ABSTRACT
Polymer-ceramic composites obtained as the result of a mineralization process hold great promise for the future of tissue engineering. Simulated body fluids (SBFs) are widely used for the mineralization of polymer scaffolds. In this work an exhaustive study with the aim of optimizing the mineralization process on a poly(L-lactic acid) (PLLA) macroporous scaffold has been performed. We observed that when an air plasma treatment is applied to the PLLA scaffold its hydroxyapatite nucleation ability is considerably improved. However, plasma treatment only allows apatite deposition on the surface of the scaffold but not in its interior. When a 5 wt % of synthetic hydroxyapatite (HAp) nanoparticles is mixed with PLLA a more abundant biomimetic hydroxyapatite layer grows inside the scaffold in SBF. The morphology, amount, and composition of the generated biomimetic hydroxyapatite layer on the pores' surface have been analyzed. Large mineralization times are harmful to pure PLLA as it rapidly degrades and its elastic compression modulus significantly decreases. Degradation is retarded in the composite scaffolds because of the faster and extensive biomimetic apatite deposition and the role of HAp to control the pH. Mineralized scaffolds, covered by an apatite layer in SBF, were implanted in osteochondral lesions performed in the medial femoral condyle of healthy sheep. We observed that the presence of biomimetic hydroxyapatite on the pore's surface of the composite scaffold produces a better integration in the subchondral bone, in comparison to bare PLLA scaffolds.
Subject(s)
Biomimetics , Hydroxyapatites , Lactic Acid , Osseointegration , Polymers , Tissue Scaffolds , Animals , Microscopy, Electron, Scanning , Polyesters , Sheep , X-Ray DiffractionABSTRACT
AIM: The aim of this work was to determine the pathways implicated in the mechanosensing of chondrocytes. METHODS: Rat chondrocytes were cultured in collagen hydrogels of different stiffness (2-20 Pa) in normoxia and hypoxia, in monolayer and embedded inside hydrogels. First, chondrocyte were cultured on hydrogels in the presence of antibodies to block integrins. Second, custom RT-PCR array plates and western blot were used to detect changes in expression of genes implicated in downstream signalling pathways. RESULTS: The results allowed us to demonstrate the mechanosensing of chondrocytes for changes in stiffness in the range of Pascals. We also identified Non-Muscle Myosin II (NMMII) and integrins α1, ß1 and ß3 as participants in the mechanosensing, since their blockade inhibits the sensing of the stiffness, and they are up-regulated in the process. RT-PCR arrays and western blot detected up-regulation of Paxillin, RhoA, Fos, Jun and Sox9. We detected no expression of Src in the monolayer cultures, but we found a role for this protein in 3D. The expression of HIF-1α was not modified under normoxia but was found to participate under hypoxia. Focal Adhesion Kinase (FAK), showed a direct relationship with the expression of Aggrecan in hypoxia and an inverse one in normoxia. Finally, immunofluorescence analysis located the expression of factors AP-1, Sox-9 and HIF-1α inside the cell nuclei and RhoA, Src, Paxillin and FAK close to the cytoplasmic membrane. CONCLUSIONS: We determined here some of the genes that are up-regulated during the process of chondrocyte mechanosensing.
Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Gene Expression/genetics , Signal Transduction/genetics , Animals , Blotting, Western , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , Genes, fos/genetics , Genes, jun/genetics , Genes, src/genetics , Hindlimb , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Integrins/genetics , Integrins/metabolism , Myosin Type II/genetics , Myosin Type II/metabolism , Paxillin/genetics , Paxillin/metabolism , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
Objetivo: analizar la reparación «in vitro» de lesiones cilíndricas de la zona avascular del menisco tratadas con factores de crecimiento y rellenas con diferentes materiales. Material y métodos: las lesiones fueron rellenadas con cilindros de menisco fresco, menisco congelado a 20 ºC, y con esponjas artificiales de colágeno cultivadas con células de cartílago. Todos los grupos fueron tratados con TGF-Beta1, IGF-1 y OP-1® y analizados a las 2, 4, 6 y 8 semanas de evolución. Resultados: el TGF-Beta1 y el IGF-1 indujeron puentes parciales de tejido entre la estructura de relleno y los bordes de la lesión, así como la expresión de colágeno de tipo II y la proliferación celular en toda la superficie del menisco. Así mismo, observamos una migración de células desde el propio menisco lesionado hacia las esponjas de colágeno utilizadas, acelerada por la presencia de TGF-Beta1 e IGF-1. La migración de células a la esponja no se corresponde con procesos de proliferación. No obtuvimos ningún efecto positivo con la proteína OP-1®. Conclusión: la utilización de IGF-1 y TGF-Beta1 y rellenando la lesión con cilindros de menisco o matrices de colágeno pueden ser de utilidad para mejorar la respuesta en modelos experimentales de reparación meniscal
Meniscus is a support tissue formed by fibrocartilage, which function is essential for the correct movement of the knee joint and for the performance of phisical activity. Outer part is vascularized, while inner is avascular and in which lesions are commonly produced. Its widely accepted that avascular lesions have the most complicated prognosis, as no regenerative processes are produced in that zone. In our study we have constructed an in vitro model of avascular lesions, by culturing complete menisci fragments, and practising them holes in their inner part. Lesions where filled up with meniscal tissue either fresh or freezed at 20 ºC, and with artificial collagen sponges injected with articular cartilage cells. All groups were treated with growth factors TGF-Beta1, IGF-1 and OP-1, and analyzed at 2, 4, 6 and 8 weeks. Results showed that both TGF-Beta1 and IGF-1 did induce the formation of tissue unions between support structure and the borders of the lesion, as well as collagen type II expression and a stimulation of proliferation in cells in the meniscal surface. On the other hand we oberved no significant effect by OP-1. We also detected a migration process of cells from the lesioned meniscus throguh collagen sponges used, that was accelerated by the presence of TGF-Beta1 and IGF-1. Results point to an effective usage of these growth factors and collagen scaffolds, in the treatment of lesions of the avascular zone of the meniscus
Subject(s)
Humans , Menisci, Tibial/surgery , Transforming Growth Factor beta/therapeutic use , Insulin-Like Growth Factor I/therapeutic use , ImmunohistochemistryABSTRACT
El cartílago articular es el tejido encargado de disminuirla fricción entre las superficies articulares durante el movimiento.Su limitada capacidad de regeneración hace quelas lesiones osteocondrales posean un mal pronóstico ypuedan acabar generando una artrosis en la articulación.La terapia génica para este tipo de patologías resulta muyprometedora, puesto que actualmente, no hay ningún procedimientocapaz de restablecer el tejido dañado. En nuestroestudio hemos puesto a punto un modelo de transferenciagénica a los tejidos de la articulación de la rodillade rata, inyectando intraarticularmente vectores derivadosde virus adeno-asociados, capaces de inducir la expresiónde luciferasa. Los resultados muestran cómo la inducciónde la expresión resulta significativa a partir de los dos mesesde evolución en todos los tejidos articulares (cartílagoarticular, menisco, membrana sinovial y hueso subcondral).La presencia de daño, tanto de tipo mecánico comoautoinmune (artritis inducida por colágeno) no modifica laexpresión de proteína por parte del vector
Articular cartilage is a tissue that decreases friction onarticular surfaces during movement. Its limited regenerativecapacity makes osteocondral lessions to extend andgenerate osteoarthritis in the joint. Gene therapy is apromising alternative to these patologies, since there areno proceedings actually, that restablishes damaged tissue.In our study, we have developed a model of gene transferto articular tissues injecting intraarticularly an adeno-associatedderived vector that induces expression of luciferase.Results obtained show a significant induction of proteinexpression two months after injecting vectors, in all articulartissues (articular cartilage, meniscus, synovium andsubcondral bone). The presence of lessions, mechanic orautoimmune (collagen induced arthritis) do not modify theexpression of protein induced by the vector
