RESUMEN

To explore the possible emergence and lived consequences of social inequality in the Atacama, we analyzed a large set (n = 288) of incredibly well preserved and contextualized human skeletons from the broad Middle Period (AD 500-1000) of the San Pedro de Atacama (Chile) oases. In this work, we explore model-based paleodietary reconstruction of the results of stable isotope analysis of human bone collagen and hydroxyapatite. The results of this modeling are used to explore local phenomena, the nature of the Middle Period, and the interaction between local situations and the larger world in which the oases were enmeshed by identifying the temporal, spatial, and biocultural correlates and dimensions of dietary difference. Our analyses revealed that: 1) over the 600-year period represented by our sample, there were significant changes in consumption patterns that may evince broad diachronic changes in the structure of Atacameño society, and 2) at/near 600 calAD, there was a possible episode of social discontinuity that manifested in significant changes in consumption practices. Additionally, while there were some differences in the level of internal dietary variability among the ayllus, once time was fully considered, none of the ayllus stood out for having a more (or less) clearly internally differentiated cuisine. Finally, sex does not appear to have been a particularly salient driver of observed dietary differences here. While we do not see any de facto evidence for complete dietary differentiation (as there is always overlap in consumption among individuals, ayllus, and time periods, and as isotopic analysis is not capable of pinpointing different foods items or preparations), there are broad aspects of dietary composition changing over time that are potentially linked to status, and foreignness. Ultimately, these stand as the clearest example of what has been termed "gastro-politics," potentially tied to the emergence of social inequality in the San Pedro oases.

Asunto(s)

Antropología Física , Arqueología , Dieta , Factores Socioeconómicos/historia , Huesos/química , Cementerios , Chile/epidemiología , Colágeno/sangre , Colágeno/aislamiento & purificación , Durapatita/química , Durapatita/aislamiento & purificación , Femenino , Historia Medieval , Humanos , Marcaje Isotópico , Masculino , Cráneo/química

RESUMEN

Nile tilapia skin, an abundant waste from fish processing, can be used for collagen extraction, which has a high aggregated value for biomedical applications. Collagen extraction was conducted under different reaction conditions (time, temperature, and concentration of acetic acid) in order to optimize the yield without compromising the integrity of the collagen. Temperature and time were responsible for increased yield. The extraction at 4 and 20 °C produced the acid-solubilized collagen (ASC) with the intact triple helix and was analysed by Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism (CD). The optimized ASC (which used 0.35 mol/L of acetic acid at 20 °C) was consumed to obtain for the first-time fish-based hydrogels with hyaluronic acid (HA) crosslinked with 1-ethyl-3-(3-dimethylaminopropryl carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The hydrogel was characterized by FT-IR, rheology, swelling, and scanning electron microscopy (SEM), confirming that cross-linking was accomplished. It possesses a robust organized network, swells 255 % in PBS and bears interconnected pores with a diameter in the range of 10-100 µm. Until now, col-HA hydrogels crosslinked with EDC/NHS have not been reported in literature with collagen from Nile Tilapia skin. Fish collagen can be a better option than those from land-based animals (cow and pig).

Asunto(s)

Ácido Hialurónico/química , Hidrogeles/química , Piel/química , Animales , Cíclidos , Colágeno/química , Colágeno/aislamiento & purificación , Tamaño de la Partícula , Propiedades de Superficie

RESUMEN

Hydrolyzed collagen (HC) is a group of peptides with low molecular weight (3-6 KDa) that can be obtained by enzymatic action in acid or alkaline media at a specific incubation temperature. HC can be extracted from different sources such as bovine or porcine. These sources have presented health limitations in the last years. Recently research has shown good properties of the HC found in skin, scale, and bones from marine sources. Type and source of extraction are the main factors that affect HC properties, such as molecular weight of the peptide chain, solubility, and functional activity. HC is widely used in several industries including food, pharmaceutical, cosmetic, biomedical, and leather industries. The present review presents the different types of HC, sources of extraction, and their applications as a biomaterial.

