On the Path to Autologous IVD Regeneration?
Authors: Caldeira J. , Molinos M. , Osório H. , Santa C. , Manadas B. , Almeida C. R. , Gonçalves R. M. , Barbosa M. A. 
Published: May 2014

Introduction

Intervertebral disc (IVD) degeneration is often the cause of low back pain. With age, extracellular matrix (ECM) depletion occurs, leading to nucleus pulpous (NP) extrusion and IVD destruction. Concomitantly, there is a decline in viable cell populations, which in turn remain poorly characterized. As current clinical treatments have not provided adequate solutions, we propose an in-depth study in terms of NP cell subpopulation characterization and ECM changes with aging, in view of regeneration.

Materials and Methods

The first step was to characterize NP cell populations harvested from bovine IVDs, to better understand IVD's potential to endogenously regenerate. We started by optimizing cell isolation procedure, still not consensual among the literature. For that, distinct digestive enzymes (collagenase type-I, collagenase type-II, and collagenase type-XI) were used at three different concentrations (0.5, 1.0, and 2.0 mg/mL, respectively), for 4 and 19 hours. To understand the cellular effect of the digestion, we evaluated the cell yield (trypan blue exclusion); cell viability/apoptosis (AnnexinV/Propidium Iodide staining, Flow Cytometry), and cell morphology (actin (phalloidin-AlexaFluor488) and nuclei (DRAQ-5) staining, Imaging Flow Cytometry). The presence of specific cell subpopulations within NP and its phenotype was then investigated, by assessing the expression of CD29, CD44, CD45, CD146, CD34, Gly-A, and Brachyury. Due to microenvironments’ growing importance for regeneration, in parallel, we compared the NP ECM proteomic profile of bovine IVDs from fetus, young and old animals by quantitative iTRAQ LC-MS/MS. The candidates differentially expressed in the three age groups are being validated by Western blot.

Results

Cell yield and viability improved, in a concentration dependent manner, with increasing collagenase activity against collagen I and II—collagenase XI rendered the highest cell yield and greatest viability. In addition, cell yield was higher for shorter digestion periods (4 hours). Furthermore, NP cells did not reveal major morphological changes dependent on the enzymes used. Interestingly, three viable subpopulations, with different sizes and autofluorescence, were consistently identified by flow cytometry, immediately after isolation. Expression of cell markers differed among these subpopulations, proving the existence of an heterogeneous cell population within the NP. Concerning iTRAQ analysis, a few interesting candidates emerged that are expressed in different proportions in fetus, young and old animals. These quantitative differences are being validated.

Conclusion

This study shows that collagenase type XI is the most efficient enzyme to be used for isolation and that different cell populations can be identified within the bovine NP. It also demonstrates that distinct ECM proteins are expressed in the IVD at different ratios depending on bovine age groups. Acknowledgments This work was financed by FEDER funds through the Programa Operacional Factores de Competitividade—COMPETE, by Portuguese funds through FCT— Fundação para a Ciência e a Tecnologia. The authors would also like to acknowledge the AO SPINE Research Network (AO SRN) for funding. Disclosure of Interest None declared