C. Cavallo (Bologna, IT)
Rizzoli Orthopaedic Institute Ramses LaboratoryPresenter Of 2 Presentations
P132 - The Inflammatory/catabolic “Signature” of IL-1 β Primed sEVs on Osteoarthritis Joint Cells
Abstract
Purpose
The last decade has seen growing efforts to exploit the effects of Adipose Derived Stromal Cells (ADSCs) in the treatment of degenerative diseases, including osteoarthritis (OA), and recent findings have suggested that ADSC therapeutic ability is related to paracrine mechanisms. Thus, scientific interest has moved to the ADSC secretome focusing on Small Extracellular Vesicles (sEVs) which can act as “biological drug carriers”. However, the behaviour of sEVs could be affected by ADSC pathophysiology that therefore represents an important issue for clinical application.
The aim of this study was to evaluate the impact of ADSC inflammatory status on their secretome and the effects of the latter on articular tissues, focusing on chondrocytes and synoviocytes derived from OA joints.
Methods and Materials
ADSCs were obtained from adipose tissue harvested during regenerative treatments; chondrocytes and synoviocytes were isolated from tissues collected during knee arthroplasty. sEVs were collected from IL-1β treated ADSC by combining precipitation and size exclusion chromatography. sEVs were quantified, characterized, and administered to chondrocytes/synoviocytes in either monoculture or in co-culture. Gene expression and protein release of cytokines/chemokines, catabolic and inflammatory molecules were analyzed at 4 and 15 h. At the same experimental times, the p65 nuclear translocation was investigated to assess the NF-κB pathway.
Results
Findings of this study demonstrated that the biological effects of ADSC secretome can be affected by the inflammatory environment. IL-1β primed sEVs were able to transfer NF-κB activation to chondrocytes and synoviocytes, inducing the transcription/release of inflammatory chemokines and cytokines, as well as the transcription of major cartilage catabolic enzymes.
Conclusion
Our findings, combined to those we recently published, underline that sEV nature and effects depend on the homeostatic or inflammatory/catabolic status of the ADSC source. Thus, for therapeutic purposes, ADSC should undergo careful characterization to avoid the transfer of an unwanted message that can further promote OA progression.
P227 - Bone Marrow Aspirate Concentrate from Tibia and Iliac Crest in Young and Old Patients: A Quality Comparison
Abstract
Purpose
The real potential of bone marrow aspirate concentrate (BMAC) remains controversial and many aspects remain to be clarified, including harvest site and age, which could play an important role in determining both the amount of contained MSCs and their quality, and consequently the biological potential of BMAC. Aim of this study was to shed some light on the characteristics of BMAC based on harvest site and patient age.
Methods and Materials
BMAC was obtained from two groups of patients based on age (n=10 per group): 19.0±2.7 years for the younger, 56.8±12.5 for the older group. In the latter, BMAC was obtained from the anterior iliac crest and proximal tibia for a comparative analysis. Mononucleated cell count and fibroblast colony-forming units (CFU-F) assay were performed, together with MSCs phenotype characterization, study of MSCs chondrogenic and osteogenic differentiation capacity, histological staining and spectrophotometric quantification, and analysis of mRNAs expression.
Results
A significantly higher number of mononuclear cells per ml was observed in younger compared to older patients (3.8±1.8×107 vs 1.2±0.8×107, p<0.0005). The number of mononuclear cells obtained from the iliac crest was higher than the 0.3±0.2×107 obtained from the proximal tibia (p<0.0005). This result was confirmed by the CFU-F analysis both at day 10 (15.9±19.4 vs 0.6±1.0, p=0.001) and day 20 (21.7±23.0 vs 2.9±4.2, p=0.006). Cells derived from iliac crest and tibia showed the same phenotypic pattern at flow cytometry, as well as similar chondrogenic and osteogenic potential.
Conclusion
Harvest site and age can affect the quality of BMAC. MSCs obtained from iliac crest and proximal tibia present comparable mesenchymal markers expression as well as osteogenic and chondrogenic differentiation potential, but iliac crest BMAC presents a four-time higher number of mononucleated cells and CFU-F compared to the tibia. Age also influences BMAC quality, with a three-time higher number of mononucleated cells in younger patients.
