Methods
We investigated growth, differentiation and extracellular matrix (ECM) protein production of human adipose-derived mesenchymal stem/stromal cells (MSCs), cultured in 5% human platelet lysate (PL) and seeded on three-dimensional polycaprolactone (PCL) scaffolds, in response to the connective-tissue related ligands fibroblast growth factor 2 (basic) (FGF2) and growth and differentiation factor-5 (GDF5). Phenotypic alterations of MSCs under different biological conditions were examined using cell viability assays, real time qPCR analysis of total RNA, as well as immunofluorescence microscopy.
Purpose
The poor healing potential of intra-articular ligament injuries drives a need for the development of novel, viable 'neo-ligament' alternatives. Ex vivo approaches combining stem cell engineering, 3-dimensional biocompatible scaffold design and enhancement of biological and biomechanical functionality via the introduction of key growth factors and morphogens, represent a promising solution to ligament regeneration.
Results
Phenotypic conversion of MSCs into ECM producing fibroblastic cells proceeds spontaneously in the presence of human platelet lysate. Administration of FGF2 and/or GDF5 enhances production of mRNAs for several ECM proteins including Collagen types I and III, as well as Tenomodulin (e.g., COL1A1, TNMD), but not Tenascin-C (TNC). Differences in the in situ deposition of ECM proteins Collagen type III and Tenascin-C were validated by immunofluorescence microscopy. Summary: Treatment of MSCs with FGF2 and GDF5 was not synergistic and occasionally antagonistic for ECM production. Our results suggest that GDF5 alone enhances the conversion of MSCs to fibroblastic cells possessing a phenotype consistent with that of connective-tissue fibroblasts.