Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures

Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21 d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E2 as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation

Morphological study of bone ingrowth in a high porous metal (TMT). A case series of human implants

Biomaterial porosity is considered one of most important proprieties required to obtain fixation of bone ingrowth and ongrowth in prostheses. Since 1998 in the USA and from 2001 in Europe a new highly porous biomaterial, Trabecular Metal Technology (TMT, ©Zimmer, USA) has been used in orthopaedic surgery. It is composed of approximately 99% tantalum and 1% vitreous carbon, by weight. Animal studies have shown that TMT supports rapid and extensive bone ingrowth. This porous metal offers several advantages over conventional materials as the high regular porosity that is considered one of most important properties in bone ingrowth and ongrowth and offers high biocompatibility and osteoconductivity, also. In bibliography there are studies that demonstrated the biomechanical properties of tantalum that are sufficient to withstand physiological load. In this study we evaluate the short-term morphological findings of TMT implanted in patients with osteonecrosis of a femoral head. Bone lamellae and cells with a lot of vascular structures are present. In particular the mineralization begin near on surface of tantalum lacunae. The bone penetrated the porous metal and many characteristics of good bio-integration were evident such as a vascularization inside the TMT, the formation of new bone, the absence of fracture and signs of implant metallosis. The presence of peri-implant medullary cisternae confirmed the functional sites of new bone formation

Method for Studying ECM Expression: In Situ RT-PCR

The gene expression of the extracellular matrix macromolecules is critical in the analysis of various pathologies. The use of a RT-PCR directly on a fixed tissue enables the recognition of the real expressing cells for any ECM molecules together with the tissue localization. The method here described is easy to perform using the same material as for common immunostaining and the same primers used for quantitative RT-PCR. Moreover, the used primers, designed with a final amplicon that spans the exon-exon junction, allow to detect the cDNA but not the gDNA sequences