Asunto(s)

Colágeno , Hidrolisados de Proteína , Animales , Colágeno/química , Colágeno/aislamiento & purificación , Colágeno/uso terapéutico , Humanos , Hidrolisados de Proteína/química , Hidrolisados de Proteína/aislamiento & purificación , Hidrolisados de Proteína/uso terapéutico

RESUMEN

Polymeric biomaterials composed of extracellular matrix components possess osteoconductive capacity that is essential for bone healing. The presence of collagen and the ability to undergo physicochemical modifications render these materials a suitable alternative in bone regenerative therapies. The objective of this study was to evaluate the osteogenic capacity of collagen-based matrices (native and anionic after alkaline hydrolysis) made from bovine intestinal serosa (MBIS). Twenty-five animals underwent surgery to create a cranial defect to be filled with native and anionic collagen matrixes, mmineralized and non mineralized. The animals were killed painlessly 6 weeks after surgery and samples of the wound area were submitted to routine histology and morphometric analysis. In the surgical area there was new bone formation projecting from the margins to the center of the defect. More marked bone neoformation occurred in the anionic matrices groups in such a way that permitted union of the opposite margins of the bone defect. The newly formed bone matrix exhibited good optical density of type I collagen fibers. Immunoexpression of osteocalcin by osteocytes was observed in the newly formed bone. Morphometric analysis showed a greater bone volume in the groups receiving the anionic matrices compared to the native membranes. Mineralization of the biomaterial did not increase its osteoregenerative capacity. In conclusion, the anionic matrix exhibits osteoregenerative capacity and is suitable for bone reconstruction therapies.

Asunto(s)

Regeneración Ósea/efectos de los fármacos , Colágeno/farmacología , Matriz Extracelular/trasplante , Intestinos/química , Membrana Serosa/química , Fracturas Craneales/tratamiento farmacológico , Animales , Biomarcadores/metabolismo , Regeneración Ósea/fisiología , Calcificación Fisiológica/fisiología , Bovinos , Colágeno/química , Colágeno/aislamiento & purificación , Expresión Génica , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Ratas , Ratas Wistar , Cráneo/efectos de los fármacos , Cráneo/lesiones , Cráneo/patología , Fracturas Craneales/patología , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

Dermatosparaxia em animais é uma doença autossômica recessiva do tecido conjuntivo caracterizada por fragilidade e hiperextensibilidade cutânea. A doença em ovinos White Dorper é provocada pela mutação c.421G>T no gene ADAMmetalopeptidase com trombospondina tipo 1 motif, 2 (ADAMTS2). O objetivo deste estudo foi descrever os achados clínicos, moleculares e histopatológicos da dermatosparaxia em ovinos White Dorper de um rebanho localizado no Centro-Oeste Paulista. [...] Dos nove animais examinados, dois apresentavam sinais clínicos compatíveis com dermatosparaxia. O exame histopatológico de amostras cutâneas das lesões destes dois animais revelou também achados compatíveis com dermatosparaxia, sendo caracterizados por epiderme e anexos cutâneos preservados e sem características atípicas; colágeno displásico arranjado em feixes pequenos, fragmentados e com focos de degeneração, anexos cutâneos proeminentes e na região da derme foco hemorrágico intenso associado a moderado infiltrado neutrofílico na derme profunda. Com o objetivo de realizar o diagnóstico molecular da enfermidade, uma PCR foi padronizada utilizando primers específicos desenhados para amplificar a região do gene ADAMTS2 que continha a mutação c.421G>T e o DNA obtido de amostras de sangue de todos os animais do rebanho. O sequenciamento direto dos produtos da PCR, comprovou que os dois animais clinicamente afetados possuíam a mutação responsável pela dermatosparaxia. A metodologia descrita neste estudo possibilitou o diagnóstico definitivo da doença. Segundo a literatura consultada, esta é a primeira vez que a dermatosparaxia é descrita em ovinos White Dorper no Brasil. A metodologia aqui descrita poderá ser empregada em estudos futuros que avaliem a prevalência desta mutação no Brasil, possibilitando a adoção de medidas que previnam a disseminação dessa mutação no rebanho brasileiro de ovinos White Dorper.

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Dermatosparaxis in animals is an autosomal recessive disorder of the connective-tissue clinically characterized by skin fragility and hiperextensibility. The disease in White Dorper sheep is caused by mutation (c.421G>T) in the ADAM metalloproteinase with thrombospondin type 1 motif, 2 (ADAMTS2) gene. This study describes the dermatological, histological and the molecular findings of the dermatosparaxis in White Dorper sheep from a herd located in the center-west of São Paulo State. [...] The herd consisted of one ram, four ewe and their lambs. In this herd two lambs had clinical signs consistent with dermatosparaxis. Histopathological evaluation of the affected skin of these two animals also revealed consistent findings with dermatosparaxis, characterized by dysplasia of the collagen, which were arranged in small and fragmented collagen bundles and with foci of degeneration of collagen. Prominent cutaneous appendages and severe hemorrhagic focus in dermis region associated with mild neutrophilic infiltrate in the deep dermis. PCR using DNA blood and specific primers to amplify the mutation region c.421G>T was optimized in order to perform molecular diagnosis of the disease. The direct sequencing of the PCR products proved that the two clinically affected animals had the mutation responsible for dermatosparaxis, previously described for this breed and allowed the definitive diagnosis of the disease. This is the first report of the dermatosparaxis in White Dorper sheep in Brazil and the methodology used to confirm the diagnosis could be used in future studies to assess the prevalence of this mutation in Brazil, allowing the adoption of measures to prevent the spread of this mutation in the Brazilian White Dorper herd.

