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Tissue‐engineered skin constructs, including bi‐layered living cellular constructs (BLCC) used in the treatment of chronic wounds, are structurally/functionally complex. While some work has been performed to understand their mechanisms, the totality of how BLCC may function in wound healing remains unknown. To this end, we have developed a delayed wound healing model to test BLCC cellular and molecular mechanisms of action. Diabetes was chemically‐induced using alloxan in Yucatan miniature pigs, and full‐thickness wounds were generated on their dorsum. These wounds were either allowed to heal by secondary intention alone (control) or treated with a single or multiple treatments of a porcine autologous BLCC. Results indicated a single treatment with porcine BLCC resulted in statistically significant wound healing at day 17, while four treatments resulted in statistically significant healing on days 10, 13, and 17 compared to control. Statistically accelerated wound closure was driven by re‐epithelialisation rather than contraction or granulation. This porcine diabetic model and the use of a porcine BLCC allowed evaluation of healing responses in vivo without the complications typically seen with either xenogenic responses of human/animal systems or the use of immune compromised animals, expanding the knowledge base around how BLCC may impact chronic wounds. [ABSTRACT FROM AUTHOR]

Protein-rich fractions inhibitory for isolated ureteric bud (UB) growth were separated from a conditioned medium secreted by cells derived from the metanephric mesenchyme (MM). Elution profiles and immunoblotting indicated the presence of members of the transforming growth factor-[beta] (TGF-[beta]) superfamily. Treatment of cultured whole embryonic kidney with BMP2, BMP4, activin, or TGF-[beta]1 leads to statistically significant differences in the overall size of the kidney, the number of UB branches, the length and angle of the branches, as well as in the thickness of the UB stalks. Thus, the pattern of the ureteric tree is altered. LIF, however, appeared to have only minimal effect on growth and development of the whole embryonic kidney in organ culture. The factors all directly inhibited, in a concentration-dependent fashion, the growth and branching of the isolated UB, albeit to different extents. Antagonists of some of these factors reduced their inhibitory effect. Detailed examination of TGF-[beta]1-treated UBs revealed only a slight increase in the amount of apoptosis in tips by TUNEL staining, but diminished proliferation throughout by Ki67 staining. These data suggest an important direct modulatory role for BMP2, BMP4, LIF, TGF-[beta]1, and activin (as well as their antagonists) on growth and branching of the UB, possibly in shaping the growing UB by playing a role in determining the number of branches, as well as where and how the branches occur. In support of this notion, UBs cultured in the presence of fibroblast growth factor 7 (FGF7), which induces the formation of globular structures with little distinction between the stalk and ampullae [Mech. Dev. 109 (2001) 123], and TGF-13 superfamily members lead to the formation of UBs with clear stalks and ampullae. This indicates that positive (i.e., growth and branch promoting) and negative (i.e., growth and branch inhibiting) modulators of UB morphogenesis can cooperate in the formation of slender arborized UB structures similar to those observed in the intact developing kidney or in whole embryonic kidney organ culture. Finally, purification data also indicate the presence of an as yet unidentified soluble non-heparin-binding activity modulating UB growth and branching. The data suggest how contributions of positive and negative growth factors can together (perhaps as local bipolar morphogenetic gradients existing within the mesenchyme) modulate the vectoral arborization pattern of the UB and shape branches as they develop, thereby regulating both nephron number and tubule/duct caliber. We suggest that TGF-[beta]-like molecules and other non-heparin-binding inhibitory factors can, in the appropriate matrix context, facilitate 'braking' of the branching program as the UB shifts from a rapid branching stage (governed by a feed-forward mechanism) to a stage where branching slows down (negative feedback) and eventually stops. Keywords: Branching morphogenesis; Kidney development; Ureteric bud; Fibroblast growth factors; Rat; Transforming growth factor-[beta]; Bone morphogenetic proteins; Activin; LIF

An experiment was performed to test whether the function of the renal anion exchanger protein, band 3, could be altered by the lipids contained in the apical and basolateral membranes of the epithelia. Using short chain sphingolipid analogues, it was discovered that sphingomyelin was targeted to the basolateral domain in intercalated cell line and ganglioside GM1 was found in the apical membrane. In conclusion, lipid structure influence the packing of transmembrane domains of band 3.

The ureteric bud cell lines from embryonic mice transgenic for SV40 T-antigen was isolated to determine the molecular mechanisms involved in the induction of metanephrogenic mesenchyme by the ureteric bud. The results suggested that the cells may serve as substitutes for the ureteric bud because they cause the not only the growth of the mesenchyme but also its differentiation into epithelia that produces that tubules.

Experimental studies of the function of Wnt genes in renal epithelial morphogenesis using metanephric mesenchymal cells of rat embryos reveal that the Wnt-1 gene encoding a embryonic spinal-expressed glycoprotein initiates nephron-inducing activity in the fibroblasts. Mesenchymal cells cocultured with the cells expressing exogenous Wnt-1 undergo differentiation to glomerular and renal tubular epithelia. Thus, embryonic spinal cord functions as an activator of nephrogenesis, through the expression of Wnt-1 protein.

Central to the process of epithelial organogenesis is branching morphogenesis into tubules and ducts. In the kidney, this can be modeled by a very simple system consisting of isolated ureteric bud (UB) cells, which undergo branching morphogenesis in response to soluble factors present in the conditioned medium of a metanephric mesenchyme cell line. By employing a targeted screen to identify transcription factors involved early in the morphogenetic program leading to UB branching, we identified the mammalian ortholog of Timeless (mTim) as a potential immediate early gene (IEG) important in this process. In the embryo, mTim was found to be expressed in patterns very suggestive of a role in epithelial organogenesis with high levels of expression in the developing lung, liver, and kidney, as well as neuroepithelium. In the embryonic kidney, the expression of mTim was maximal in regions of active UB branching, and a shift from the large isoform of mTim to a smaller isoform occurred as the kidney developed. Selective down-regulation of mTim resulted in profound inhibition of embryonic kidney growth and UB morphogenesis in organ culture. A direct effect on the branching UB was supported by the observation that down-regulation of mTim in the isolated UB (cultured in the absence of mesenchyme) resulted in marked inhibition of morphogenesis, suggesting a key role for Tim in the epithelial cell morphogenetic pathway leading to the formation of branching tubules.