Donald I. Brown

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.

Photoperiodic modulation of GnRH mRNA in the male Syrian hamster

Male Syrian hamsters (Mesocricetus auratus) are seasonal breeders. They show marked testicular regression when exposed to short autumnal photoperiods, and then remain sexually quiescent for several months. By mid-winter, however, they show a loss in responsiveness to the inhibitory influence of short photoperiods and their testes begin to recrudesce. To shed light on the neuroendocrine mechanism responsible for mediating these reproductive changes, we examined the influence of photoperiod on the expression of GnRH mRNA in the hamster forebrain. Adult males were either exposed to short photoperiods (6L:18D) for 16 weeks or were maintained under long photoperiods (14L:10D); additional animals were exposed to short or long photoperiods for 22 weeks. As expected, exposure to short photoperiods for 12 weeks resulted in a marked decrease (P<0.01) in testicular mass and serum testosterone levels, but after 22 weeks these reproductive parameters were once again significantly elevated (P<0.01). In contrast, quantitative in situ hybridization histochemistry revealed no difference (P>0.05) between the GnRH mRNA levels of the short-photoperiod hamsters and their aged-matched long-photoperiod controls, although an age-related decrease (P<0.05) was evident in both photoperiod-treatment groups. These data emphasize that GnRH mRNA is highly expressed in hamsters even when their reproductive axis has been rendered sexually quiescent by exposure to short photoperiods, and that photoperiod-induced changes in GnRH secretion, rather than synthesis, are more likely to regulate the timing of the breeding season. On the other hand, the data indicate that GnRH mRNA levels show an aging-related decrease, regardless of photoperiod, suggesting that in the long term a decrease in GnRH gene expression may contribute to the reduced fertility of old hamsters.

Using RGB Image Processing for Designing an Alginate Edible Film

The use of edible films to coat food products is a technique that allows for an extended shelf-life. One of the most widely used polymers is calcium alginate. However, this polymer can modify the original food color and the perception by consumers. The objective was to design an alginate film based principally on color changes using a RGB color model. Edible films were prepared with sodium alginate and glycerol as plasticizer, cross-linking the polymer with calcium. Dry and hydrated states of the edible films were studied. Film thickness was directly proportional to surface concentration and increased with hydration. There is a zone in which the color does not change with alginate surface concentration and another where the color is directly proportional to it. This latter scenario is not a consequence of structural changes or the degree of hydration. Results showed a range where the color was not modified by the alginate concentration; hence, an optimal surface concentration was determined as a design parameter. Edible films made using the optimal surface concentration would not mask microbial contamination and have good physical properties (water vapor transmission and swelling) compared with other surface concentrations. In addition, it was possible to model alginate surface concentration as a function of surface color using mathematical tools (clustering, linear regression, and support vector machine), allowing one to study the optimal use of the edible films.

Designing a gelatin/chitosan/hyaluronic acid biopolymer using a thermophysical approach for use in tissue engineering.

Cell culture on biopolymeric scaffolds has provided treatments for tissue engineering. Biopolymeric mixtures based on gelatin (Ge), chitosan (Ch) and hyaluronic acid (Ha) have been used to make scaffolds for wound healing. Thermal and physical properties of scaffolds prepared with Ge, Ch and Ha were characterized. Thermal characterization was made by using differential scanning calorimetry (DSC), and physical characterization by gas pycnometry and scanning electron microscopy. The effects of Ge content and cross-linking on thermophysical properties were evaluated by means of a factorial experiment design (central composite face centered). Gelatin content was the main factor that affects the thermophysical properties (microstructure and thermal transitions) of the scaffold. The effect of Ge content of the scaffolds for tissue engineering was studied by seeding skin cells on the biopolymers. The cell attachment was not significantly modified at different Ge contents; however, the cell growth rate increased linearly with the decrease of the Ge content. This relationship together with the thermophysical characterization may be used to design scaffolds for tissue engineering.

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.

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.

Microscopic Anatomy of the Male Reproductive System in Echinolittorina peruviana (Mollusca: Caenogastropoda)

Echinolittorina peruviana (Lamarck, 1822), representante gonocorico de Littorinidae en el Pacifico Sur, tiene un sistema reproductor masculino adaptado para la fecundacion interna, que en este estudio se describe a niveles macrocoscopico y microscopico; enfatizando la organizacion compartimentalizada de la gonada y la morfologia del pene. El sistema reproductor masculino se presenta como un complejo conico gonada-glandula digestiva abigarrado. La gonada presenta tres compartimientos: 1) gametogenico acinar entre los acinos glandulares, 2) periacinar con una capa de celulas somaticas fusiformes y 3) interacinar con celulas almacenadoras de glicogeno, compartido con los acinos glandulares. En el compartimiento gametogenico acinar ocurre la espermatogenesis con la linea germinal organizada en forma centripeta hacia el lumen. Hacia anterior, la vesicula seminal almacena los productos de la espermatogenesis; en su region cefalica los euespermatozoides se unen al epitelio y los paraespermatozoides se distribuyen en el lumen. Este organo se conecta con un conducto corto a la glandula prostatica, abierta hacia la cavidad paleal en toda su longitud. La zona anterior de la glandula prostatica se une al surco espermatico cervical, que recorre el cuello del animal por la region paleal derecha; este continua como surco espermatico peniano ascendiendo desde su base hasta la punta. El pene es conico aguzado no pigmentado; hacia anterior adyacente a su base, hay un complejo glandular con un proceso mamiliforme y una region glandular discoidal. Su producto de secrecion seria una espicula de funcion desconocida, cuya histologia es descrita por primera vez. En este complejo glandular, el proceso mamiliforme esta constituido por la proyeccion espicular y una banda conjuntivo-muscular que lo rodea y separa de la region glandular folicular discoidal; su secrecion atraviesa dicha banda, incorporandose al epitelio de revestimiento interno del proceso.

