Amniotic membrane induces epithelialization in massive posttraumatic wounds.

Large-surface or deep wounds often become senescent in the inflammatory or proliferation stages and cannot progress to reepithelialization. This failure makes intervention necessary to provide the final sealing epithelial layer. The best current treatment is autologous skin graft, although there are other choices such as allogenic or autologous skin substitutes and synthetic dressings. Amniotic membrane (AM) is a tissue of interest as a biological dressing due to its biological properties and immunologic characteristics. It has low immunogenicity and beneficial reepithelialization effects, with antiinflammatory, antifibrotic, antimicrobial, and nontumorigenic properties. These properties are related to its capacity to synthesize and release cytokines and growth factors. We report the use of AM as a wound dressing in two patients with large and deep traumatic wounds. Negative pressure wound therapy followed by AM application was capable of restoring skin integrity avoiding the need for skin graft reconstruction. AM induced the formation of a well-structured epidermis. To understand this effect, we designed some assays on human keratinocyte-derived HaCaT cells. AM treatment of HaCaT induced ERK1/2 and SAP/JNK kinases phosphorylation and c-jun expression, a gene critical for keratinocytes migration; however, it did not affect cell cycle distribution. These data suggest that AM substantially modifies the behavior of keratinocytes in chronic wounds, thereby allowing effective reepithelialization.

In vitro determination of zinc dialyzability from duplicate hospital meals: influence of other nutrients.

OBJECTIVES: We determined zinc (Zn) dialyzability as an indicator of Zn bioavailability in the in vitro gastrointestinal digests of 108 duplicate meals. The interaction exerted by levels of 51 nutrients and energy on total and dialyzable Zn fractions and on Zn dialyzability was also assessed. METHODS: Total and dialyzable Zn levels were measured by flame atomic absorption spectrometry. The Zn dialyzability is expressed as the percentage of dialyzed Zn in relation to the total Zn content in duplicate meals (dialysis Zn percentages). RESULTS: The mean total and dialyzable Zn fractions and Zn dialyzability were 2.180 +/- 1.806 mg, 0.478 +/- 0.556 mg, and 25.23 +/- 15.05%, respectively. The dialysis Zn levels increased significantly with total Zn content in duplicate meals (P < 0.001, r = 0.690). Total and dialyzable Zn fractions found in breakfasts were significantly lower (P < 0.001). The Zn dialyzability was significantly correlated only with total chromium contents, dialyzable copper (Cu) and manganese (Mn) fractions, and Cu and Mn dialyzabilities (P < 0.05). Zn dialyzabilities decreased significantly with increased daily Zn intakes (P = 0.010, r = 0.423). The total Zn supply by meals was directly and significantly (P < 0.001) correlated with their macronutrient contents (carbohydrates, protein, and fiber). The mean daily Zn intake determined was 6.541 mg. CONCLUSION: Duplicate diets studied are moderately low Zn-bioavailability diets. The protein and derivate amino acids act as formers of soluble complexes with Zn in the gastrointestinal tract. Daily Zn levels supplied by hospital meals are low.

Total and dialyzable levels of manganese from duplicate meals and influence of other nutrients: Estimation of daily dietary intake.

Both total and dialyzable Mn levels were determined in 108 duplicate meals during 36 consecutive days. Both mineral fractions were measured by a graphite furnace atomic absorption spectrometry (GFAAS) method previously optimized. A total mean Mn fraction of 1.03±0.49mg was found in the meals. The Mn supplied by the meals is directly and significantly (p<0.001) correlated with macronutrient content (carbohydrates, fibre and protein). The mean Mn fraction dialyzed through the dialysis membrane was 0.23±0.17mg (22.0±8.93% as bioaccessible fraction). The total and dialyzable Mn fractions found for breakfasts were significantly lower (p<0.001). Nevertheless, the Mn bioavailabilities expressed as the percentage of dialyzable element, were not significantly different among the three primary meals (breakfast, lunch and dinner). A significant correlation between the total and the dialyzable fraction of Mn in meals was found (p<0.001, r=0.78, r(2)=0.61). The dialyzed element fractions present in meals were significantly correlated mainly with carbohydrates, protein and several amino acid levels (p <0.01). Foods with higher carbohydrate and therefore energy contents, e.g. cereals, legumes, vegetables and fruits, would be primary sources of bioaccessible Mn in the diet. The bioaccessibility of Mn was only significant influenced by energy, carbohydrates and Se levels present in meals. The mean Mn daily dietary intake (DDI) was 3.05±0.61mgday(-1).