Asunto(s)

Animales , Colágeno/aislamiento & purificación , Cutis Laxo/veterinaria , Enfermedades Cutáneas Genéticas/diagnóstico , Enfermedades Cutáneas Genéticas/veterinaria , Enfermedades de las Ovejas/genética , Trastornos de los Cromosomas/veterinaria , Mutación/genética , Reacción en Cadena de la Polimerasa/veterinaria

RESUMEN

Dermatosparaxia em animais é uma doença autossômica recessiva do tecido conjuntivo caracterizada por fragilidade e hiperextensibilidade cutânea. A doença em ovinos White Dorper é provocada pela mutação c.421G>T no gene ADAMmetalopeptidase com trombospondina tipo 1 motif, 2 (ADAMTS2). O objetivo deste estudo foi descrever os achados clínicos, moleculares e histopatológicos da dermatosparaxia em ovinos White Dorper de um rebanho localizado no Centro-Oeste Paulista. [...] Dos nove animais examinados, dois apresentavam sinais clínicos compatíveis com dermatosparaxia. O exame histopatológico de amostras cutâneas das lesões destes dois animais revelou também achados compatíveis com dermatosparaxia, sendo caracterizados por epiderme e anexos cutâneos preservados e sem características atípicas; colágeno displásico arranjado em feixes pequenos, fragmentados e com focos de degeneração, anexos cutâneos proeminentes e na região da derme foco hemorrágico intenso associado a moderado infiltrado neutrofílico na derme profunda. Com o objetivo de realizar o diagnóstico molecular da enfermidade, uma PCR foi padronizada utilizando primers específicos desenhados para amplificar a região do gene ADAMTS2 que continha a mutação c.421G>T e o DNA obtido de amostras de sangue de todos os animais do rebanho. O sequenciamento direto dos produtos da PCR, comprovou que os dois animais clinicamente afetados possuíam a mutação responsável pela dermatosparaxia. A metodologia descrita neste estudo possibilitou o diagnóstico definitivo da doença. Segundo a literatura consultada, esta é a primeira vez que a dermatosparaxia é descrita em ovinos White Dorper no Brasil. A metodologia aqui descrita poderá ser empregada em estudos futuros que avaliem a prevalência desta mutação no Brasil, possibilitando a adoção de medidas que previnam a disseminação dessa mutação no rebanho brasileiro de ovinos White Dorper.(AU)

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Dermatosparaxis in animals is an autosomal recessive disorder of the connective-tissue clinically characterized by skin fragility and hiperextensibility. The disease in White Dorper sheep is caused by mutation (c.421G>T) in the ADAM metalloproteinase with thrombospondin type 1 motif, 2 (ADAMTS2) gene. This study describes the dermatological, histological and the molecular findings of the dermatosparaxis in White Dorper sheep from a herd located in the center-west of São Paulo State. [...] The herd consisted of one ram, four ewe and their lambs. In this herd two lambs had clinical signs consistent with dermatosparaxis. Histopathological evaluation of the affected skin of these two animals also revealed consistent findings with dermatosparaxis, characterized by dysplasia of the collagen, which were arranged in small and fragmented collagen bundles and with foci of degeneration of collagen. Prominent cutaneous appendages and severe hemorrhagic focus in dermis region associated with mild neutrophilic infiltrate in the deep dermis. PCR using DNA blood and specific primers to amplify the mutation region c.421G>T was optimized in order to perform molecular diagnosis of the disease. The direct sequencing of the PCR products proved that the two clinically affected animals had the mutation responsible for dermatosparaxis, previously described for this breed and allowed the definitive diagnosis of the disease. This is the first report of the dermatosparaxis in White Dorper sheep in Brazil and the methodology used to confirm the diagnosis could be used in future studies to assess the prevalence of this mutation in Brazil, allowing the adoption of measures to prevent the spread of this mutation in the Brazilian White Dorper herd.(AU)

Asunto(s)

