Acid Ceramidase Maintains the Chondrogenic Phenotype of Expanded Primary Chondrocytes and Improves the Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells

Calogera M Simonaro,Yi Ge,Hui B Sun,Jeffrey B Mason,Efrat Eliyahu,Sylvain Sachot,Xingxuan He,Daniel J Leong,Dean W Richardson,Victor A Deangelis,Edward H Schuchman,Mark E Haskins

doi:10.1371/journal.pone.0062715

Abstract

Acid ceramidase is required to maintain the metabolic balance of several important bioactive lipids, including ceramide, sphingosine and sphingosine-1-phosphate. Here we show that addition of recombinant acid ceramidase (rAC) to primary chondrocyte culture media maintained low levels of ceramide and led to elevated sphingosine by 48 hours. Surprisingly, after three weeks of expansion the chondrogenic phenotype of these cells also was markedly improved, as assessed by a combination of histochemical staining (Alcian Blue and Safranin-O), western blotting (e.g., Sox9, aggrecan, collagen 2A1), and/or qPCR. The same effects were evident in rat, equine and human cells, and were observed in monolayer and 3-D cultures. rAC also reduced the number of apoptotic cells in some culture conditions, contributing to overall improved cell quality. In addition to these effects on primary chondrocytes, when rAC was added to freshly harvested rat, equine or feline bone marrow cultures an ∼2-fold enrichment of mesenchymal stem cells (MSCs) was observed by one week. rAC also improved the chondrogenic differentiation of MSCs, as revealed by histochemical and immunostaining. These latter effects were synergistic with TGF-beta1. Based on these results we propose that rAC could be used to improve the outcome of cell-based cartilage repair by maintaining the quality of the expanded cells, and also might be useful in vivo to induce endogenous cartilage repair in combination with other techniques. The results also suggest that short-term changes in sphingolipid metabolism may lead to longer-term effects on the chondrogenic phenotype.

Highlights

Cell-based therapy for cartilage repair has gained increasing popularity since the first reports of successful autologous chondrocyte implantation (ACI) over 10 years ago [1]
We propose that recombinant acid ceramidase (rAC) may be an important supplement to include in chondrocyte and mesenchymal stem cells (MSCs) growth media that improves the production and chondrogenic potential of cells used for cartilage repair
These results demonstrated that rAC was taken up by rat chondrocytes and retained biological activity by hydrolyzing ceramide and producing sphingosine and S1P

Summary

Introduction

Cell-based therapy for cartilage repair has gained increasing popularity since the first reports of successful autologous chondrocyte implantation (ACI) over 10 years ago [1]. In ACI, primary chondrocytes are obtained from small biopsies of healthy articular cartilage, expanded, and placed onto 3-D scaffolds for subsequent use in cartilage repair surgery (for review see [2]). ACI has been used in veterinary medicine to improve the outcome of cartilage repair surgery in large (equine) and small (dog) animals [4,5]. There have been many reports documenting the improved clinical effectiveness of ACI as compared to other cartilage repair procedures, and several large, multi-site clinical studies are currently underway [6]. An important limitation of this procedure is the requirement of two invasive surgeries, the first of which requires extraction of cells from healthy cartilage tissue, and the second to implant the cells that have been expanded ex vivo. Recent research has focused on the use of alternative chondrocyte sources where the cells can be obtained less invasively (e.g., nasoseptal) [7], the generation of chondrocytes from adult stem cells (e.g., mesenchymal stem cells [MSC] from the bone marrow or adipose tissue), and/or the use of MSCs directly for transplantation [8,9,10]

Methods

Results

Conclusion