RESUMEN
OBJECTIVE: Despite new strategies in tissue engineering, cartilage repair remains a major challenge. Our aim is to treat patients with focal lesions of articular cartilage with autologous hyaline cartilage implants using a scaffold-free approach. In this article, we describe experiments to optimize production of scaffold-free cartilage discs. DESIGN: Articular chondrocytes were expanded in vitro, seeded in transwell inserts and redifferentiated using established chondrogenic components. Experimental variables included testing 2 different expansion media, adding bone morphogenetic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), growth/differentiation factor 5 (GDF5), or fibroblast growth factor 18 (FGF18) to the differentiation medium and allowing the disc to float freely in large wells. Cartilage discs were analyzed by weight and thickness, real-time RT-qPCR (reverse transcriptase qualitative polymerase chain reaction), fluorescence immunostaining, transmission electron microscopy, second harmonic generation imaging, and measurement of Young's modulus. RESULTS: Addition of BMP2 to the chondrogenic differentiation medium (CDM) was essential for stable disc formation, while IGF1, GDF5, and FGF18 were redundant. Allowing discs to float freely in CDM on a moving platform increased disc thickness compared with discs kept continuously in transwell inserts. Discs cultured for 6 weeks reached a thickness of almost 2 mm and Young's modulus of >200 kPa. There was abundant type II collagen. Collagen fibrils were 25 nm thick, with a tendency to be organized perpendicular to the disc surface. CONCLUSION: Scaffold-free engineering using BMP2 and providing free movement in CDM produced firm, elastic cartilage discs with abundant type II collagen. This approach may potentially be used in clinical trials.
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Cartílago Articular/cirugía , Condrocitos , Ingeniería de Tejidos , Células Cultivadas , Condrogénesis , Colágeno Tipo II , HumanosRESUMEN
A method for measuring three-dimensional (3-D) direction images of collagen fibers in biological tissue is presented. Images of the 3-D directions are derived from the measured transmission Mueller matrix images (MMIs), acquired at different incidence angles, by taking advantage of the form birefringence of the collagen fibers. The MMIs are decomposed using the recently developed differential decomposition, which is more suited to biological tissue samples than the common polar decomposition method. Validation of the 3-D direction images was performed by comparing them with images from second-harmonic generation microscopy. The comparison found a good agreement between the two methods. It is envisaged that 3-D directional imaging could become a useful tool for understanding the collagen framework for fibers smaller than the diffraction limit.
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Colágeno/ultraestructura , Imagenología Tridimensional/métodos , Microscopía/métodos , Acústica , Animales , Pollos , Reproducibilidad de los Resultados , Tendones/química , Tendones/ultraestructuraRESUMEN
The collagen meshwork in articular cartilage of chicken knee is characterized using Mueller matrix imaging and multiphoton microscopy. Direction and degree of dispersion of the collagen fibers in the superficial layer are found using a Fourier transform image-analysis technique of the second-harmonic generated image. Mueller matrix images are used to acquire structural data from the intermediate layer of articular cartilage where the collagen fibers are too small to be resolved by optical microscopy, providing a powerful multimodal measurement technique. Furthermore, we show that Mueller matrix imaging provides more information about the tissue compared to standard polarization microscopy. The combination of these techniques can find use in improved diagnosis of diseases in articular cartilage, improved histopathology, and additional information for accurate biomechanical modeling of cartilage.
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Cartílago Articular/anatomía & histología , Cartílago Articular/química , Diagnóstico por Imagen/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía de Polarización/métodos , Procesamiento de Señales Asistido por Computador , Animales , Pollos , Microscopía de Fluorescencia por Excitación MultifotónicaRESUMEN
Cardiovascular disease is the primary cause of death in the United States; the majority of these deaths are caused by the rupture of vulnerable plaques. An important feature of vulnerable plaques is the thickness of the fibrous cap that covers the necrotic core. A thickness of less than 65 microm has been proposed as a value that renders the plaque prone to rupture. This work shows that multiphoton microscopy (MPM) can image the plaque with microm resolution to a depth deeper than 65 microm. The fibrous cap emits primarily second harmonic generation due to collagen, in contrast to the necrotic core and healthy artery, which emits primarily two-photon excited fluorescence from elastin. This gives a good demarcation of the fibrous cap from underlying layers, facilitating the measurement of the fibrous cap thickness. Based on a measure of the collagen/elastin ratio, plaques were detected with a sensitivity of 65% and specificity of 81%. Furthermore, the technique gives detailed information on the structure of the collagen network in the fibrous cap. This network ultimately determines the mechanical strength of the plaque. A mechanical model based on this information could yield a measure of the propensity of the plaque to rupture.