Animales , Enfermedades de las Ovejas/genética , Trastornos de los Cromosomas/veterinaria , Enfermedades Cutáneas Genéticas/veterinaria , Enfermedades Cutáneas Genéticas/diagnóstico , Cutis Laxo/veterinaria , Colágeno/aislamiento & purificación , Reacción en Cadena de la Polimerasa/veterinaria , Mutación/genética

RESUMEN

This study evaluated alternative methods for extracting collagen from the tunica albuginea of pig testes and characterized the functional properties of the isolated collagen. Using the statistical tools of factorial design (24⁻¹) and a central composite rotatable design (2³), it was concluded that the best conditions were 0.83 mol L⁻¹ acetic acid, 0.24% pepsin and 28 h of hydrolysis to isolate 82.54 g of collagen per 100g of sample. This purified collagen had improved functional properties in relation to bovine skin collagen, including water solubility, water-holding capacity, emulsifying capacity and emulsion stability. These results suggest that isolated collagen from the tunica albuginea can be used in pharmaceutical and food products.

Asunto(s)

Castración , Colágeno/aislamiento & purificación , Porcinos , Testículo/química , Ácido Acético , Animales , Castración/métodos , Bovinos , Colágeno/química , Emulsiones , Hidrólisis , Masculino , Pepsina A , Piel , Solubilidad , Agua

RESUMEN

This paper describes the preparation and characterization of collagen films extracted from swim bladders of three species of tropical fishes: Arius parkeri (Gurijuba), Cynoscion acoupa (Pescada Amarela) and Cynoscion leiarchus (Pescada Branca). Collagen was extracted under acidic conditions (CH(3)COOH, 2.5 pH) and precipitated by the addition of NaCl up to 3.0 mol L(-1). The films were prepared in acrylic containers and dried in a vacuum atmosphere. The collagen films were characterized by hydroxyproline contents, thermal analysis, scanning electron microscopy and impedance spectroscopy. The determined values of 4-hydroxiproline and collagens in the films were: 105.23+/-4.48 and 873.2; 102.94+/-4.42 and 854.1; 100.65+/-4.80 and 835.8 mg g(-1) for A. parkeri, C. acoupa and C. leiarchus, respectively. Differential scanning calorimetry revealed high denaturation temperature peaks at temperatures ranging from 65.9 to 74.8 degrees C. The micrographs showed no fibrillar organization along the material, but spongy structure, with cavity diameters relatively uniform, at around 2 microm. The impedance spectroscopy presented a distributed relaxation process. A. parkeri's films showed piezoelectricity.

Asunto(s)

Sacos Aéreos/química , Colágeno/química , Colágeno/aislamiento & purificación , Animales , Rastreo Diferencial de Calorimetría , Bagres , Impedancia Eléctrica , Microscopía Electrónica de Rastreo , Perciformes

RESUMEN

Introdução: Dentre as diversas pesquisas realizadas na área de bioengenharia de tecidos, a criação in vitro de tecidos vivos tornase uma das mais promissoras formas de atuação científica. Neste caminho, cada vez mais têm sido descritos novos substitutos cutâneos para o tratamento de grandes lesões da pele. Bell et al. descreveram um modelo de matriz colágena tipo I contrátil após a adição de fibroblastos. Método: O presente estudo propõe a elaboração in vitro de um equivalente dermo-epidérmico composto por uma “derme” de colágeno povoada por fibroblastos, acompanhado de um epitélio de queratinócitos cultivados em sua superfície anterior no papel de “epiderme”. Além disso, estuda as alterações histológicas encontradas nos dias de cultura 4, 7, 14 e 21, avaliando a influência da interface ar-líquido na estratificação do epitélio. Resultados: Os resultados mostraram que houve a criação de um substituto dermo-epidérmico in vitro, com a formação de um epitélio com boa estratificação, sendo que o contato com o ar aumenta esta estratificação

Asunto(s)

Femenino , Adulto , Queratinocitos , Colágeno/aislamiento & purificación , Fibroblastos , Técnicas In Vitro , Piel Artificial , Medios de Cultivo , Técnicas Histológicas , Métodos , Pruebas Cutáneas

RESUMEN

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P < 0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

Asunto(s)

Proteínas de la Matriz Extracelular/metabolismo , Sporothrix/patogenicidad , Adhesión Celular , Colágeno/aislamiento & purificación , Proteínas de la Matriz Extracelular/fisiología , Fibronectinas , Laminina , Sporothrix/fisiología , Esporotricosis/microbiología , Trombospondinas

RESUMEN

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P<0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus

Asunto(s)

