Development of a three-dimensional transmigration assay for testing cell--polymer interactions for tissue engineering applications.

The ability of synthetic or natural scaffolds to support invasion of cells from surrounding tissue is a key parameter for tissue engineering (TE). In this study, the migration of fibroblasts, chondrocytes, and osteoblasts into biodegradable polymer scaffolds was evaluated using a novel, three-dimensional (3-D) transmigration assay. This assay is based on a cell-populated contracted collagen lattice with a biodegradable polymer scaffold implanted at the center of the collagen gel. Cell migration into the scaffolds was assessed both quantitatively and qualitatively following various time lengths in culture using image analysis. Chondrocytes, incorporated within the collagen lattice, migrated into polymer scaffolds, when cultured both statically or in a rotating bioreactor. However, the bioreactor cultures resulted in a significantly greater cell invasion as compared to static cultures. There was a cell density-dependent osteoblast migration from collagen lattice into polymer scaffold, when tested in the transmigration assay. In addition, polymer scaffolds, treated with or without recombinant human platelet-derived growth factor (rh-PDGF-BB) were evaluated for fibroblast migration. The presence of rh-PDGF-BB resulted in significantly greater fibroblast invasion as compared to untreated scaffolds. Our studies suggest that the transmigration model provides a rapid system for testing cell invasion of potential scaffolds for tissue engineering applications.     

Characterization of a macrophage-based system for studying the activiation of latent TGF-β

TGF- has been implicated in scarring and tissue fibrosis. Most cells secrete TGF- as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor- II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF- 1 activation, which could be used for screening potential anti- fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor andtransglutaminase. The activation of latent TGF- in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor- II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF- activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF- activation, thus providing a screening method for potential anti-scarring molecules.     

Differential expression and regulation of extracellular matrix-associated genes in fetal and neonatal fibroblasts

Adults and neonates heal wounds by a repair process associated with scarring in contrast to scar-free wound healing in the fetus. In the present study, human dermal fetal fibroblasts, representing the scarless phenotype, and neonatal human dermal fibroblasts, representing scar-forming phenotype, were examined for potential differences that might influence the wound healing process. Fetal fibroblasts secreted four- to tenfold more latent transforming growth factor-beta1 depending on the cell strains compared. Fetal fibroblasts also produced higher levels of collagen protein and mRNA for most types of collagen (particularly type III) as compared to neonatal cells. Interestingly, mRNA for type V collagen was significantly reduced in fetal cells. Neonatal fibroblasts expressed significantly higher levels of latent transforming growth factor-beta1 binding protein mRNA, in contrast to almost undetectable levels in fetal fibroblasts. By ligand blot analysis, the levels of insulin-like growth factor binding protein-3, a reported mediator of transforming growth factor-beta1 activity, was eightfold higher in neonatal versus fetal fibroblasts. Approximately 20 other mRNAs for various cytokines, matrix molecules and receptors were examined and found to be similar between the two cell types. The phenotypic differences described in this article may represent potentially important mechanisms to explain the differences in the quality of wound repair observed in fetal versus adult/neonatal tissues.     

Synchronization of somatic embryogenesis at high frequency using carrot suspension cultures: model systems and application in plant development

Materials and methods for the high frequency induction and synchronous somatic embryogenesis from cultured cells of higher plants are described, using carrot suspension cultures as a model system of higher plants. The following four synchronous systems of somatic embryogenesis, which were established in our laboratories, are reported: (1) Somatic embryogenesis from single cells. a) Small spherical single cells, obtained from suspension cultures in the presence of 2,4-D, zeatin and mannitol by sieving, density gradient centrifugation in Percoll solutions and manual picking up, form embryogenic cell clusters, which differentiate to embryos at high frequency, when embryogenic cell clusters are transferred to a medium lacking 2,4-D. b) Explants of hypocotyls of regenerated plantlets from somatic embryos were cultured after treatment with 2,4-D for 12--24 h, and then transferred into a fresh medium lacking 2,4-D. Single cells are released from hypocotyl explants and differentiated into embryos at high frequency. In this system, a large number of single cells and embryogenic cells can be collected. (2) Somatic embryogenesis from embryogenic cell clusters, which are obtained from suspension cultures by sieving, density gradient centrifugation in Ficoll solutions, and subsequent centrifugation at a low speed, differentiate synchronously to globular embryos at high frequency. Plantlets are formed from globular embryos. (3) Embryogenic cell clusters obtained according to the procedure described in (2) are cultured at cell densities of 2 × 10³ cell clusters ml^–1. Globular embryos differentiate to torpedo-shaped embryos and subsequently to plantlets at high frequency when they are cultured at densities below 150 globular embryos ml^–1.     

Effects of aging and xerosis on the amino acid composition of human skin

Amino acid compositions of skin samples from young and old subjects and from age-matched donors with dry skin syndrome (xerosis) were examined. The amino acid contents of the free amino acid (FAA) fraction, soluble hydrolysate (SH) fraction, and whole cell hydrolysate (WCH) were determined. The greatest differences were observed between the FAA compositions of the young and old normal subjects. Xerosis did not appear to affect the amino acid compositions of samples from young subjects as much as old subjects. Overall, the effect of aging on the amino acid contents was more pronounced than the effect of xerosis. The amino acid composition of the FAA showed a high degree of similarity to filaggrin, whereas the WCH showed a similarity to keratin.