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

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.

The molecular medicine of acid ceramidase

“…Another potential in vitro use of rhAC recently explored is for cell-based cartilage repair (Simonaro et al, 2013). Joint replacement surgeries are among the most common surgical procedures carried out worldwide, however outcomes are often severely limited because of the inability of cartilage to regenerate and repair.…”

“…We therefore examined the use of rhAC to improve the quality of cells used for cellbased cartilage repair, with the goal of improving the clinical outcome of cartilage repair surgery (Simonaro et al, 2013). Supplementation of primary chondrocyte culture media with the recombinant enzyme led to significantly fewer apoptotic cells after a 3-week expansion.…”

Acid ceramidase treatment enhances the outcome of autologous chondrocyte implantation in a rat osteochondral defect model

“…The results confirmed previous in vitro findings [8], and documented for the first time positive biochemical and histological outcomes of the implanted cells in vivo . In recent years ACI has attracted considerable attention [2,21], however in vitro expansion and de-differentiation of chondrocytes prior to implantation limits the utility of this approach [4, 7]. Classical methods to prevent de-differentiation use chondrogenic medium containing TGFβ homologues [3, 25], although other factors have been shown to have similar effects, including BMP2 [5], FGF2 [6] and, recently, rhAC [7].…”

“…Two rats were used per isolation, and cells were pooled to obtain enough for proper culture and expansion. Cells were isolated according to previously reported methods [7]. Briefly, animals were euthanized using CO 2 and the meniscus from each knee joint was removed along with scraping of the articular surface.…”

“…In our lab we have previously reported the effects of exogenous recombinant human acid ceramidase (rhAC) on the de-differentiation of chondrocytes by observing specific chondrocyte markers at the protein level, and also showed that this enzyme treatment enhanced survival of the expanded cells [7]. …”