Fibroblast Growth Factor-2 Primes Human Mesenchymal Stem Cells for Enhanced Chondrogenesis

Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG

A cartilage tissue engineering approach combining starch-polycaprolactone fibre mesh scaffolds with bovine articular chondrocytes

In the present work we originally tested the suitability of corn starch-polycaprolactone (SPCL) scaffolds for pursuing a cartilage tissue engineering approach. Bovine articular chondrocytes were seeded on SPCL scaffolds under dynamic conditions using spinner flasks (total of 4 scaffolds per spinner flask using cell suspensions of 0.5×106 cells/ml) and cultured under orbital agitation for a total of 6 weeks. Poly(glycolic acid) (PGA) non-woven scaffolds and bovine native articular cartilage were used as standard controls for the conducted experiments. PGA is a kind of standard in tissue engineering approaches and it was used as a control in that sense. The tissue engineered constructs were characterized at different time periods by scanning electron microscopy (SEM), hematoxylin-eosin (H&E) and toluidine blue stainings, immunolocalisation of collagen types I and II, and dimethylmethylene blue (DMB) assay for glycosaminoglycans (GAG) quantification assay. SEM results for SPCL constructs showed that the chondrocytes presented normal morphological features, with extensive cells presence at the surface of the support structures, and penetrating the scaffolds pores. These observations were further corroborated by H&E staining. Toluidine blue and immunohistochemistry exhibited extracellular matrix deposition throughout the 3D structure. Glycosaminoglycans, and collagen types I and II were detected. However, stronger staining for collagen type II was observed when compared to collagen type I. The PGA constructs presented similar features toSPCLat the end of the 6 weeks. PGA constructs exhibited higher amounts of matrix glycosaminoglycans when compared to the SPCL scaffolds. However, we also observed a lack of tissue in the central area of the PGA scaffolds. Reasons for these occurrences may include inefficient cells penetration, necrosis due to high cell densities, or necrosis related with acidic by-products degradation. Such situation was not detected in the SPCL scaffolds, indicating the much better biocompatibility of the starch based scaffolds

Bone tissue engineering by using a combination of polymer/Bioglass composites with human adipose-derived stem cells.

Translational research in bone tissue engineering is essential for “bench to bedside” patient benefit. However, the ideal combination of stem cells and biomaterial scaffolds for bone repair/regeneration is still unclear. The aim of this study is to investigate the osteogenic capacity of a combination of poly(DL-lactic acid) (PDLLA) porous foams containing 5 wt% and 40 wt% of Bioglass particles with human adipose-derived stem cells (ADSCs) in vitro and in vivo. Live/dead fluorescent markers, confocal microscopy and scanning electron microscopy showed that PDLLA/Bioglass porous scaffolds supported ADSC attachment, growth and osteogenic differentiation, as confirmed by enhanced alkaline phosphatase (ALP) activity. Higher Bioglass content of the PDLLA foams increased ALP activity compared with the PDLLA only group. Extracellular matrix deposition after 8 weeks in the in vitro cultures was evident by Alcian blue/Sirius red staining. In vivo bone formation was assessed by using scaffold/ADSC constructs in diffusion chambers transplanted intraperitoneally into nude mice and recovered after 8 weeks. Histological and immunohistochemical assays indicated significant new bone formation in the 40 wt% and 5 wt% Bioglass constructs compared with the PDLLA only group. Thus, the combination of a well-developed biodegradable bioactive porous PDLLA/Bioglass composite scaffold with a high-potential stem cell source (human ADSCs) could be a promising approach for bone regeneration in a clinical setting