Specific biochemical and physical cues in tissue extracellular matrices play a critical role in regulating cellular growth processes and their fate. We report initial responses of bone stem cells induced by collagen-derived DGEA-peptides on nanofibrous M13 phage tissue matrices. We constructed genetically engineered M13 phage with DGEA-peptide displayed in high density on the major coat proteins and biomimetic nanofibrous tissue-like matrices in two and three dimensions. We investigated the effects of biochemical cues, specifically DGEA-peptides on preosteoblast (MC3T3) morphologies. The preosteoblasts grown on the top of the DGEA-incorporated phage matrices exhibited significant outgrown morphology with early bone cell marker protein expression. Through soluble peptide competition assays and control experiments, we verified that the observed cellular morphologies and osteogenic protein marker expression were specifically caused by the DGEA-peptides. We confirmed that the outgrown morphologies are linked with the early phase of osteogenic protein expression through mRNA quantification and bone cell protein marker expression. Additionally, we demonstrated that the phage-based tissue matrix systems could work as a good cell culture platform to investigate the specific effect of biochemical cues, which can be tuned precisely at a single amino acid level with little change in other physical and chemical properties of the environment. Our study advances the understanding of osteogenic differentiation and our phage-based tissue matrices have the potential for future bone regeneration therapy and systemic investigation of specific cellular responses to biochemical ligand stimulation.
Background & AimsIntestinal microfold (M) cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer’s patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs), and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting.MethodsHuman intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium.ResultsFunctional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2) in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells.ConclusionsHuman intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an in vitro setting. We anticipate that this model can be used to generate large numbers of M cells for further functional studies of these key cells of intestinal immune induction and their impact on controlling enteric pathogens and the intestinal microbiome.
Controlling cellular alignment is critical in engineering intestines with desired structure and function. Although previous studies have examined the directional alignment of cells on the surface (x-y plane) of parallel fibers, quantitative analysis of the cellular alignment inside implanted scaffolds with oriented fibers has not been reported. This study examined the cellular alignment in the x-z and y-z planes of scaffolds made with two layers of orthogonally oriented fibers. The cellular orientation inside implanted scaffolds was evaluated with immunofluorescence. Quantitative analysis of coherency between cell orientation and fiber direction confirmed that cells aligned along the fibers not only on the surface (x-y plane) but also inside the scaffolds (x-z & y-z planes). Our study demonstrated that two layers of orthogonally aligned scaffolds can generate the histological organization of cells similar to that of intestinal circular and longitudinal smooth muscle.
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