Manuel E. Young

The effect of an autologous cellular gel-matrix integrated implant system on wound healing

BackgroundThis manuscript reports the production and preclinical studies to examine the tolerance and efficacy of an autologous cellular gel-matrix integrated implant system (IIS) aimed to treat full-thickness skin lesions.MethodsThe best concentration of fibrinogen and thrombin was experimentally determined by employing 28 formula ratios of thrombin and fibrinogen and checking clot formation and apparent stability. IIS was formed by integrating skin cells by means of the in situ gelification of fibrin into a porous crosslinked scaffold composed of chitosan, gelatin and hyaluronic acid. The in vitro cell proliferation within the IIS was examined by the MTT assay and PCNA expression. An experimental rabbit model consisting of six circular lesions was utilized to test each of the components of the IIS. Then, the IIS was utilized in an animal model to cover a 35% body surface full thickness lesion.ResultsThe preclinical assays in rabbits demonstrated that the IIS was well tolerated and also that IIS-treated rabbit with lesions of 35% of their body surface, exhibited a better survival rate (p = 0,06).ConclusionIIS should be further studied as a new wound dressing which shows promising properties, being the most remarkable its good biological tolerance and cell growth promotion properties.

Volatile organic compounds produced by human skin cells.

Skin produces volatile organic compounds (VOCs) released to the environment with emission patterns characteristic of climatic conditions. It could be thought that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, using gas chromatography, we answered the question of whether VOC profiles of primary cultures of human dermal fibroblasts were affected by the type of culture conditions. VOCs were determined for different types of culture, finding significant differences between skin cells grown in classical monolayer culture -2D- compared with 3D matrix immobilized cultures. This indicates that VOC profiles could provide information on the physiological state of skin cells or skin.

Volatile profiles of human skin cell cultures in different degrees of senescence

It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.

A Comparative Study of Stout Beer Batch Fermentation Using Free and Microencapsulated Yeasts

Because many questions arise regarding the use of immobilization technology to consistently produce a high quality beer, this work focuses on the effects of using an immobilization matrix in the fermentation process. The aim of this study was to explore the feasibility and potential uses of immobilization on sensorial characteristics such as color, flavor, and headspace compounds of stout beer, when using batch fermentation. Batch production of beer was conducted as a standard ale process for stout beer production. For the immobilized yeasts fermentation, cells were microencapsulated in alginate, by using the Thiele modulus procedure for microcapsule design. Glucose concentration, cell multiplication, cell viability, specific gravity, pH, Brix, and ethanol were monitored throughout the fermentation process. Both, sensorial analysis (statistic triangle tests) and instrumental methods (gas chromatography to measure headspace compounds and visible spectrophotometer to quantify the color) were used to evaluate characteristics of the beer that was produced from immobilized and free yeast fermentations. Free and immobilized yeasts fermentation showed no significant difference (p > 0.05) for all variables of interest. The profile of headspace compounds was different, perhaps because of changes in yeast’s behavior and the presence of secondary metabolites. However, immobilization did not have a significant impact on the beer flavor, as detected by the sensorial triangle test.

Modeling Volatile Organic Compounds Released by Bovine Fresh Meat Using an Integration of Solid Phase Microextraction and Databases

The influence of volatile organic compounds is often regarded as a critical factor of food flavor quality. Nevertheless, information about the biochemical origin of odorants is not totally available. Quantification of volatile compounds in meat is not easy, but a new approach that helps is the use of GC/MS–SPME. This technique can generate large-scale data sets that allow for the development of computational technologies for better integration and biochemical interpretation. Volatile organic compounds of bovine fresh meat samples were measured by GC/MS–SPME, using four SPME fibers. The compounds were analyzed using an integration of chemical and biological database systems (NIST and KEGG, respectively). Twelve metabolic pathways were identified. A metabolic network model was constructed using graph elements, linking pathways through a central branch mediated by glycolysis. Experimental data indicated that post-mortem glycolysis is associated with the release of volatile compounds in fresh meat. The proposed technique could be used to study possible sources of biochemical compounds in meat to be applied in food analysis.

