Notochordal Cells In Treating Intervertebral Disc Degeneration; Cell Markers And Regenerative Capacity.

Abstract

Delineating markers to identify notochordal cells in the canine nucleus pulposus.

Background: Intervertebral disc (IVD) degeneration in humans and chondrodystrophic (CD) dogs is a naturally occurring process. It often starts with a shift in cell type from large vacuolated notochordal cells to small non-vacuolated cells. When developing a therapy to either retain or restore the notochordal cell (NC) population, readout parameters are needed to determine the specific phenotype of NCs. A specific marker set to identify these cells in the (canine) nucleus pulposus (NP) is currently lacking. Objective: Investigating the expression of several NP/progenitor markers in the canine NP to identify the specific phenotype of NCs. Methods: Immunohistochemical stainings for several markers were performed on canine NPs of different ages and breeds (stillborn pups, mature non-chondrodystrophic and chondrodystrophic dogs). The markers that were tested included proposed NC makers brachyury, TIE2, CD24, CK8 and 19 and mesenchymal progenitor marker CD73. Results: CK19 was present in both the puppy and the non-chondrodystrophic (NCD) dog NPs, but more abundant in the puppy. There was a high expression of CD24, brachyury, TIE2 and CD73 in the NP of the puppy, whereas the NCD and CD NP only showed some intranuclear staining of TIE2. Conclusion: CD24, CK19, Brachyury and CD73 seem to be useful canine NC markers. Further research is necessary to confirm the findings in this study, to be able to perform statistical analysis and to determine which markers select for functional NCs.

The effect of NCM on the expression of cytokeratin 19 and aggrecan in degenerated canine intervertebral discs

Background: Intervertebral disc (IVD) degeneration in dogs often starts with the disappearance of notochordal cells (NC) from the nucleus pulposus (NP). NC secreted substances are known to have a regenerative effect on the NP. Without them and their matrix, the jelly-like NP converts into a more rigid structure uncapable of fulfilling its biomechanical function. NC conditioned medium (NCCM) and NC derived matrix (NCM) have been proposed as a possible therapeutic strategy for IVD degeneration. Objective: The aim of this study is to determine whether intradiscal treatment with NCM stimulates the production of aggrecan and upregulates the expression of cytokeratin 19 (CK19) in mildly and moderately degenerated canine IVDs. Methods: Six beagles were used in this study. Three IVDs of each beagle were subjected to partial removal of the NP to induce moderate IVD degeneration (NX-IVD). Three other mildly degenerated IVDs of each beagle were also included (NoNX-IVD). Each mildly and moderately degenerated IVD of each beagle received a different treatment: no treatment, a onetime injection with 50 µL 10 mg/mL porcine NCM or two injections with 50 µL 10 mg/mL three months apart. The beagles were euthanized 6 months after the initial injection and an aggrecan and a CK19 immunohistochemical staining was done on each IVD. Results: In the noNX-IVDs as well as the NX-IVDs, a lot of donor variation was seen with regard to the aggrecan content. However, the noNX-IVDs all show more aggrecan content in the NCM treated IVDs in comparison with the not treated IVDs. No CK19 immunopositivity is seen in the noNX-IVDs, apart from 1 IVD that was not treated with NCM. Although a visible response to the NCM treatment is seen in the NX-IVDs regarding the CK19 expression, these differences were not statistically significant. Conclusion: In vivo treatment of canine mildly and moderately degenerated IVDs with porcine NCM results in more aggrecan in the noNX and NX-IVDs and more CK19 expression in the NX-IVDs. More research is necessary to confirm the therapeutic potential of NCM.