Magnesium and calcium contents in foods from SE Spain: influencing factors and estimation of daily dietary intakes.

The magnesium and calcium content (fresh weight basis) of 243 food, 69 beverages and 11 potable water samples were determined using flame atomic absorption spectrometry. Analyses of NIST and CBR-CEC reference materials demonstrated the reliability and accuracy of this technique. The highest magnesium and calcium levels corresponded to molluscs and crustacea, and dairy products, respectively. Magnesium concentrations found in different food groups were significantly and linearly related with corresponding calcium levels (P<0.001) with the exception of fish products. Significant linear relationships among magnesium and calcium concentrations, and fiber content for cereals, legumes and dry-fruits were found (P<0.001); however, total fat contents of considered foods were not related (P>0.05) with levels of elements considered. Significant linear relationships among magnesium and calcium concentrations in cereals, legumes and fruits, and protein content were found (P<0.001). In meat and by-products, magnesium concentrations found in organ meats were significantly lower (P<0.01); for calcium, levels measured in sausages were significantly higher (P<0.001) than those found in meats and organ meats. In fish products, magnesium levels in molluscs and crustacea were significantly higher than those measured in fish and cephalopoda (P<0.001); for calcium, concentrations in cephalopoda were significantly lower (P<0.001). Mean magnesium and calcium concentrations analyzed in cheese were statistically higher than those determined in other dairy products (P<0.01). In alcoholic drinks, magnesium and calcium levels measured in distilled beverages (whisky, gin, rum, brandy and alcoholic liquors) were statistically lower than those found in fermented types (wine and beer) (P<0.01). The daily intake of magnesium and calcium in the Spanish diet was 366.1 and 1266.6 mg/day per person, respectively. Results revealed that the intake of analyzed elements was similar to recommended dietary allowances.

Autocrine TGF-ß induces epithelial to mesenchymal transition in human amniotic epithelial cells.

Human amniotic epithelial cells (hAECs) have been the object of intense research due to their potential therapeutic use. In this paper, we present molecular evidence of a bona fide epithelial to mesenchymal transition (EMT) undergone by hAECs. Amniotic membrane (AM)-derived hAECs showed the presence of typical epithelial markers such as E-cadherin and cytokeratins. hAECs in culture, however, underwent morphological changes acquiring a mesenchymal shape. Epithelial cell markers were lost and typical mesenchymal markers, such as vimentin and α-SMA, appeared. Several genes associated with EMT, such as SNAI1, MMP9, PAI1, or ACTA2, increased their expression. The expression of the transcription activators KLF4 or MTA3 was consistent with the downregulation of CDH1. We have shown that hAECs undergo EMT due to the autocrine production of TGF-ß. Furthermore, the addition of the TGF-ß receptor I (ALK5) inhibitor SB-431542 or TGF-ß neutralizing antibody to hAECs prevented EMT and preserved the hAECs' epithelial phenotype. Altogether, these results suggest that cultured hAECs undergo EMT through the autocrine production of TGF-ß.

Isolation and characterization of mesenchymal stem cells from the fat layer on the density gradient separated bone marrow.

The density gradient centrifugation method was originally designed for the isolation of mononuclear peripheral blood cells and rapidly adapted to fractionate bone marrow (BM) cells. This method involves the use of gradient density solutions with low viscosity and low osmotic pressure that allows erythrocytes and more mature cells gravitate to the bottom at a density fraction superior to 1.080 g/dL; mononuclear cells (MNCs) held in the plasma-solution to interphase at a density between 1.053 and 1.073 g/dL; plasma, dilution medium and anticoagulant to occupy a density less than 1.050 g/dL and the fat cells to float due to their very low density. BM-mesenchymal stem cells (MSCs) are usually obtained after the separation and cultures of BM-MNCs from the plasma-solution interphase, which is traditionally considered the only source of progenitor cells (hematopoietic and nonhematopoietic). In this study evidences that MSCs could be isolated from the very low-density cells of the fat layer are presented. In addition, we demonstrated that the MSCs obtained from these cells have similar immunophenotypic characteristics, and similar proliferative and differentiation potential to those obtained from the MNCs at plasma-solution interphase. The method represents a simple and cost effective way to increase the MSCs yield from each BM donor, without the need to look for other sources, additional manipulation of cells, and risks of contamination or disturbances of the potential of differentiation. These cells might serve as a complementary source of MSCs to facilitate preclinical and clinical application in tissue engineering and cell therapy.

