A Reproducible Papain-Induced Disk Degeneration Model of Reduced GAG and Hydration

Abstract

Introduction: Nucleus pulposus (NP) digestion using chymopapain is a clinical procedure to release pressure of a herniated disc. It reduces matrix content and hence offers a method to simulate a condition of disc degeneration with reduced glycosaminoglycans (GAG) and water content. The aims of the study is first to characterize a set of in-vitro disc degeneration model (DDM) of different severity of GAG and water loss by using various concentration of papain and second to determine the initial response of mesenchymal stem cells (MSC) introduced in these papain-induced disc degeneration model. Materials and Methods: Disc Degeneration Model: Bovine caudal IVDs with endplates were isolated as previously described which is readily for in-vitro culture in a multi-loading bioreactor. Various concentration of papain (3, 15, 30, 60, 150 U/mL, N=4 for each group) or phosphate buffer saline (PBS control) were injected into the center of the disc. Tens days after the injection of papain, cell viability was assessed by staining the tissue with Live/Dead stain and visualized by confocal microscopy. The other half of the tissue was evaluated by histology. Tissues were embedded in methylmetaacrylate (MMA) for histology sectioning. Overall matrix composition was evaluated by staining the 6 μm section with safranin O and fast green, and haematoxylin and eosin. Disc hydration of the bovine tail disc injected with papain and PBS after 2 days was assessed by magnetic resonance imaging (MRI) using T2∗ protocol. MSC Injection: Human MSCs were isolated from bone marrow obtained from spinal surgery. MSC were labeled with a fluorescence cell membrane tracker DiO (Invitrogen, Basel, Switzerland) and then suspended in culture medium and injected into the nucleus of the DDM (30 U/mL) or a PBS control disc. MSC distribution and viability were evaluated after 2 days of free swelling culture by staining the tissue with DAPI and calcein AM and imaging with confocal microscopy. The presence of live injected cells were visualized as colocalization of orange DiL dye and calcein AM live cell stain. Results: Disc Degeneration Model: Cell viability of the papain digested NP was maintained at ∼75% after 10 days in all the injected papain concentration. In the mild DDM (3 U/mL), small amount of GAG was maintained in the center of the disc. In the medium DDM (30- 60 /mL), GAG was completely lost in the entire disc with a small cavity in the center while AF maintained intact. In the severer DDM (150 U/mL), GAG was completely lost in the entire disc with a large cavity present in the center of the disc and the AF was collapsed. MRI result showed that there was a mild decrease in disc hydration by injecting 3 U/mL papain, and half of the disc hydration was lost when injecting 150 U/mL papain. There was a striking analogy using T2∗ mapping to the Watanabe grading found (Figure 1). MSC Injection: At day 2, MSC injected into a control disc (injected with PBS) formed a cluster and largely decreased the MSC viability. While MSC injected in the DDM dispread inside the NP and a significantly higher cell viability was resulted when MSC were injected into this papain-induced DDM (40.64 ±10.81 %) than in the PBS control (14.50 ± 6.08 %) (N=4, p= 0.0329). Conclusion: A reproducible disc degeneration model of various severities of reduced GAG and hydration can be achieved by injecting various dosage of papain without compromising the disc cell viability. MSCs injected as cell suspension have a poorchance of survival in a healthy or degenerated disc. Biomaterials would possibly provide even better support for the survival of MSC. (Figure Presented) .