Senescence during early differentiation reduced the chondrogenic differentiation capacity of mesenchymal progenitor cells

Introduction: Mesenchymal stromal/progenitor cells (MSCs) are promising for cartilage cell-based therapies due to their chondrogenic differentiation capacity. However, MSCs can become senescent during in vitro expansion, a state characterized by stable cell cycle arrest, metabolic alterations, and substantial changes in the gene expression and secretory profile of the cell. In this study, we aimed to investigate how senescence and the senescence-associated secretory phenotype (SASP) affect chondrogenic differentiation of MSCs.Methods: To study the effect of senescence, we exposed MSCs to gamma irradiation during expansion or during chondrogenic differentiation (the pellet culture). Western blot analysis was used to evaluate MSCs response to the chondrogenic inductor TGF-β.Results: When senescence was induced during expansion or at day 7 of chondrogenic differentiation, we observed a significant reduction in the cartilage matrix. Interestingly, when senescence was induced at day 14 of differentiation, chondrogenesis was not significantly altered. Moreover, exposing chondrogenic pellets to the medium conditioned by senescent pellets had no significant effect on the expression of anabolic or catabolic cartilage markers, suggesting a neglectable paracrine effect of senescence on cartilage generation in our model. Finally, we show that senescent MSCs showed lower phosphorylated SMAD2 levels after TGFβ1 stimulation than control MSCs.Conclusion: Overall, these results suggest that the occurrence of senescence in MSCs during expansion or early differentiation could be detrimental for cartilage tissue engineering.Biomaterials & Tissue Biomechanic

The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo

Objective: Cartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo. Design: We evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors. Results: RNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed. Conclusion: Using multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.Biomaterials & Tissue Biomechanic