RESUMEN
With the increase in popularity of 3D printing, an important question arises as to the equivalence between devices manufactured by standard methods vs. those presenting with identical bulk specifications, but manufactured via fused deposition modeling (FDM) printing. Using thermal imaging in conjunction with electron and atomic force microscopy, we demonstrate that large thermal gradients, whose distribution is difficult to predict, are associated with FDM printing and result in incomplete fusion and sharkskin of the printing filament. Even though these features are micro or submicron scale, and hence may not interfere with the intended function of the device, they can have a profound influence if the device comes in contact with living tissue. Dental pulp stem cells were cultured on substrates of identical dimensions, which were either printed or molded from the same PLA stock material. The cultures exhibited significant differences in plating efficiency, migration trajectory, and morphology at early times stemming from attempts by the cells to minimize cytoplasm deformation as they attempt to adhere on the printed surfaces. Even though biomineralization without dexamethasone induction was observed in all cultures at later times, different gene expression patterns were observed on the two surfaces. (Osteogenic markers were upregulated on molded substrates, while odontogenic markers were upregulated on the FDM printed surfaces.) Our results clearly indicate that the method of manufacturing is an important consideration in comparing devices, which come in contact with living tissues.
RESUMEN
Retinol and its metabolites modulate epithelial differentiation and serve as cellular UV sensors through changes in retinoid status. Of note is the dehydroretinol family which may serve functions distinct from parental retinol. This study focuses on the metabolism of this family and its potential participation in the response of normal epidermal human keratinocytes to UV irradiation. There were three findings. First, keratinocytes contain two pools of dehydroretinyl esters, one of which is shielded from UVB-, but not from UVA-induced decomposition. Second, using a novel in vitro assay we demonstrated that both UVA and UVB promote dehydroretinol biosynthesis in keratinocytes, but only UVB exposure promotes retinoid ester accretion by enhancing the activity of at least one acyl transferase. Finally, dehydroretinol sufficiency reduces UVA/B driven apoptosis more effectively than retinol sufficiency. This may in part be due to differences in the expression of Fas ligand, which we found to be upregulated by retinoic acid, but not dehydroretinoic acid. These observations implicate a role of dehydroretinol and its metabolites in UVA/B adaptation. Thus, the keratinocyte response to UV is jointly shaped by both the retinoids and dehydroretinoids.
Asunto(s)
Activación Enzimática/efectos de la radiación , Ésteres/metabolismo , Protectores contra Radiación/metabolismo , Rayos Ultravioleta , Vitamina A/análogos & derivados , Vitamina A/biosíntesis , Vitamina A/metabolismo , Aciltransferasas/metabolismo , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Digitonina/farmacología , Proteína Ligando Fas/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Protectores contra Radiación/efectos de la radiaciónRESUMEN
A combination of fused deposition modeling printing with atomic layer deposition (ALD) of titania was designed to achieve templated biomineralization and terminal odontogenic differentiation of dental pulp stem cells on three-dimensional (3D) printed polylactic acid (PLA) scaffolds. In the absence of the ALD-deposited titania coating, we had previously shown that both plating efficiency and differentiation are adversely impacted when scaffolds are produced by 3D printing rather than traditional polymer molding. These differences were removed when both printed and molded structures were coated with ALD of titania, which improved the outcomes regardless of the manufacturing method. In this case, on all titania-coated substrates, the plating efficiency increased, copious mineral deposition was observed, and RT-PCR indicated a significant upregulation of osteocalcin, a gene associated with mineral deposition. The influence of additional coatings of collagen, gelatin, or fibronectin on the ALD titania-coated and uncoated PLA-printed and molded scaffolds was also investigated. Upregulation of the odontogenic late-stage differentiation sibling protein, dentin sialoprotein, was observed on the collagen ALD-titania-coated scaffolds and to a lesser extent on the gelatin ALD-titania-coated scaffolds.
