RESUMEN
A method was developed to isolate extracellular matrix from the human placenta (pECM). The isolated material is composed primarily of collagen, in addition to, elastin, fibronectin, laminin, and glycosaminoglycans (GAGs). The pECM is isolated as a water insoluble paste. This paste can be molded into sheets, tubes, and other 3-D structures that are stable at room temperature. This report describes the interaction of the pluripotent progenitor cells (PDACs) with the isolated pECM. The stem cells used in this study are of human placental origin (placenta derived adherent cells or PDACs) and have a phenotype described as CD200+, CD105+, CD10+, CD34-, and CD45-. The PDACs bind to and proliferate on the pECM, and are stimulated to secrete soluble fibronectin. They actively assemble the soluble fibronectin into a complex network of detergent-insoluble extracellular matrix fibrils. While proliferating on the pECM, PDACs secrete key cytokines at levels well above that observed on tissue-treated tissue culture plates. These cytokines included monocyte chemoattractant protein (MCP-1), IL-6, and IL-8, all of which are important participants in wound healing processes. These results suggest the feasibility of designing a combination product of pECM with PDACs to augment repair processes in nonhealing deep wounds and in diabetic ulcers.
RESUMEN
Tissue engineering aims to regenerate new biological tissue for replacing diseased or injured tissues. We propose a new approach to accelerate the deposition of cell-secreted matrix proteins into extracellular matrix fibrils. We examined whether dynamic substrates with nanoscale ligand features allowing for alpha5beta1 integrin recruiting, cellular tension generation, and alpha5beta1 integrin mobility would enhance fibronectin matrix assembly in a ligand model system that is routinely not sufficient for its induction. To this end, we developed biodynamic substrates consisting of cell adhesive fragment from the 9th and 10th type repeats of fibronectin (FNf ) functionalized to 100 nm prefabricated albumin nanoparticles (ANPs). FNf-ANPs modulated cellular spreading processes, promoting the development of stellate or dendritic morphologies. Concomitant with the spreading, FNf-ANPs rapidly recruited beta1 integrins to focal contacts and promoted the migration of beta1 integrins centripetally from the cell periphery toward the center. FNf-ANPs stimulated the deposition of secreted fibronectin into matrix fibrils; FNf, the key ligand alone, was not sufficient for fibronectin fibrillogenesis. When FNf-ANPs were displayed from "immobilized" substrates, abolishing any mobility of ligated beta1 integrins, fibronectin matrix assembly was abrogated, implicating the role of dynamic matrix display on matrix assembly. Receptor ligation of FNf-ANPs via noncontractile adhesions was not sufficient to stimulate fibrillogenesis, and Rho-kinase inhibitors abolished fibronectin matrix deposition. Our approach highlights the possibility of engineering integrin-based extracellular matrix assembly using nanotechnology, which may have implications for improved biomaterials for wound repair and basic understanding of matrix remodeling within pathogenesis and biomedicine.
Asunto(s)
Matriz Extracelular , Nanopartículas , Ingeniería de Tejidos , Albúminas , Adhesión Celular/fisiología , Células Cultivadas , Fibroblastos , Fibronectinas , Humanos , MasculinoRESUMEN
ACELAGRAFT™ (Celgene Cellular Therapeutics, Cedar Knolls, NJ) was developed as a decellularized and dehydrated human amniotic membrane product (DDHAM). The product has demonstrated potential as a wound healing product with several ongoing preclinical and clinical studies in the area of acute and chronic ulcers. Although the mechanism of action of such a decellularized product has not been examined, a detailed study of the ability of fibroblasts to interact with DDHAM and subsequent cellular responses are presented. These studies indicate that the composition of DDHAM is that of an extracellular matrix (ECM)-like material with high collagen content, retaining key bioactive molecules, such as fibronectin, laminin, glycosaminoglycans (GAGs), and elastin. No cytokines or growth factors were identified as one might expect in a nondecellularized amniotic membrane product. Cell assays show that fibroblasts can recognize fibronectin in DDHAM and bind to it via typical integrin-fibronectin interactions. Fibroblasts secrete fibronectin and can actively assemble the soluble fibronectin into a complex extracellular matrix on DDHAM. Fibroblasts are also stimulated by DDHAM to secrete key proinflammatory(IL-1 and IL-6) and chemotactic cytokines or chemokines (proand IL-8) involved in regulating and enhancing wound repair processes. Microarray gene expression studies on fibroblasts bound to DDHAM show increased expression of key wound healing cytokines. Together, these studies provide insight into the mechanisms by which DDHAM may augment the wound healing process.
