Expansion in the presence of FGF-2 enhances the functional development of cartilaginous tissues engineered using infrapatellar fat pad derived MSCs

Abstract

A bstractMSCs from non-cartilaginous knee joint tissues such as the infrapatellar fat pad (IFP) and synovium possess significant chondrogenic potential and provide a readily available and clinically feasible source of chondroprogenitor cells. Fibroblast growth factor-2 (FGF-2) has been shown to be a potent mitotic stimulator during ex vivo expansion of MSCs, as well as regulating their subsequent differentiation potential.The objective of this study was to investigate the longer term effects of FGF-2 expansion on the functional development of cartilaginous tissues engineered using MSCs derived from the IFP. IFP MSCs were isolated and expanded to passage 2 in a standard media formulation with or without FGF-2 (5ng/ml) supplementation.Expanded cells were encapsulated in agarose hydrogels, maintained in chondrogenic media for 42 days and analysed to determine their mechanical properties and biochemical composition. Culture media, collected at each feed, was also analysed for biochemical constituents.MSCs expanded in the presence of FGF-2 proliferated more rapidly, with higher cell yields and lower population doubling times. FGF-2 expanded MSCs generated the most mechanically functional tissue. Matrix accumulation was dramatically higher after 21 days for FGF-2 expanded MSCs, but decreased between day 21 and 42. By day 42, FGF-2 expanded MSCs had still accumulated ~1.4 fold higher sGAG and ~1.7 fold higher collagen compared to control groups. The total amount of sGAG synthesised (retained in hydrogels and released into the media) was ~ 2.4 fold higher for FGF-2 expanded MSCs, with only ~25% of the total amount generated being retained within the constructs. Further studies are required to investigate whether IFP derived MSCs have a diminished capacity to synthesise other matrix components important in the aggregation, assembly and retention of proteoglycans. In conclusion, expanding MSCs in the presence of FGF-2 rapidly accelerates chondrogenesis in 3D agarose cultures resulting in superior mechanical functionality.

Controlled release of transforming growth factor-β3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells

“…A ratio of 4 ml of collagenase (750 U/ml) per gram of tissue was found to be optimal based on previous work [15][16][17][18].…”

“…Each experiment used FPSCs from different donors. Scaffolds were maintained in chemically defined chondrogenic medium (CDM), as previously described, for 28 days (at 5% O 2 and 37˚C) [15]. CDM consisted of DMEM GlutaMAX TM supplemented with penicillin (100 U/ml)-streptomycin (100 µg/ml) (both GIBCO, Biosciences, Ireland), 100 µg/ml sodium pyruvate, 40 µg/ml L-proline, 50 µg/ml L-ascorbic acid-2-phosphate, 1.5 mg/ml BSA, 1x insulin-transferrin-selenium, 100 nM dexamethasone (all from Sigma-Aldrich, Ireland) and 10 ng/ml recombinant human growth factor-β3 (TGF-β3; ProSpec-Tany TechnoGene Ltd, Israel).…”

“…Mesenchymal stem cells or multipotent stromal cells (MSCs) isolated from various tissues such as bone marrow [1][2][3][4][5][6][7], subcutaneous fat [8][9][10][11], infrapatellar fat pad (IFP) [6,[12][13][14][15][16][17][18][19][20][21] and synovium [9,[22][23][24][25][26], combined with various scaffolds and hydrogels, have been explored for tissue regeneration. The IFP is a particularly attractive source of MSCs for this type of application as it is easy to access and provides a large number of cells with the potential to generate cartilaginous tissue [12,13,20].…”

In vitro extracellular matrix accumulation of nasal and articular chondrocytes for intervertebral disc repair

“…When subconfluent (~80%), cells were trypsinised and counted using trypan blue exclusion. The population doubling time was defined as the culture expansion time divided by the number of doublings during the expansion phase (Buckley and Kelly, 2012).…”

Incorporation of TGF‐Beta 3 within Collagen–Hyaluronic Acid Scaffolds Improves their Chondrogenic Potential

“…The focus of research over the last decade has been in developing methods of incorporating these therapeutic biomolecules within biomaterials. These biomolecules include growth factors such as fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-6 (BMP-6) and transforming growth factor-beta (TGF-β) isoforms [2][3][4][5] . TGF-β is a chondro-inductive growth factor that has been shown to play a key modulatory role in mesenchymal stem cell (MSC) differentiation towards a chondrocytic lineage [6] .…”