Presenter Of 2 Presentations
P132 - The Inflammatory/catabolic “Signature” of IL-1 β Primed sEVs on Osteoarthritis Joint Cells
Abstract
Purpose
The last decade has seen growing efforts to exploit the effects of Adipose Derived Stromal Cells (ADSCs) in the treatment of degenerative diseases, including osteoarthritis (OA), and recent findings have suggested that ADSC therapeutic ability is related to paracrine mechanisms. Thus, scientific interest has moved to the ADSC secretome focusing on Small Extracellular Vesicles (sEVs) which can act as “biological drug carriers”. However, the behaviour of sEVs could be affected by ADSC pathophysiology that therefore represents an important issue for clinical application.
The aim of this study was to evaluate the impact of ADSC inflammatory status on their secretome and the effects of the latter on articular tissues, focusing on chondrocytes and synoviocytes derived from OA joints.
Methods and Materials
ADSCs were obtained from adipose tissue harvested during regenerative treatments; chondrocytes and synoviocytes were isolated from tissues collected during knee arthroplasty. sEVs were collected from IL-1β treated ADSC by combining precipitation and size exclusion chromatography. sEVs were quantified, characterized, and administered to chondrocytes/synoviocytes in either monoculture or in co-culture. Gene expression and protein release of cytokines/chemokines, catabolic and inflammatory molecules were analyzed at 4 and 15 h. At the same experimental times, the p65 nuclear translocation was investigated to assess the NF-κB pathway.
Results
Findings of this study demonstrated that the biological effects of ADSC secretome can be affected by the inflammatory environment. IL-1β primed sEVs were able to transfer NF-κB activation to chondrocytes and synoviocytes, inducing the transcription/release of inflammatory chemokines and cytokines, as well as the transcription of major cartilage catabolic enzymes.
Conclusion
Our findings, combined to those we recently published, underline that sEV nature and effects depend on the homeostatic or inflammatory/catabolic status of the ADSC source. Thus, for therapeutic purposes, ADSC should undergo careful characterization to avoid the transfer of an unwanted message that can further promote OA progression.
P227 - Bone Marrow Aspirate Concentrate from Tibia and Iliac Crest in Young and Old Patients: A Quality Comparison
Abstract
Purpose
The real potential of bone marrow aspirate concentrate (BMAC) remains controversial and many aspects remain to be clarified, including harvest site and age, which could play an important role in determining both the amount of contained MSCs and their quality, and consequently the biological potential of BMAC. Aim of this study was to shed some light on the characteristics of BMAC based on harvest site and patient age.
Methods and Materials
BMAC was obtained from two groups of patients based on age (n=10 per group): 19.0±2.7 years for the younger, 56.8±12.5 for the older group. In the latter, BMAC was obtained from the anterior iliac crest and proximal tibia for a comparative analysis. Mononucleated cell count and fibroblast colony-forming units (CFU-F) assay were performed, together with MSCs phenotype characterization, study of MSCs chondrogenic and osteogenic differentiation capacity, histological staining and spectrophotometric quantification, and analysis of mRNAs expression.
Results
A significantly higher number of mononuclear cells per ml was observed in younger compared to older patients (3.8±1.8×107 vs 1.2±0.8×107, p<0.0005). The number of mononuclear cells obtained from the iliac crest was higher than the 0.3±0.2×107 obtained from the proximal tibia (p<0.0005). This result was confirmed by the CFU-F analysis both at day 10 (15.9±19.4 vs 0.6±1.0, p=0.001) and day 20 (21.7±23.0 vs 2.9±4.2, p=0.006). Cells derived from iliac crest and tibia showed the same phenotypic pattern at flow cytometry, as well as similar chondrogenic and osteogenic potential.
Conclusion
Harvest site and age can affect the quality of BMAC. MSCs obtained from iliac crest and proximal tibia present comparable mesenchymal markers expression as well as osteogenic and chondrogenic differentiation potential, but iliac crest BMAC presents a four-time higher number of mononucleated cells and CFU-F compared to the tibia. Age also influences BMAC quality, with a three-time higher number of mononucleated cells in younger patients.