Adhesión Celular , Proteínas de la Matriz Extracelular/metabolismo , Sporothrix/patogenicidad , Colágeno/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Matriz Extracelular/fisiología , Fibronectinas , Laminina , Sporothrix/fisiología , Esporotricosis/microbiología , Trombospondinas

RESUMEN

We have investigated the effects of nontoxic doses of vitamin A on the hepatic contents of collagen and sulfated glycosaminoglycans (SGAGs) in rats chronically treated with CCl4. When the animals were treated with this retinoid before the intoxication with CCl4, liver collagen level was significantly reduced as compared with that in rats that received only CCl4 (3.31 +/- 0.40 vs 5.00 +/- 0.61 mg/gm wet liver, mean +/- SD, respectively), although no significant differences were found for the relative proportion of type III collagen related to type I collagen. The absolute increment in the total amount of liver SGAG in the vitamin A--pretreated group was followed by a more important increase in the concentration of dermatan sulfate as compared with the CCl4 group (dermatan sulfate-to-heparan sulfate ratio: 1.15 for the CCl4 group vs 1.70 for the vitamin A--pretreated group). A significant proportion of the dermatan sulfate from this last group was of higher molecular weight when compared with the dermatan sulfate found in the liver of rats that received only CCl4. Our results indicate that the pretreatment with vitamin A modifies hepatic collagen and SGAG deposition and can inhibit or delay the development of liver cirrhosis in rats chronically treated with CCl4. We speculate that this effect could be due to the changes in the fat-storing (Ito) cells phenotype induced by vitamin A.

Asunto(s)

Intoxicación por Tetracloruro de Carbono/metabolismo , Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Cirrosis Hepática Experimental/metabolismo , Hígado/metabolismo , Vitamina A/uso terapéutico , Animales , Colágeno/aislamiento & purificación , Dermatán Sulfato/aislamiento & purificación , Dermatán Sulfato/metabolismo , Electroforesis en Gel de Poliacrilamida , Glicosaminoglicanos/aislamiento & purificación , Heparitina Sulfato/aislamiento & purificación , Heparitina Sulfato/metabolismo , Hígado/efectos de los fármacos , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/prevención & control , Masculino , Peso Molecular , Ratas , Ratas Endogámicas , Vitamina A/farmacología

Asunto(s)

Colágeno/aislamiento & purificación , Tendones/química , Adolescente , Anciano , Animales , Electroforesis en Gel de Poliacrilamida , Humanos , Ratas , Hombro

Asunto(s)

Humanos , Animales , Adolescente , Anciano , Ratas , Colágeno/aislamiento & purificación , Tendones/química , Electroforesis en Gel de Poliacrilamida , Hombro

RESUMEN

Se analizan las características de las membranas amnióticas de partos con ruptura prematura de membranas (RPM) y con ruptura tardía de las membranas fetales (RTM), en cuanto a su composición aminoacídica y relación prolina/hidroxiprolina y la importancia de la situación dietética y nutricional de las gestantes, con respecto a la vitamina C en relación con este accidente obstétrico, así como demostrar, mediante el uso de un modelo experimental, que la membrna fetal es también un sitio sensible de la deficiencia de vitamina C. A las mujeres estudiadas se les determinó acido ascórbico en plasma en el segundo y tercer trimestre de gestación, cortisol en plasma en las mismas etapas, acido ascórbico en leucocitos al final de la gestación, en las membranas amnióticas colectadas durante el trabajo de parto. Se determinó prolina, hidroxiprolina y aminoacidos totales individuales y se aisló el colágeno en el cual se realizaron las mismas determinaciones que en la membrana. Se realizó una encuesta dietétic. El modelo experimental utilizado fue la curiela gestante, los animales se dividieron en 3 grupos, uno de los cuales ingirió una dieta escorbutigénica durante la última etapa de la gestación, al fianl de la misma los animales fueron sacrificados y en las membranas fetales se realizaron las mismas determinaciones descritas para humanos. Se encontró una mayor incidencia de RPM en mujeres con bajos niveles de vitamina C

Asunto(s)

Embarazo , Adulto , Cobayas , Animales , Humanos , Femenino , Ácido Ascórbico/sangre , Aminoácidos/análisis , Deficiencia de Ácido Ascórbico , Membranas Extraembrionarias/análisis , Rotura Prematura de Membranas Fetales/etiología , Hidrocortisona/sangre , Hidroxiprolina/análisis , Leucocitos/análisis , Líquido Amniótico/análisis , Prolina/análisis , Colágeno/aislamiento & purificación