Spermatozoon ultrastructure in seven South American species of Tegula Lesson, 1835 (Mollusca: Vetigastropoda) and the phylogenetic implications for the subfamily Tegulinae

Summary This study presents results of the examination of the ultrastructure of the spermatozoon of seven South American species of Tegula using light and transmission electron microscopy. In all cases the spermatozoa were of the primitive or ect-aquaspermatozoon type, common in species which employ external fertilization as part of their reproductive strategy. The spermatozoon of Tegula, from anterior to posterior, are composed of: (a) a bullet-shaped head with an anterior acrosome and a basal nucleus, (b) a mid-piece with a prevalent number of five mitochondria plus proximal and distal centrioles, and (c) a flagellum with a 9+2 arrangement of microtubules. The ultrastructural dimensions of the spermatozoa and the characteristics of the acrosome and the nucleus of the Tegula species studied here, as well as those previously published in the literature, showed that each species produces male gametes having a species-specific morphology. The results also suggest that the ultr structure of the spermatozoon could be potentially useful for recognizing subgenera within Tegula. The comparative study showed that some characters of the spermatozoon of the Tegulinae (e.g. length of the head, acrosome shape, acrosome length as percentage of total head length) agree with fossil, morphological and molecular evidence which suggest that members of this subfamily are more closely related to representative of the family Turbinidae.

Structure of the spermatozoa of two sympatric species of Fissurella Brugière, 1789 (Mollusca: Archaeogastropoda) from the southeast coast of South America

Summary This study analyzes the structure of the spermatozoa of Fissurella latimarginata and Fissurella cumingi, two sympatric keyhole limpet species from the coast of Chile. Observations were made using light and scanning and transmission electron microscopy. The spermatozoa of these species are of the ect-aquasperm type, typical of molluscs having external fertilization. They are composed of a conical acrosome, with a deep posterior subacrosomal imagination, a truncated conical nucleus, and a mid-piece containing a ring of five mitochondria, from which emerges a flagellum. The spermatozoa of the two species have quantitative and qualitative differences between different structural components. The total mean length of the head of F. latimarginata spermatozoa was significantly longer than that of F. cumingi. Components of the heads of the spermatozoa including the acrosomes and nuclei also showed interspecific differences. The morphology of the spermatozoa of these species constitutes a valid taxonomic character which supports the proposal that F. latimarginata and F. cumingi are valid species.

Design of a hybrid biomaterial for tissue engineering: Biopolymer-scaffold integrated with an autologous hydrogel carrying mesenchymal stem-cells

Biologically active biomaterials as biopolymers and hydrogels have been used in medical applications providing favorable results in tissue engineering. In this research, a wound dressing device was designed by integration of an autologous clot hydrogel carrying mesenchymal stem-cells onto a biopolymeric scaffold. This hybrid biomaterial was tested in-vitro and in-vivo, and used in a human clinical case. The biopolymeric scaffold was made with gelatin, chitosan and hyaluronic acid, using a freeze-drying method. The scaffold was a porous material which was designed evaluating both physical properties (glass transition, melting temperature and pore size) and biological properties (cell viability and fibronectin expression). Two types of chitosan (120 and 300kDa) were used to manufacture the scaffold, being the high molecular weight the most biologically active and stable after sterilization with gamma irradiation (25kGy). A clot hydrogel was formulated with autologous plasma and calcium chloride, using an approach based on design of experiments. The optimum hydrogel was used to incorporate cells onto the porous scaffold, forming a wound dressing biomaterial. The wound dressing device was firstly tested in-vitro using human cells, and then, its biosecurity was evaluated in-vivo using a rabbit model. The in-vitro results showed high cell viability after one week (99.5%), high mitotic index (19.8%) and high fibronectin expression. The in-vivo application to rabbits showed adequate biodegradability capacity (between 1 and 2weeks), and the histological evaluation confirmed absence of rejection signs and reepithelization on the wound zone. Finally, the wound dressing biomaterial was used in a single human case to implant autologous cells on a skin surgery. The medical examination indicated high biocompatibility, partial biodegradation at one week, early regeneration capacity at 4weeks and absence of rejection signs.