RESUMEN
Cell-adhesive ligands organized on nanoscale synthetic biomaterials can potentially recapitulate the nanoscale organization of extracellular matrix and the consequent effects of cell dynamics. In this study, 100 nm albumin nanocarriers (ANC) were fabricated to serve as nanoscale organizational units for a well-defined ligand, the recombinant fragment from fibronectin comprised of the RGD-containing module 10 and the synergy-region-containing module 9. Conventional protein conjugation chemistry was employed to fabricate nanocarriers with increasing levels of displayed ligand. Presentation of ligand-functionalized ANCs adsorbed onto substrates was found to enhance keratinocyte attachment when compared to equivalent levels of adsorbed ligands, supported by ELISA data that the display of ligand on ANCs essentially increased the accessibility of the cell-binding domain and AFM data that the ligand was likely exposed due to ligand-ANC repulsion. The ligand presentation from ANCs converted the cellular morphology from a stationary phenotype to a motile phenotype, with the expression of filopodia-like microextensions, and a decrease in focal adhesions, indicating decreased cell adhesion strength. Consequently, cell motility was found to be significantly elevated on ligand-ANC substrates relative to substrates with equivalent levels of ligand. Overall, the ligand-functionalized albumin nanocarriers offer a unique model platform with two distinct properties: enhanced ligand exposure for enhancement of cell attachment to ligands at low concentrations; and enhanced cell detachment, motile phenotype, and migration kinetics.
Asunto(s)
Portadores de Fármacos , Nanoestructuras , Albúmina Sérica , Materiales Biocompatibles , Adhesión Celular , Movimiento Celular , Fibronectinas , Humanos , Técnicas In Vitro , Queratinocitos/citología , Queratinocitos/fisiología , Ligandos , Ensayo de Materiales , Nanotecnología , Tamaño de la Partícula , Fragmentos de Péptidos , Fenotipo , Proteínas RecombinantesRESUMEN
Leptospirosis is a zoonosis that affects humans, domestic animals, and wildlife. Carnivores are at the top of the feeding chain, thus being exposed to pathogens through their preys. From June 2004 to April 2007, we analyzed for evidences of contact with 14 serovars of Leptospira interrogans Sensu Lato serum (analyzed by indirect Microscopic Agglutination Test) and urine or kidney samples (analyzed by microscopic observation, immunostaining and culture) collected from 201 wild and domestic carnivores, including 26 free-living Iberian lynx (Lynx pardinus), 33 red foxes (Vulpes vulpes), 33 Egyptian mongooses (Herpestes ichneumon), 25 common genets (Genetta genetta), two Eurasian badgers (Meles meles) and one Eurasian otter (Lutra lutra), and 53 free-roaming cats and 28 rural dogs in protected areas in Andalusia (southern Spain). Twenty-three percent of the animals presented evidences of contact, being the prevalence similar among wild (23.5%) and domestic species (22.2%). Contact with Lesptospira was detected in all the species but the otter. Prevalence was: lynx (11% by bacteriological detection, 32% by serology), fox (0%, 47%), mongoose (5%, 20%), genet (0%, 12%), badger (0%, 50%), cat (20%, 14%), dog (only serology: 36%). Serovar Icterohemorragiae accounted for 2/3 of the cases. Serovar Canicola was detected in half of the positive dogs and one lynx. Other serovars detected were Ballum, Sejroë, and Australis. No macroscopic lesions were observed in necropsied animals that showed evidence of contact with the agent, although histopathologic lesions (chiefly chronic interstitial nephritis) were observed in 7 out of the 11 microscopically analyzed individuals. Thus, L. interrogans may cause previously unrecorded disease in wild carnivores in Spain. Wild and free-roaming carnivores may not act as reservoir of L. interrogans but as a dead-end hosts, though the dog may act as reservoir of serovar Canicola. Carnivores are apparently good sentinels for the epidemiological monitorization of leptospirosis.
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Carnívoros , Leptospirosis/veterinaria , Animales , Animales Salvajes , Leptospira/clasificación , Leptospirosis/epidemiología , EspañaRESUMEN
Fibrinogen is a soluble protein produced by hepatocytes and secreted into plasma, where it functions in hemostasis. During inflammation, the hepatic synthesis of fibrinogen is induced 2-10 fold. Recent studies demonstrate that after an inflammatory stimulus, fibrinogen gene expression and protein production is upregulated in lung epithelial cells, where it is secreted basolaterally and consequently deposited into the extracellular matrix in fibrils that extensively colocalize with fibronectin fibrils. In this study, we show that the deposition of fibrinogen into the matrix of fibroblasts occurred rapidly and in a Rho-dependent manner in response to serum or lysophosphatidic acid; RhoA GTPase signaling is also required for fibronectin matrix assembly. Using mouse embryonic fibronectin-null cells, we show that incorporation of exogenous fibrinogen into matrix fibrils occurred only in the presence of exogenous fibronectin, which is also assembled into matrix fibrils. Furthermore, treatment of fibroblasts and fibronectin-null cells with an antibody that inhibits fibronectin matrix assembly impaired incorporation of fibrinogen into matrix fibrils. Collectively, these data suggest that incorporation of fibrinogen into the extracellular matrix requires active fibronectin polymer elongation into matrix fibrils. From these data, we hypothesize that fibrinogen deposition rapidly changes the topology of the extracellular matrix to provide a surface for cell migration and matrix remodeling during tissue repair.