Nucleus pulposus (NP) digestion using chymopapain is a clinical procedure to release pressure of a herniated disk. It reduces matrix content and hence offers a method to simulate a condition of disk degeneration with reduced glycosaminoglycans (GAG) and water content.1 The aims of the study is to characterize a set of in vitro disk 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 DDM.
Materials and Methods
Disk Degeneration Model. Bovine caudal IVDs (diameter 16 to 22 mm) with endplates were isolated as previously described which is readily for in vitro culture in a multiloading bioreactor.2 Various concentration of papain (3, 15, 30, 60, 150 U/mL, n = 4 for each group) (Sigma-Aldrich, Switzerland) or phosphate buffer saline (PBS control) were injected into the center of the disk using a 25 G. Ten days after the injection of papain, cell viability was assessed by staining the tissue with Live/Dead stain (Fluka, Sigma-Aldrich, Switzerland) and visualized by confocal microscopy.3 The other half of the tissue was evaluated by histology for overall matrix organization. Nondecalcified tissues were embedded in methylmethacrylate (MMA) for histology sectioning. Overall matrix composition was evaluated by staining the 6 µm section with safranin O and fast green, and hematoxylin and eosin. Disk hydration of the bovine tail disk 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 disk. 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 Disk 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 disk. In the medium DDM (30 to 60 U/mL), GAG was completely lost in the entire disk with a small cavity in the center while AF maintained intact. In the DDM (150 U/mL), GAG was completely lost in the entire disk with a large cavity present in the center of the disk and the AF was collapsed. MRI result showed that there was a mild decrease in disk hydration by injecting 3 U/mL papain, and half of the disk hydration was lost when injecting 150 U/mL papain. (Fig. 1)
MSC Injection. At day 2, MSC injected into a control disk (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).
MRI image of the bovine tail DDM as caused by injecting increasing dosage of papain.
Conclusion
A reproducible DDM of various severities of reduced GAG and hydration can be achieved by injecting various dosage of papain without compromising the disk cell viability. This model is ready for the testing of injectable tissue engineering strategies inside a controlled loading and culture environment of a bioreactor. MSCs injected as cell suspension have a poor-chance of survival in a healthy or degenerated disk. This indicated that matrix content of the disk affect the initial survival of the introduced cells and revealed that the use of cell carrier/scaffold will be needed for cell therapy.
Acknowledgements This project was supported by the Swiss National Science Foundation (SNF # 310030–127586/1).
I confirm having declared any potential conflict of interest for all authors listed on this abstract
Yes
Disclosure of Interest
None declared
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Chan S et al. JoVE, In press, 2011
Gantenbein-Ritter B et al. Tissue Engineering Part C 2008;14:353–358
