RESUMEN
Injured tendons have limited repair ability after full-thickness lesions. Tendon regeneration properties and adverse reactions were assessed ex vivo in an experimental animal model using a new collagen I membrane. The multilamellar membrane obtained from purified equine Achilles tendon is characterized by oriented collagen I fibers and has been shown to sustain cell growth and orientation in vitro. The central third of the patellar tendon (PT) of 10 New Zealand White rabbits was sectioned and grafted with the collagen membrane; the contralateral PT was cut longitudinally (sham-operated controls). Animals were euthanized 1 or 6 months after surgery, and tendons were subjected to histological and Synchrotron Radiation-based Computed Microtomography (SRµCT) examination and 3D structure analysis. Histological and SRµCT findings showed satisfactory graft integration with native tendon. Histological examination also showed ongoing angiogenesis. Adverse side-effects (inflammation, rejection, calcification) were not observed. The multilamellar collagen I membrane can be considered as an effective tool for tendon defect repair and tendon augmentation.
Asunto(s)
Colágeno Tipo I/fisiología , Ligamento Rotuliano/cirugía , Traumatismos de los Tendones/fisiopatología , Traumatismos de los Tendones/cirugía , Andamios del Tejido , Tendón Calcáneo/fisiología , Tendón Calcáneo/trasplante , Animales , Colágeno Tipo I/aislamiento & purificación , Modelos Animales de Enfermedad , Imagenología Tridimensional , Masculino , Ligamento Rotuliano/diagnóstico por imagen , Ligamento Rotuliano/fisiología , Conejos , Procedimientos de Cirugía Plástica , Regeneración/fisiología , Traumatismos de los Tendones/diagnóstico por imagen , Cicatrización de Heridas/fisiología , Microtomografía por Rayos XRESUMEN
The aim of this work was to study the in vitro biocompatibility of glass-ceramic scaffolds based on 45S5 Bioglass, using a human osteosarcoma cell line (HOS-TE85). The highly porous scaffolds were produced by the foam replication technique. Two different types of scaffolds with different porosities were analysed. They were coated with a biodegradable polymer, poly(3-hydroxybutyrate) (P(3HB)). The scaffold bioactivity was evaluated by soaking in a simulated body fluid (SBF) for different durations. Compression strength tests were performed before and after immersion in SBF. These experiments showed that the scaffolds are highly bioactive, as after a few days of immersion in SBF a hydroxyapatite-like layer was formed on the scaffold's surface. It was also observed that P(3HB)-coated samples exhibited higher values of compression strength than uncoated samples. Biocompatibility assessment was carried out by qualitative evaluation of cell morphology after different culture periods, using scanning electron microscopy, while cell proliferation was determined by using the AlamarBlue assay. Alkaline phosphatase (ALP) and osteocalcin (OC) assays were used as quantitative in vitro indicators of osteoblast function. Two different types of medium were used for ALP and OC tests: normal supplemented medium and osteogenic medium. HOS cells were seeded and cultured onto the scaffolds for up to 2 weeks. The AlamarBlue assay showed that cells were able to proliferate and grow on the scaffold surface. After 7 days in culture, the P(3HB)-coated samples had a higher number of cells on their surfaces than the uncoated samples. Regarding ALP- and OC-specific activity, no significant differences were found between samples with different pore sizes. All scaffolds containing osteogenic medium seemed to have a slightly higher level of ALP and OC concentration. These experiments confirmed that Bioglass/P(3HB) scaffolds have potential as osteoconductive tissue engineering substrates for maintenance and normal functioning of bone tissue.
Asunto(s)
Materiales Biocompatibles , Cerámica , Vidrio , Hidroxibutiratos , Poliésteres , Fosfatasa Alcalina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo , Humanos , Técnicas In Vitro , Microscopía Electrónica de Rastreo , Osteocalcina/metabolismo , Osteosarcoma/patología , Espectrometría de Fluorescencia , Tensión SuperficialRESUMEN
Synchrotron radiation micro-computed tomography (SRmuCT) revealed the microstructure of a CEL2 glass-ceramic scaffold with macropores of several hundred microns characteristic length, in terms of the voxel-by-voxel 3D distribution of the attenuation coefficients throughout the scanned space. The probability density function of all attenuation coefficients related to the macroporous space inside the scaffold gives access to the tomograph-specific machine error included in the SRmuCT measurements (also referred to as instrumental resolution function). After Lorentz function-based clearing of the measured CT data from the systematic resolution error, the voxel-specific attenuation information of the voxels representing the solid skeleton is translated into the composition of the material inside one voxel, in terms of the nanoporosity embedded in a dense CEL2 glass-ceramic matrix. Based on voxel-invariant elastic properties of dense CEL2 glass-ceramic, continuum micromechanics allows for translation of the voxel-specific nanoporosity into voxel-specific elastic properties. They serve as input for Finite Element analyses of the scaffold structure. Young's modulus of a specific CT-scanned macroporous scaffold sample, predicted from a Finite Element simulation of a uniaxial compression test, agrees well with the experimental value obtained from an ultrasonic test on the same sample. This highlights the satisfactory predictive capabilities of the presented approach.
Asunto(s)
Huesos/diagnóstico por imagen , Tomografía Computarizada por Rayos X/métodos , Fenómenos Biomecánicos , Elasticidad , Modelos Biológicos , Nanoestructuras , Porosidad , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
The aim of this study was the preparation and characterization of bioactive glass-ceramic scaffolds for bone tissue engineering. For this purpose, a glass belonging to the system SiO2-P2O5-CaO-MgO-Na2O-K2O (CEL2) was used. The sponge-replication method was adopted to prepare the scaffolds; specifically, a polymeric skeleton was impregnated with a slurry containing CEL2 powder, polyvinyl alcohol (PVA) as a binding agent and distilled water. The impregnated sponge was then thermally treated to remove the polymeric phase and to sinter the inorganic one. The obtained scaffolds possessed an open and interconnected porosity, analogous to cancellous bone texture, and with a mechanical strength above 2 MPa. Moreover, the scaffolds underwent partial bioresorption due to ion-leaching phenomena. This feature was investigated by X-ray computed microcomputed tomography (micro-CT). Micro-CT is a three-dimensional (3-D) radiographic imaging technique, able to achieve a spatial resolution close to 1 microm(3). The use of synchrotron radiation allows the selected photon energy to be tuned to optimize the contrast among the different phases in the investigated samples. The 3-D scaffolds were soaked in a simulated body fluid (SBF) to study the formation of hydroxyapatite microcrystals on the scaffold struts and on the internal pore walls. The 3-D scaffolds were also soaked in a buffer solution (Tris-HCl) for different times to assess the scaffold bioresorption according to the ISO standard. A gradual resorption of the pores walls was observed during the soakings both in SBF and in Tris-HCl.