RESUMEN
The anterior cruciate ligament (ACL) is necessary for normal knee stability and movement. Unfortunately the ACL is also the most frequently injured ligament of the knee with severe disruptions requiring surgical intervention. In response to this, tissue engineering has emerged as an option for ACL replacement and repair. In this study we present a novel hydrogel-fibrous scaffold as a potential option for ACL replacement. The scaffold was composed of PLLA fibers, in a previously evaluated braid-twist structure, combined with a polyethylene glycol diacrylate (PEGDA) hydrogel to improve viscoelastic properties. Both hydrogel concentration (10%, 15%, and 20%) and amount of hydrogel (soaking the fibrous scaffold in hydrogel solution or encasing the scaffold in a block of hydrogel) were evaluated. It was found that the braid-twist scaffold had a greater porosity and larger number of pores above 100 µm than braided scaffolds with the same braiding angle. After testing for their effects on swelling, fiber degradation, and protein release, as well as viscoelastic and tensile testing (when combined with fibrous scaffolds), it was found that the composite scaffold soaked in 10% hydrogel had the best chemical release and mechanical properties. The optimized structure behaved similarly to natural ligament in tension with the addition of the hydrogel decreasing the ultimate tensile stress (UTS), but the UTS was still comparable to natural ACL. In addition, cellular studies showed that the hydrogel-PLLA fiber composite supported fibroblast growth.
Asunto(s)
Ligamento Cruzado Anterior/crecimiento & desarrollo , Fibroblastos/citología , Fibroblastos/fisiología , Hidrogeles/síntesis química , Ácido Láctico/síntesis química , Polímeros/síntesis química , Ingeniería de Tejidos/instrumentación , Andamios del Tejido , Animales , Ligamento Cruzado Anterior/citología , Ligamento Cruzado Anterior/cirugía , Materiales Biocompatibles/síntesis química , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Fuerza Compresiva , Diseño de Equipo , Análisis de Falla de Equipo , Dureza , Ensayo de Materiales , Poliésteres , Porosidad , ConejosRESUMEN
The anterior cruciate ligament (ACL) is the most commonly injured ligament of the knee; it also contributes to normal knee function and stability. Due to its poor healing potential severe ACL damage requires surgical intervention, ranging from suturing to complete replacement. Current ACL replacements have a host of limitations that prevent their extensive use. Investigators have begun to utilize tissue-engineering techniques to create new options for ACL repair, regeneration and replacement. In this study we tested novel braid-twist scaffolds, as well as braided scaffolds, twisted fiber scaffolds and aligned fiber scaffolds, for use as ACL replacements composed of poly(L-lactic acid) fibers. Scaffolds were examined using stress relaxation tests, cell viability assays and scanning electron microscopy. The behaviors of the braid-twist scaffolds were modeled with Maxwell and quasi-linear viscoelastic (QLV) models. In stress relaxation tests, the braid-twist scaffolds behaved similarly to native ACL tissue, with final normalized stresses of 87% and 83% after an 8 N load. There was agreement between the experimental data and the Maxwell model when the model included an element for each structural element in the scaffold. There was also agreement between the experimental data and QLV model, scaffolds with similar braiding angles shared constants. In cell proliferation studies no differences were found between fibroblast growth on the braided scaffolds and the braid-twist scaffolds. SEM images showed the presence of new extracellular matrix. Data from this and previous tensile studies demonstrate that the braid-twist scaffold design may be effective in scaffolds for ACL tissue regeneration.
Asunto(s)
Ligamento Cruzado Anterior/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Sustancias Viscoelásticas/farmacología , Animales , Ligamento Cruzado Anterior/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Modelos Lineales , Ensayo de Materiales , Modelos Biológicos , Conejos , Estrés Mecánico , Factores de TiempoRESUMEN
IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, beta-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5-50 microM K(d)) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP1(2-210), which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP1(2-210) was found to be monomeric, to bind F-actin with a K(d) of approximately 47 microM, to saturate F-actin at a molar ratio of one IQGAP1(2-210) per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD.
Asunto(s)
Actinas/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Células COS , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Chlorocebus aethiops , Dimerización , Células HeLa , Humanos , Proteínas de Microfilamentos , Microscopía Fluorescente , Peso Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Transfección , Proteínas Activadoras de ras GTPasa/química , CalponinasRESUMEN
IQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and beta-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca(2+) and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAP1 much more efficiently in the presence of Ca(2+) than EGTA, and all 8 IQ motifs in each IQGAP1 dimer could bind calmodulin simultaneously. In live cells, IQGAP1-YFP localized to the cell cortex, but elevation of intracellular Ca(2+) reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in free intracellular Ca(2+) promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments.