# In Vitro Model for Lumbar Disc Herniation to Investigate Regenerative Tissue Repair Approaches
## 2 Materials and Methods
### New Physiological Loading Protocol  
A new physiological loading protocol was developed to simulate typical daily in vivo activities associated with lumbar disc herniations. Therefore, activities were chosen that either consist of extreme flexion like tying shoes or with additional loading like lifting boxes. Furthermore, the influence of activities with complex motion patterns on the risk of lumbar disc herniations was investigated by simulating sweeping the floor or lifting boxes while turning. For each activity, physiological in vivo segmental motion [37] and intradiscal pressures (IDP, [38]) were replicated in a dynamic disc loading simulator [39]. This dynamic disc loading simulator is able to deform the specimens in six degrees of freedom and thereby allows the replication of complex motion in all anatomical planes, as well as additional axial loading or compression to simulate resultant muscle forces or body weight. In order to monitor the IDP that results from this complex deformation and loading of the specimen, an IDP sensor (Mammendorfer Institut fur Physik und Medizin GmbH, Hattenhofen, Germany) was implanted into the nucleus pulposus from the right lateral side of the disc. From a neutral position, the starting activity of the patient in a standing position was approached. Then, the dynamic activities tying shoes, sweeping the floor, lifting boxes (20 kg) and the corresponding complex activity lifting boxes while turning were performed subsequently (Supplementary Materials: Video S1). Each activity consisted either of pure axial loading (standing) or in combination with bending. For the simulation of tying shoes and lifting boxes, the specimens were bent in flexion; for the simulation of sweeping the floor or lifting boxes while turning in flexion, lateral bending and axial rotation were applied simultaneously (Table 2). The motion values that were applied for each activity resulted from an in vivo study performed by [PERSON] in 1985 [37]. The amount of the necessary axial load for every individual specimen was determined from in vivo IDP measurements from the study by [PERSON] et al. [38]. During a quasistatic calibration cycle prior to testing, the equivalent motion of each activity was applied and axial compression was subsequently adjusted (Table 2) until the equivalent in vivo IDP value had been created within the disc. However, in vivo IDP was known for the activities of standing, tying shoes, and lifting boxes. For the simulation of the complex activities (sweeping the floor and \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Defect Group** & **Donor** & **Sex** & **Age in Years** & **Segment** &
\begin{tabular}{c} **Degeneration** \\ **(acc. to Pfirrmann [36])** \\ \end{tabular} & **Relevant Diagnoses** \\ \hline vertical cut & 2130 & male & 26 & L2-L3 & 2 \\  & 2133 & female & 24 & L4-L5 & 3-4 & disc herniation 1 \\  & 2134 & male & 24 & L2-L3 & 1 \\  & 2134 & male & 26 & L4-L5 & 1 \\  & 2138 & male & 26 & L5-L4 & 1 \\  & 2138 & male & 33 & L2-L3 & 1 \\  & & & & L4-L5 & 2 \\ \hline  & median (range) & & 26 (24, 33) & & 1 (1, 2) 2  \\ \hline round hole & 2036 & male & 40 & L2-L3 & 1 \\  & 2072 & male & 53 & L4-L5 & 1 \\  & & & & L3-L4 & 1-2 \\  & & & & L5-L5 & 1-2 \\  & 2129 & n.a. & 19 & L2-L3 & 1 \\  & 2132 & male & 31 & L2-L3 & 1 \\  & & & & L4-L5 & 1 \\ \hline  & median (range) & & 35.5 (19, 53) & & 1 (1, 1-2) \\ \hline overall & median (range) & & 35.5 (19, 53) & & 1 (1, 2) \\ \hline \hline \end{tabular}  
* The diagnosis disc herniation led to exclusion of those specimens. 2 Median (range) based on specimens that were included in the study.  
\end{table} 
Table 1: Motion segments from human donors that were used for this study for the different defect groups. Only segments with hardly no or low degeneration (degree of degeneration according to Pfirrmann [36] 1–2) and no relevant diagnoses such as present disc herniation that led to exclusion of the specimen were chosen. lifting boxes while turning), rotations in all motion directions were applied.