Materials chemistries for hydrogel scaffolds that are capable of programming temporal (4D) attributes of cellular decision‐making in supported 3D microcultures are described. The scaffolds are fabricated using direct‐ink writing (DIW)—a 3D‐printing technique using extrusion to pattern scaffolds at biologically relevant diameters (≤ 100 µm). Herein, DIW is exploited to variously incorporate a rheological nanoclay, Laponite XLG (LAP), into 2‐hydroxyethyl methacrylate (HEMA)‐based hydrogels—printing the LAP–HEMA (LH) composites as functional modifiers within otherwise unmodified 2D and 3D HEMA microstructures. The nanoclay‐modified domains, when tested as thin films, require no activating (e.g., protein) treatments to promote robust growth compliances that direct the spatial attachment of fibroblast (3T3) and preosteoblast (E1) cells, fostering for the latter a capacity to direct long‐term osteodifferentiation. Cell‐to‐gel interfacial morphologies and cellular motility are analyzed with spatial light interference microscopy (SLIM). Through combination of HEMA and LH gels, high‐resolution DIW of a nanocomposite ink (UniH) that translates organizationally dynamic attributes seen with 2D gels into dentition‐mimetic 3D scaffolds is demonstrated. These analyses confirm that the underlying materials chemistry and geometry of hydrogel nanocomposites are capable of directing cellular attachment and temporal development within 3D microcultures—a useful material system for the 4D patterning of hydrogel scaffolds.
Somatic and testis-specific cytochromes c were localized ultrastructurally in the seminiferous epithelium by immunocytochemistry using monospecific antibodies. Cytochrome cS was lost from the mitochondria as spermatogenesis advanced, while there was a relative increase in cytochrome cT during the zygotene-to-pachytene transition; this was in agreement with other studies that have suggested activation of the cytochrome cT gene during prophase of the first meiotic division. Cytochrome cT was highly concentrated in mitochondria that were being degraded within cytoplasmic lobes of spermatids and in residual bodies that were phagocytized by Sertoli cells. The two isoforms were found to coexist within the same mitochondrion during the transitional period from cytochrome cS to cytochrome cT predominance. In addition, both cytochromes c were present in the chromatoid bodies of spermatocytes and round spermatids; this suggests that the chromatoid body may be involved in the storage of these isozymes and possibly in their differential expression within germ cell mitochondria. Apocytochrome c was concentrated in mitochondria and chromatoid bodies of the germ cells and also scattered in the cytoplasm. The presence of the holoprotein and apoprotein immunoprobes within the chromatoid bodies of spermatocytes and spermatids was an interesting observation that raises questions regarding the precise location of the synthesis of cytochromes c in spermatogenic cells.
The cyanobacterial toxin microcystin-LR (MCLR) is a potent inhibitor of protein phosphatases 1 and 2A, and is selectively toxic to the liver in vivo and to isolated hepatocytes in vitro. This selectivity is believed to be due to toxin uptake via bile acid carriers. We investigated at the light and ultrastructural levels the effects of high concentrations of MCLR and long incubation times to determine in vitro whether fibroblasts and kidney cells (non-target cells) respond in the same manner as do hepatocytes (target cells) at low concentrations and short incubation times. Cultured rat skin fibroblasts (ATCC 1213) and rat kidney epithelial cells (ATCC 1571) were incubated with with MCLR at 133 microM for 1-24 hr. Lesions in these cells were compared with those in cultured hepatocytes incubated MCLR at 13.3 microM from 1 to 32 min. Lesions in hepatocytes, kidney cells, and fibroblasts were noted at 4 min, 1 hr, and 8 hr, respectively, after initial exposure to MCLR. Lesions in all three cell types progressed and included plasma membrane blebbing, loss of cell-to-cell contact, clumping and rounding of cells, cytoplasmic vacuolization, and redistribution of cytoplasmic organelles. Loss of microvilli, whorling of rough endoplasmic reticulum, dense staining and dilated cristae in mitochondria, and pinching off of membrane blebs were noted only in hepatocytes. Nuclear changes typical of apoptosis were observed only in fibroblasts and kidney cells. Similarities in responses of different cell types to MCLR exposure probably reflect a common biochemical mechanism of action, i.e., inhibition of protein phosphatases 1 and 2A as described by others.(ABSTRACT TRUNCATED AT 250 WORDS)
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