Influence on Chondrogenesis of Human Osteoarthritic Chondrocytes in Co-Culture with Donor-Matched Mesenchymal Stem Cells from Infrapatellar Fat Pad and Subcutaneous Adipose Tissue

Abstract

Co-culture of mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) has been proposed for autologous cartilage cell-based therapies, to overcome the issues associated to limited availability of articular chondrocytes (ACs). To evaluate the potentiality of a co-culture approach in aged osteoarthritic patients, MSCs from infrapatellar fat pad (IFP-MSCs) and knee subcutaneous adipose tissue (ASCs) were co-cultured with donor-matched osteoarthritic, expanded and cryopreserved, ACs in a 75%/25% ratio. Co-cultures were prepared also from nasal chondrocytes (NCs) to evaluate their possible use as an alternative to ACs. Pellets were differentiated for 14 days, using mono-cultures of each cell type as reference. Chondrogenic genes SOX9, COL2Al, ACAN were less expressed in co-cultures compared to ACs and NCs. Total GAGs content in co-cultures did not differ significantly from values predicted as the sum of each cell type contribution corrected for the co-culture ratio, as confirmed by histology. No significant differences were observed for GAGslDNA in mono-cultures, demonstrating a reduced chondrogenic potential of ACs and NCs. In conclusion, a small percentage of expanded and cryopreserved ACs and NCs did not lead to IFP-MSCs and ASCs chondro-induction. Our results suggest that chondrogenic potential and origin of chondrocytes may playa relevant role in the outcome of co-cultures, indicating a need for further investigations to demonstrate their clinical relevance in the treatment of aged osteoarthritic patients.

Fabrication of multi‐well chips for spheroid cultures and implantable constructs through rapid prototyping techniques

“…A similar reduction was observed in standard spheroids cultured in polypropylene tubes. This observation is coherent with literature data obtained for the chondrogenic 3D culture of mesenchymal stem cells and articular chondrocytes [Giovannini et al, ; Lopa et al, ] and indicates that the increase in spheroid area observed in our experiments over time was due to extracellular matrix deposition and not to cell proliferation.…”

“…As hypothesized for the PDMS chips, the multi‐well construct produced in this study can be used for the co‐culture of different cell types, which is usually performed by more standard 3D models, such as spheroids [Giovannini et al, ; Lopa et al, ; Wu et al, ] and random cell dispersion in 3D scaffolds [Hildner et al, ; Sabatino et al, ]. The controlled distribution of mesenchymal stem cells and bovine chondrocytes in an implantable multi‐well scaffold has been performed in a recent study screening the effect of several extracellular matrix components on the chondrogenesis of MSCs co‐cultured with bovine chondrocytes [Higuera et al, ].…”

“…Samples of articular cartilage were harvested from patients affected by osteoarthritis undergoing total hip replacement (five males and five females, mean age 64 ± 8 years), with patients' informed written consent and with the approval of the Institutional Review Board. ACs were isolated from cartilage by enzymatic digestion using 0.15% type II collagenase (Worthington Biochemical Corp., Lakewood, NJ) for 22 h at 37°C, according to a previously established protocol [Lagana et al, ; Lopa et al, ]. The cells were plated at a density of 10 4 cells/cm 2 and cultured in expansion medium.…”

Strategies to Mitigate Variability in Engineering Human Nasal Cartilage

“…Lopa et al . previously demonstrated no positive effect of co-culture with adipose derived MSCs 25 . That study used cryogenically preserved cells and had expanded the cells with the use of transforming growth factor- β1 (TGF-β1), which may have resulted in their negative finding.…”

Enhancing adipose stem cell chondrogenesis: A study on the roles of dexamethasone, transforming growth factor β3 and ascorbate supplements and their combination

“…Total mRNA (up to 2.5 μg) was reverse-transcribed into cDNA using SuperScript® VILO™ Master Mix. cDNA was ampli ied with the TaqMan® Gene Expression Master Mix (Applied Biosystems by Life Technologies, Grand Island, NY, USA) on an ABI 7900HT Sequence Detection System (Applied Biosystems) and probes speci ic for GAPDH, a housekeeping gene that has served as a baseline in several studies on chondrocyteprogenitor interaction and MSC-chondrocyte co-culture [32][33][34][35][36][37], sex-determining region Y (SRY)-box 9 (SOX9), ACAN, Lubricin, Col XI, Col X, VEGF-A, Col I, alkaline phosphatase (ALP), and osterix were used. Known primer sequences for these species appear in Table 1, and where primer sequences are proprietary, the company name and catalogue number are given.…”