C. Davis (Ann Arbor, US)
University of MichiganPresenter Of 1 Presentation
P005 - High-level of expansion combined with IL-1β treatment accelerates development of chondrosenescent phenotype
Abstract
Purpose
Chronic, low-grade inflammation (inflammaging) contributes to the age-dependent deterioration of chondrocytes, or chondrosenescence. The chondrosenescent phenotype includes the development of a senescence associated secretory phenotype (SASP). Using both simulated aging (monolayer expansion) and inflammation, we sought to create an in vitro model that replicates the chondrosenescent phenotype found in patient samples. We hypothesized that treatment of young human articular chondrocytes (HACs) with inflammatory cytokines during monolayer expansion will induce biomarker profiles and ECM production levels similar to that of HACs isolated from aged/OA cartilage.
Methods and Materials
HACs (30 ± 10 years) were treated with 1 ng/mL IL-1β during monolayer expansion. Expression of senescence markers (SA-β-gal and p16) and RNAseq for SASP were measured at each passage. High passage, chondrosenescent HACs were then redifferentiated in pellet culture for 28 days and sGAG production was measured using the DMMB assay and Alcian blue staining.
Results
IL-1β treatment during expansion reduced proliferation (Figure 1), increased expression of SASP factors (Figure 2), and increased expression of senescence markers SA-β-gal and p16 (Figure 1) after 7 passages. SASP markers including MMPs, ILs and CXCLs were higher in IL-1β treated samples at P8 (Figure 2a). Expression of SASP markers decreased over time but remined 2-3 orders of magnitude higher in IL-1β treated samples. (Figure 2b). When redifferentiated in pellet culture, matrix production was lower in treated and untreated P7 chondrocytes compared to P4. IL-1β treatment reduced ECM production to levels comparable to OA chondrocytes (not shown).
Conclusion
Treatment with IL-1β during monolayer expansion decreased proliferative potential of HACs after the 4th passage and accelerated expression of SASP and other senescence markers, making it a viable system to model and study chondrosenescence.
Presenter Of 1 Presentation
P005 - High-level of expansion combined with IL-1β treatment accelerates development of chondrosenescent phenotype
Abstract
Purpose
Chronic, low-grade inflammation (inflammaging) contributes to the age-dependent deterioration of chondrocytes, or chondrosenescence. The chondrosenescent phenotype includes the development of a senescence associated secretory phenotype (SASP). Using both simulated aging (monolayer expansion) and inflammation, we sought to create an in vitro model that replicates the chondrosenescent phenotype found in patient samples. We hypothesized that treatment of young human articular chondrocytes (HACs) with inflammatory cytokines during monolayer expansion will induce biomarker profiles and ECM production levels similar to that of HACs isolated from aged/OA cartilage.
Methods and Materials
HACs (30 ± 10 years) were treated with 1 ng/mL IL-1β during monolayer expansion. Expression of senescence markers (SA-β-gal and p16) and RNAseq for SASP were measured at each passage. High passage, chondrosenescent HACs were then redifferentiated in pellet culture for 28 days and sGAG production was measured using the DMMB assay and Alcian blue staining.
Results
IL-1β treatment during expansion reduced proliferation (Figure 1), increased expression of SASP factors (Figure 2), and increased expression of senescence markers SA-β-gal and p16 (Figure 1) after 7 passages. SASP markers including MMPs, ILs and CXCLs were higher in IL-1β treated samples at P8 (Figure 2a). Expression of SASP markers decreased over time but remined 2-3 orders of magnitude higher in IL-1β treated samples. (Figure 2b). When redifferentiated in pellet culture, matrix production was lower in treated and untreated P7 chondrocytes compared to P4. IL-1β treatment reduced ECM production to levels comparable to OA chondrocytes (not shown).
Conclusion
Treatment with IL-1β during monolayer expansion decreased proliferative potential of HACs after the 4th passage and accelerated expression of SASP and other senescence markers, making it a viable system to model and study chondrosenescence.