Background We aimed at determining whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be employed in postnatal sternal repair. Methods Leporine aMSCs were isolated, identified, transfected with green fluorescent protein (GFP), expanded, and seeded onto biodegradable electrospun nanofibrous scaffolds (n=6). Constructs were dynamically maintained in an osteogenic medium and equally divided into two groups with respect to time in vitro, namely 14.6 or 33.9 weeks. They were then used to repair full thickness sternal defects spanning 2–3 intercostal spaces in allogeneic kits (n=6). Grafts were submitted to multiple analyses 2 months thereafter. Results Chest roentgenograms showed defect closure in all animals, confirmed at necropsy. Graft density as assessed by micro-CT scans increased significantly in vivo, yet there were no differences in mineralization by extracellular calcium measurements pre- and post-implantation. There was a borderline increase in alkaline phosphatase activity in vivo, suggesting ongoing graft remodeling. Histologically, implants contained GFP-positive cells and few mononuclear infiltrates. There were no differences between the two construct groups in any comparison. Conclusions Engineered osseous grafts derived from amniotic mesenchymal stem cells may become a viable alternative for sternal repair. The amniotic fluid can be a practical cell source for engineered chest wall reconstruction.
We sought to compare engineered cartilaginous constructs derived from different perinatal mesenchymal progenitor cell (MPC) sources. Ovine MPCs isolated from amniotic fluid (AF, n = 8), neonatal bone marrow (BM, n = 6), and preterm umbilical cord blood (CB, n = 12) were expanded and comparably seeded onto synthetic scaffolds. Constructs were maintained in chondrogenic media containing transforming growth factor-beta. After 12-15 weeks, specimens were compared with native fetal hyaline and elastic cartilage by gross inspection, histology, immunohistochemistry, and quantitative extracellular matrix (ECM) assays. MPCs from AF proliferated significantly faster ex vivo when compared to MPCs from the other sources. Chondrogenic differentiation was evident in all groups, as shown by toluidine blue staining and expression of aggrecan, cartilage proteoglycan link protein, and collagen type II. Quantitatively, all engineered specimens had significantly lower levels of glycosaminoglycans than native hyaline cartilage. Elastin levels in AF-based constructs (156.0 +/- 120.4 microg/mg) were comparable to that of native elastic cartilage (235.8 +/- 54.2 microg/mg), both of which were significantly higher than in BM- and CB-based specimens. We conclude that the ECM profile of cartilage engineered from perinatal MPCs is highly dependent on cell source. ECM peculiarities should be considered when designing the optimal cartilaginous bioprosthesis for use in perinatal surgical reconstruction.
Diverse progenitor cell populations, including mesenchymal, hematopoietic, trophoblastic, and possibly more primitive stem cells can be isolated from the amniotic fluid and the placenta. At least some of the amniotic and placental cells share a common origin, namely the inner cell mass of the morula. Indeed, most types of progenitor cells that can be isolated from these two sources share many characteristics. This unit will focus solely on the mesenchymal stem cells, the most abundant progenitor cell population found therein and, unlike some of the other stem cell types, present all through gestation. Protocols for isolation, expansion, freezing, and thawing of these cells are presented. Preference is given to the simplest methods available for any given procedure. Curr. Protoc. Stem Cell Biol. 1:1E.2.1‐1E.2.12. © 2007 by John Wiley & Sons, Inc.
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