Dialysability of magnesium and calcium from hospital duplicate meals: influence exerted by other elements.

Total and dialysable magnesium and calcium levels and corresponding dialysabilities were measured in duplicate meals (n = 108) during 36 consecutive days. The interaction exerted by other nutrients and energy on them was also performed. Total mean magnesium and calcium fractions of 113.9 +/- 98.3 and 337.2 +/- 278.9 mg/meal respectively, were found. The Mg and Ca levels supplied by meals are positively (p < 0.05) correlated with macronutrient contents (carbohydrates and proteins). The mean dialysable Mg and Ca fractions were 56.9 +/- 36.3 and 127.4 +/- 112.3 mg/meal (50.4 +/- 13.2 and 37.8 +/- 10.7% as dialysabilities, respectively). Total Mg and Ca levels are significantly correlated with corresponding element dialysabilities (p < 0.05). For both minerals, significant correlations between their total and dialysable fractions and between their dialysable level and dialysabilities were noted (p < 0.01). The mean Mg and Ca daily dietary intakes (DDI) were 341.7 +/- 68.0 and 1,011.6 +/- 424.4 mg/day, respectively. For Ca and Mg the existence of similarities in their behaviour in meals and absorptive processes has been found. Duplicate meals with raw vegetables are good sources of bioaccessible Mg. High Ca dialysability has been found in the analysed meals. The fish and products constitute a good source of bioaccessible Ca. Mg, Ca, zinc, and chromium levels enhanced significantly the Mg dialysability. The Ca dialysability rose significantly with dialysable Ca and chromium fractions (p < 0.05).

Iron dialyzability from hospital duplicate meals: daily intake.

Both total and dialyzable iron levels and corresponding dialyzability were determined in 108 duplicate meals during 36 consecutive days. Total mean iron fraction of 5.90 +/- 4.97 mg was found in the meals. The iron supplied by the meals is directly and significantly (p < 0.05) correlated with macromicronutrient content (carbohydrates, fiber, and protein). The mean iron dialyzability (4.81 +/- 3.25%) was low and not significantly different among the three primary meals (breakfast, lunch, and dinner). Significant interactions of several minerals on iron levels were found (p < 0.05). Iron dialyzability was only statistically influenced by zinc dialyzability in meals (p < 0.05). The dialyzed iron fraction present in meals was significantly correlated with protein and ascorbic acid levels (p < 0.01). The mean iron daily dietary intake was 17.7 +/- 6.91 mg. The hospital meals provided enough iron. Foods of animal origin are primary sources of iron in diet.

Determination of daily dietary intake of chromium by duplicate diet sampling: in vitro availability study.

Intake of chromium was estimated using a duplicate diet sampling method of 108 meals (36 breakfasts, 36 lunches and 36 dinners) from the restaurant of the Hospital of Motril (S.E. Spain), corresponding to 36 consecutive days. Total and dialyzable Cr levels were measured by a validated electro-thermal atomic absorption spectrometry (ETAAS) method. A mean Cr fraction of 26 +/- 12 microg meal (-1) was found. The Cr uptake from meals was directly and significantly (p < 0.001) correlated with their macronutrient (carbohydrates, fibre and protein) content. Cereals and cereal by-products, legumes, dry fruits, meat, potatoes, dairy products and seafood are the primary sources of Cr. The mean Cr fraction dialyzed through dialysis tubing was 1.2 +/- 1.1 microg meal(-1) (4.6 +/- 3.8% as mean Cr dialysability). Cr intake for breakfasts was significantly lower (p < 0.001). A correlation between the logarithmic data of total and dialyzable fraction of Cr in meals (p = 0.020) was found and dialysis ratio enhancement and, therefore, bioavailability increased with total Cr. The dialysed element content present in meals was significantly correlated with fibre, protein, Fe, Na, I, F, sodium, ascorbic acid and vitamin A levels (p < 0.05). At Fe contents in meals higher than congruent with7.5 mg meal(-1) the net absorption of Cr decreased significantly. The mean Cr daily dietary intake (DDI) was 77 +/- 17 microg day (-1) which indicates that no adverse effects in relation to Cr nutrition (deficiency or toxicity) should occur in individuals from the area.