Luis Freitas Mendes

Combinatorial Analysis of Growth Factors Reveals the Contribution of Bone Morphogenetic Proteins to Chondrogenic Differentiation of Human Periosteal Cells.

Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-β1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (μMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of μMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-β1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control μMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated μMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches.

Definition of a Critical Size Osteochondral Knee Defect and its Negative Effect on the Surrounding Articular Cartilage in the Rat

Summary Background Joint trauma is predisposing to the incidence of osteoarthritis (OA) of the knee. There is a limited knowledge on the impact of posttraumatic osteochondral defects on the whole joint. This study was designed to define a critical size osteochondral defect in the knee of rats and to investigate a possible association between osteochondral defects and degeneration of the surrounding joint surface. Methods Cylindrical osteochondral defects of different sizes were created in the knee joint of rats. The natural course of these lesions was studied by macroscopic observation, histology, and immunohistochemistry. Gene expression of the articular cartilage surrounding the defects in vivo and of articular chondrocytes cultured in vitro in IL1β and fibroblast growth factor 2 (FGF2) supplemented media was evaluated by quantitative polymerase chain reaction (qPCR). Results In defects of 0.9 mm diameter, spontaneous joint surface healing was observed but also upward advancing of the subchondral bone plate at 16 weeks. Larger 1.4 mm diameter defects were critical size, not resulting in successful healing at any time point. Importantly, the articular cartilage surrounding the defects expressed FGF2 and IL1β, but not ACAN and Col2. Chondrocytes cultured in IL1β and FGF2 supplemented media lost the natural fibroblast growth factor receptors – FGFr1/FGFr3 balance and showed decreased viability. Conclusions A critical size osteochondral defect was defined as 1.4 mm in diameter in rat. Subchondral bone plate advancement occured rapidly. The articular cartilage surrounding osteochondral defects showed catabolic activity with expression of IL1β, FGF2 and a disturbed FGFr1/FGFr3 balance, potentially initiating a process of early osteoarthritic disease.

Fine-tuning pro-angiogenic effects of cobalt for simultaneous enhancement of vascular endothelial growth factor secretion and implant neovascularization

Rapid neovascularization of a tissue-engineered (TE) construct by the host vasculature is quintessential to warrant effective bone regeneration. This process can be promoted through active induction of angiogenic growth factor secretion or by implementation of in vitro pre-vascularization strategies. In this study, we aimed at optimizing the pro-angiogenic effect of Cobalt (Co2+) to enhance vascular endothelial growth factor (VEGF) expression by human periosteum-derived mesenchymal stem cells (hPDCs). Simultaneously we set out to promote microvascular network formation by co-culturing with human umbilical vein endothelial cells (HUVECs). The results showed that Co2+ treatments (at 50, 100 or 150 µM) significantly upregulated in vitro VEGF expression, but inhibited hPDCs growth and HUVECs network formation in co-cultures. These inhibitory effects were mitigated at lower Co2+ concentrations (at 5, 10 or 25 µM) while VEGF expression remained significantly upregulated and further augmented in the presence of Ascorbic Acid and Dexamethasone possibly through Runx2 upregulation. The supplements also facilitated HUVECs network formation, which was dependent on the quantity and spatial distribution of collagen type-1 matrix deposited by the hPDCs. When applied to hPDCs seeded onto calcium phosphate scaffolds, the supplements significantly induced VEGF secretion in vitro, and promoted higher vascularization upon ectopic implantation in nude mice shown by an increase of CD31 positive blood vessels within the scaffolds. Our findings provided novel insights into the pleotropic effects of Co2+ on angiogenesis (i.e. promoted VEGF secretion and inhibited endothelial network formation), and showed potential to pre-condition TE constructs under one culture regime for improved implant neovascularization in vivo. STATEMENT OF SIGNIFICANT Cobalt (Co2+) is known to upregulate vascular endothelial growth factor (VEGF) secretion, however it also inhibits in vitro angiogenesis through unknown Co2+-induced events. This limits the potential of Co2+ for pro-angiogenesis of tissue engineered (TE) implants. We showed that Co2+ upregulated VEGF expression by human periosteum-derived cells (hPDCs) but reduced the cell growth, and endothelial network formation due to reduction of col-1 matrix deposition. Supplementation with Ascorbic acid and Dexamethasone concurrently improved hPDCs growth, endothelial network formation, and upregulated VEGF secretion. In vitro pre-conditioning of hPDC-seeded TE constructs with this fine-tuned medium enhanced VEGF secretion and implant neovascularization. Our study provided novel insights into the pleotropic effects of Co2+ on angiogenesis and formed the basis for improving implant neovascularization.