Growth factor production from fibrin-encapsulated human keratinocytes.

Fibrin has been used extensively in cell encapsulation because it has important biological properties. Keratinocyte encapsulation in fibrin is a widely used technique in skin tissue engineering. The production of growth factors (EGF, TGF-β1 and PDGF-BB) was evaluated when keratinocytes are encapsulated in fibrin. Secretions of TGF-β1 and PDGF-BB increased more than five times compared to monolayer cultures. Encapsulated cells secreted about 80% active form of TGF-β1 (monolayer cells only secreted inactive form). An enhanced secretion of TGF-β1 and PDGF-BB was found in encapsulated cells, showing that fibrin capsules are favourable for the production of these growth factors.

Prediction correlation of vapor pressure for methyl jasmonate

Abstract A vapor pressure correlation based on Antoine’s equation was proposed for methyl jasmonate. Equilibrium data was obtained in the range of 3–100 °C and analyses were performed with GC–MS by solid phase microextraction technique. The correlation gives an acceptable extrapolation for the boiling point at atmospheric pressure, allowing evaluation of vapor pressure at temperatures usually found in food and agricultural process applications.

Senescent cultures of human dermal fibroblasts modified phenotype when immobilized in fibrin polymer.

One of the limitations in tissue engineering is the restricted ability to expand the number of cells, because somatic cells can duplicate a limited number of times before they lose the ability to divide, leading to a senescent state. Here we report that the interaction of senescent fibroblasts with fibrin polymer can modify the senescent phenotype and partially restore the ability of growth-arrested cells to continue replicating. Primary human dermal fibroblasts were grown to >90% SA/β-Gal (senescence associated β-galactosidase). The senescent cells were immobilized in fibrin-polymers by mixing fibrinogen and thrombin solutions. Immobilized senescent cell cultures grew, however, their growth arrested after 24 h of immobilization. The percentage of cells with a positive reaction at SA/β-Gal did not decrease significantly after immobilization, but the intensity of the stain decreased. The glycolytic activity in immobilized senescent fibroblast was re-established at pre-senescent levels. In conclusion, fibrin induces changes in the phenotype of senescent human fibroblasts. This simple procedure could complement available tissue-engineering techniques to increase the amount of biomass seeded on a fibrin scaffold, which could be beyond senescence.

TGFβ3 secretion by three‐dimensional cultures of human dental apical papilla mesenchymal stem cells

Mesenchymal stem cells (MSCs) can be isolated from dental tissues, such as pulp and periodontal ligament; the dental apical papilla (DAP) is a less‐studied MSC source. These dental‐derived MSCs are of great interest because of their potential as an accessible source for cell‐based therapies and tissue‐engineering (TE) approaches. Much of the interest regarding MSCs relies on the trophic‐mediated repair and regenerative effects observed when they are implanted. TGFβ3 is a key growth factor involved in tissue regeneration and scarless tissue repair. We hypothesized that human DAP‐derived MSCs (hSCAPs) can produce and secrete TGFβ3 in response to micro‐environmental cues. For this, we encapsulated hSCAPs in different types of matrix and evaluated TGFβ3 secretion. We found that dynamic changes of cell–matrix interactions and mechanical stress that cells sense during the transition from a monolayer culture (two‐dimensional, 2D) towards a three‐dimensional (3D) culture condition, rather than the different chemical composition of the scaffolds, may trigger the TGFβ3 secretion, while monolayer cultures showed almost 10‐fold less secretion of TGFβ3. The study of these interactions is provided as a cornerstone in designing future strategies in TE and cell therapy that are more efficient and effective for repair/regeneration of damaged tissues. Copyright

Measuring b-Galactosidase activity at pH 6 with a differential pH sensor

The β-Galactosidase activity at pH 6 is used as a cellular marker to identify senescent cell cultures. The classic method to identify this enzymatic activity is using cytochemical staining with X-Gal after 16 hrs. In this work, a differential pH sensor was used to measure β-Galactosidase activity at pH 6. The measurement is easy and only takes 3 min.