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.

SummaryBackgroundJoint 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.MethodsCylindrical 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).ResultsIn 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.ConclusionsA 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.

In recent years several studies have been supporting the existence of a close relationship in terms of function and progeny between Mesenchymal Stem Cells (MSCs) and Pericytes. This concept has opened new perspectives for the application of MSCs in Tissue Engineering (TE), with special interest for the pre-vascularization of cell dense constructs. In this work, cell sheet technology was used to create a scaffold-free construct composed of osteogenic, endothelial and perivascular-like (CD146+) cells for improved in vivo vessel formation, maturation and stability. The CD146 pericyte-associated phenotype was induced from human bone marrow mesenchymal stem cells (hBMSCs) by the supplementation of standard culture medium with TGF-β1. Co-cultured cell sheets were obtained by culturing perivascular-like (CD146+) cells and human umbilical vein endothelial cells (HUVECs) on an hBMSCs monolayer maintained in osteogenic medium for 7 days. The perivascular-like (CD146+) cells and the HUVECs migrated and organized over the collagen-rich osteogenic cell sheet, suggesting the existence of cross-talk involving the co-cultured cell types. Furthermore the presence of that particular ECM produced by the osteoblastic cells was shown to be the key regulator for the singular observed organization. The osteogenic and angiogenic character of the proposed constructs was assessed in vivo. Immunohistochemistry analysis of the explants revealed the integration of HUVECs with the host vasculature as well as the osteogenic potential of the created construct, by the expression of osteocalcin. Additionally, the analysis of the diameter of human CD146 positive blood vessels showed a higher mean vessel diameter for the co-cultured cell sheet condition, reinforcing the advantage of the proposed model regarding blood vessels maturation and stability and for the in vitro pre-vascularization of TE constructs.

Cobalt (Co) is known to upregulate vascular endothelial growth factor (VEGF) secretion, however it also inhibits in vitro angiogenesis through unknown Co-induced events. This limits the potential of Co for pro-angiogenesis of tissue engineered (TE) implants. We showed that Co 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 Co on angiogenesis and formed the basis for improving implant neovascularization.

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