenetik

Für die Zweite Pandemie vom 14. bis zum 17. Jahrhundert sind historisch zahlreiche Pestausbrüche für die Schweiz belegt. Anthropologisch und archäologisch waren die Opfer dieser Pestwellen allerdings lange Zeit nicht fassbar, allenfalls Mehrfachbestattungen und Massengräber wurden gelegentlich als potenzielle Pestgräber interpretiert. Mithilfe der Paläogenetik ist es uns heute jedoch möglich, den Pesterreger Yersinia pestis direkt nachzuweisen. Bisher stellen Stans (Spyrou, Keller et al. 2019, Nat. Comm.) und Domat/Ems (Keller, Guellil, Scheib 2020, Archäologie Graubünden) die einzigen Schweizer Fundorte dar, für die paläogenetische Pestnachweise publiziert wurden. In diesem Vortrag werden die Ergebnisse in Kürze vorgestellt und deren Implikationen für die Interpretation der archäologischen und anthropologischen Befunde diskutiert

Intramembranous bone formation after callus distraction is augmented by increasing axial compressive strain.

The mechanical environment is a primary factor in the success of distraction osteogenesis. It is known that the interfragmentary movement during the distraction and maturation phase effects the callus formation. In addition to cyclic compression, other movements like shear and bending influence the bone formation process as shown in previous callus distraction studies. Reports of cartilage presence and endochondral ossification in the regenerative zone have been associated with a lack of fixation stability and delayed healing. So far the effects of the direction of interfragmentary movements could not be studied separately. By means of a unique lateral callus distraction model, we investigated the effects of small (0.1 mm) and moderate (0.6 mm), purely axial compression on ossification during callus maturation in sheep. A distraction device incorporating a mobile titanium plate was mounted on the tibia. Following lateral callus distraction, electromechanically controlled movements allowed purely axial cyclic compression of the tissue regenerate. Seven weeks post-operatively, the tissue regenerates were investigated using μCT, histology and immunohistochemistry. The larger amplitude significantly increased bone formation (Fractional bone volume: 19.4% vs. 5.2%, p = 0.03; trabecular thickness: 0.1 mm vs. 0.06 mm, p = 0.006; mean spicule height: 2.6 mm vs. 1.1 mm, p = 0.02) however, no endochondral ossification occurred. The elimination of shear movement, unimpaired neovascularization as well as the tensile strain stimuli during the distraction phase suppressing chondrogenic differentiation may all contribute to the absence of cartilage. In clinical application of distraction osteogenesis, moderate axial interfragmentary movement augments intramembranous ossification provided shear strain is minimized

In Vitro and in Silico Investigations of Disc Nucleus Replacement

Currently, numerous hydrogels are under examination as potential nucleus replacements. The clinical success, however, depends on how well the mechanical function of the host structure is restored. This study aimed to evaluate the extent to and mechanisms by which surgery for nucleus replacements influence the mechanical behaviour of the disc. The effects of an annulus defect with and without nucleus replacement on disc height and nucleus pressure were measured using 24 ovine motion segments. The following cases were considered: intact; annulus incision repaired by suture and glue; annulus incision with removal and re-implantation of nucleus tissue repaired by suture and glue or plug. To identify the likely mechanisms observed in vitro, a finite-element model of a human disc (L4–L5) was employed. Both studies were subjected to physiological cycles of compression and recovery. A repaired annulus defect did not influence the disc behaviour in vitro, whereas additional nucleus removal and replacement substantially decreased disc stiffness and nucleus pressure. Model predictions demonstrated the substantial effects of reductions in replaced nucleus water content, bulk modulus and osmotic potential on disc height loss and pressure, similar to measurements. In these events, the compression load transfer in the disc markedly altered by substantially increasing the load on the annulus when compared with the nucleus. The success of hydrogels for nucleus replacements is not only dependent on the implant material itself but also on the restoration of the environment perturbed during surgery. The substantial effects on the disc response of disruptions owing to nucleus replacements can be simulated by reduced nucleus water content, elastic modulus and osmotic potential

Schober test is not a valid assessment tool for lumbar mobility

Abstract The Schober test is considered reliable in evaluating lumbar mobility and its impairment. Especially in patients with chronic low back pain (cLBP) identification of functional restriction is important. We aimed to investigate whether the 5 cm Schober cut-off provides a valid distinction between unrestricted and restricted mobility in participants with and without cLBP (18–65 years). cLBP is defined as LBP persisting for ≥ 12 weeks. We analyzed agreement between the Schober test with two measurement devices (Epionics SPINE®; Idiag M360®) and the influence of lumbar lordosis (LL) on their agreement. Also, the sensitivity and specificity of the Schober test was evaluated. For 187 participants (49.6%) Epionics SPINE® RoF and Schober test matched (either ≥ 5 cm; > 40.8° RoF or ≤ 5 cm;  46.0° RoF or ≤ 5 cm; < 46.0° RoF). Non-agreement was seen in 187 participants (49.6%). LL differed significantly in the Epionics SPINE® cohort (p < 0.001). Regarding the Epionics SPINE® cohort, Schober test showed a sensitivity of 79.6% with a specificity of 36.1%. For the Idiag M360® cohort, Schober test showed a sensitivity of 68.2% and a specificity of 46.6%. Our results do not establish a consistent matching between Schober test and the device measurements. Therefore, Schober test may not be valid to predict impairment of lumbar mobility. We recommend Schober test as an add-on in monitoring of an individual relative to its case