Asunto(s)
Gelatina , Andamios del Tejido , Técnicas de Cultivo de Célula , Colágeno/química , Pulpa Dental , Dentina , Poliésteres/química , Impresión Tridimensional , Andamios del Tejido/químicaRESUMEN
Transcriptional repression often depends on the action of recruited co-repressor complexes with intrinsic enzymatic activities. The composition of these complexes depends on the nicotine amide dinucleotide co-factors and is thus directly reflective of the metabolic state of the cells. This study provides evidence that an enzyme, hRoDH-E2, with cytoplasmic phosphorylated and reduced forms of NAD-dependent retinol dehydrogenase activity may function in the nucleus as a transcriptional repressor. By using the promoter of the epidermal late differentiation marker profilaggrin as a model, we show that both in vivo and in vitro the protein is recruited over the promoter. hRoDH-E2 represses profilaggrin promoter activity by altering the function of other activators, such as Sp1. The repressive function is associated with the ability of nuclear hRoDH-E2 to modulate the acetylation/deacetylation activity in the vicinity of transcription initiation site. These findings add hRoDH-E2 to the small group of metabolic enzymes, which, by being recruited over promoter regions, could directly link the cytoplasmic and nuclear functions within the cell.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Queratinocitos/enzimología , Familia de Multigenes/fisiología , Regiones Promotoras Genéticas/fisiología , Transcripción Genética/fisiología , Acetilación , Secuencia de Bases , Núcleo Celular/enzimología , Cromatina/genética , Cromatina/metabolismo , Células Epidérmicas , Proteínas Filagrina , Silenciador del Gen/fisiología , Células HeLa , Humanos , Proteínas de Filamentos Intermediarios/genética , Queratinocitos/citología , Datos de Secuencia Molecular , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Transcripción Sp1/metabolismoRESUMEN
Reversible protein acetylation modulates higher-order chromatin structure and transcription activity of the genome. The reversible acetylation is executed by the intrinsic acetylase and deacetylase activities of co-regulators associated with the regulatory regions. Compounds capable of inhibiting deacetylase activity are a powerful tool for dissecting the role of protein acetylation in gene function. The ability of the deacetylase inhibitors to preferentially affect the homeostasis of transformed cells has also prompted studies for their clinical application. We present evidence that deacetylase inhibition with trichostatin A (TSA) affects the normal epidermal tissue architecture and pattern of expression by a mechanism(s) that does not correlate directly with the hyperacetylated histone status. While promoting abnormal differentiation, TSA specifically represses transcription initiation of the differentiation marker profilaggrin. Multiple factors, among which we have identified decreased Sp1 binding, a local decrease in acetylation activity, and enhanced synthesis and recruitment of a repressor histone demethylase, alter the chromatin configuration over the promoter, ultimately blocking its activation by c-jun. As compromised profilaggrin production leads to epidermal and consequently allergic disorders, our findings emphasize the need for a detailed investigation of the role deacetylase inhibitors may play in the maintenance of epidermal homeostasis in order to optimize their clinical applicability.
Asunto(s)
Diferenciación Celular/fisiología , Epidermis/fisiología , Inhibidores de Histona Desacetilasas , Histonas/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/fisiología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Células Epidérmicas , Proteínas Filagrina , Regulación de la Expresión Génica/efectos de los fármacos , Histona Desacetilasas/fisiología , Histona Demetilasas , Homeostasis/fisiología , Humanos , Ácidos Hidroxámicos/farmacología , Proteínas de Filamentos Intermediarios/genética , Queratinocitos/citología , Masculino , Oxidorreductasas N-Desmetilantes/fisiología , Proteínas Quinasas/fisiología , Proteínas Proto-Oncogénicas c-jun/fisiologíaRESUMEN
The function of many enzymes that regulate metabolism and transcription depends critically on the nicotinamide pyridine dinucleotides. To understand the role of NAD(P)(H) in physiology and pathophysiology, it is imperative to estimate both their amount and ratios in a given cell type. In human epidermis and in cultured epidermal keratinocytes, we found that the total dinucleotide content is in the low millimolar range. The dinucleotide pattern changes during proliferation and maturation of keratinocytes in culture. Differences in the concentrations of NAD(P)(H) of 1.5- to 12-fold were observed. This resulted in alteration of the NAD(P)H/NAD(P) ratio, which could impact the differential regulation of both transcriptional and metabolic processes. In support of this notion, we provide evidence that the two-step oxidation of retinol to retinoic acid, a nuclear hormone critical for epidermal homeostasis, can be regulated by the relative physiological amounts of the pyridine dinucleotides.