RESUMEN
A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T(2)) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T(2) values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T(2) values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 +/- 0.02 for control strips to a maximum value of 0.31 +/- 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils.
Asunto(s)
Colágeno/química , Colágeno/ultraestructura , Cristalografía/métodos , Imagen por Resonancia Magnética/métodos , Microscopía/métodos , Minerales/químicaRESUMEN
We present a three-dimensional mineralizing model based on a hollow fiber bioreactor (HFBR) inoculated with primary osteoblasts isolated from embryonic chick calvaria. Using non-invasive magnetic resonance microscopy (MRM), the growth and development of the mineralized tissue around the individual fibers were monitored over a period of 9 weeks. Spatial maps of the water proton MRM properties of the intact tissue, with 78 microm resolution, were used to determine changes in tissue composition with development. Unique changes in the mineral and collagen content of the tissue were detected with high specificity by proton density (PD) and magnetization transfer ratio (MTR) maps, respectively. At the end of the growth period, the presence of a bone-like tissue was verified by histology and the formation of poorly crystalline apatite was verified by selected area electron diffraction and electron probe X-ray microanalysis. FTIR microspectroscopy confirmed the heterogeneous nature of the bone-like tissue formed. FTIR-derived phosphate maps confirmed that those locations with the lowest PD values contained the most mineral, and FTIR-derived collagen maps confirmed that bright pixels on MTR maps corresponded to regions of high collagen content. In conclusion, the spatial mapping of tissue constituents by FTIR microspectroscopy corroborated the findings of non-invasive MRM measurements and supported the role of MRM in monitoring the bone formation process in vitro.
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Reactores Biológicos , Huesos/citología , Huesos/metabolismo , Animales , Calcificación Fisiológica , Células Cultivadas , Embrión de Pollo , Colágeno/metabolismo , Imagen por Resonancia Magnética , Minerales/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Fosfatos/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Ingeniería de TejidosRESUMEN
Paramagnetic manganese (II) can be employed as a calcium surrogate to sensitize magnetic resonance microscopy (MRM) to the processing of calcium during bone formation. At high doses, osteoblasts can take up sufficient quantities of manganese, resulting in marked changes in water proton T(1), T(2) and magnetization transfer ratio values compared to those for untreated cells. Accordingly, inductively coupled plasma mass spectrometry (ICP-MS) results confirm that the manganese content of treated cell pellets was 10-fold higher than that for untreated cell pellets. To establish that manganese is processed like calcium and deposited as bone, calvaria from the skull of embryonic chicks were grown in culture medium supplemented with 1 mM MnCl(2) and 3 mM CaCl(2). A banding pattern of high and low T(2) values, consistent with mineral deposits with high and low levels of manganese, was observed radiating from the calvarial ridge. The results of ICP-MS studies confirm that manganese-treated calvaria take up increasing amounts of manganese with time in culture. Finally, elemental mapping studies with electron probe microanalysis confirmed local variations in the manganese content of bone newly deposited on the calvarial surface. This is the first reported use of manganese-enhanced MRM to study the process whereby calcium is taken up by osteoblasts cells and deposited as bone.
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Calcificación Fisiológica , Cloruros/química , Espectroscopía de Resonancia Magnética/métodos , Compuestos de Manganeso/química , Osteoblastos/metabolismo , Animales , Embrión de Pollo , PollosRESUMEN
One of the intents of tissue engineering is to fabricate biological materials for the augmentation or replacement of impaired, damaged, or diseased human tissue. In this context, novel models of the human phalanges have been developed recently through suturing of polymer scaffolds supporting osteoblasts, chondrocytes, and tenocytes to mimic bone, cartilage, and tendon, respectively. Characterization of the model constructs has been accomplished previously through histological and biochemical means, both of which are necessarily destructive to the constructs. This report describes the application of two complementary, non-destructive, non-invasive techniques, magnetic resonance microscopy (MRM) and X-ray microtomography (XMT or quantitative computed tomography), to evaluate the spatial and temporal growth and developmental status of tissue elements within tissue-engineered constructs obtained after 10 and 38 weeks of implantation in athymic (nude) mice. These two times represent respective points at which model middle phalanges are comprised principally of organic components while being largely unmineralized and later become increasingly more mineralized. The spatial distribution of mineralized deposits within intact constructs was readily detected by XMT (qCT) and was comparable to low intensity zones observed on MRM hydration maps. Moreover, the MRM-derived hydration values for mineralized zones were inversely correlated with mineral densities measured by XMT. In addition, the MRM method successfully mapped fat deposits, collagenous tissues, and the hydration state of the soft tissue elements comprising the specimens. These results support the application of non-destructive, non-invasive, quantitative MRM and XMT for the evaluation of constituent tissue elements within complex constructs of engineered implants.
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Órganos Bioartificiales , Falanges de los Dedos de la Mano , Imagen por Resonancia Magnética , Ingeniería de Tejidos , Tomografía Computarizada por Rayos X , Animales , Animales Recién Nacidos , Cartílago Articular/citología , Bovinos , Técnicas de Cultivo de Célula , Células Cultivadas , Condrocitos/citología , Medios de Cultivo , Humanos , Implantes Experimentales , Ácido Láctico/química , Ratones , Ratones Desnudos , Osteoblastos/citología , Poliésteres , Ácido Poliglicólico/química , Polímeros/química , Factores de TiempoRESUMEN
Invasive "body-opening" autopsy represents the traditional means of postmortem investigation in humans. However, modern cross-sectional imaging techniques can supplement and may even partially replace traditional autopsy. Computed tomography (CT) is the imaging modality of choice for two- and three-dimensional documentation and analysis of autopsy findings including fracture systems, pathologic gas collections (eg, air embolism, subcutaneous emphysema after trauma, hyperbaric trauma, decomposition effects), and gross tissue injury. Various postprocessing techniques can provide strong forensic evidence for use in legal proceedings. Magnetic resonance (MR) imaging has had a greater impact in demonstrating soft-tissue injury, organ trauma, and nontraumatic conditions. However, the differences in morphologic features and signal intensity characteristics seen at antemortem versus postmortem MR imaging have not yet been studied systematically. The documentation and analysis of postmortem findings with CT and MR imaging and postprocessing techniques ("virtopsy") is investigator independent, objective, and noninvasive and will lead to qualitative improvements in forensic pathologic investigation. Future applications of this approach include the assessment of morbidity and mortality in the general population and, perhaps, routine screening of bodies prior to burial.
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Autopsia/métodos , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Imagen por Resonancia Magnética/métodos , Programas Informáticos , Tomografía Computarizada por Rayos X/métodos , Interfaz Usuario-Computador , Gráficos por Computador , Humanos , Aumento de la Imagen/métodos , Procedimientos Quirúrgicos Mínimamente InvasivosRESUMEN
Magnetic resonance microscopy (MRM) and X-ray microtomography (XMT) were used to investigate de novo bone formation in porous poly(ethyl methacrylate) (PEMA) scaffolds, prepared by a novel co-extrusion process. PEMA scaffolds were seeded with primary chick calvarial osteoblasts and cultured under static conditions for up to 8 weeks. Bone formation within porous PEMA scaffolds was confirmed by the application of histologic stains to intact PEMA disks. Disks were treated with Alizarin red to visualize calcium deposits and with Sirius red to visualize regions of collagen deposition. DNA analysis confirmed that cells reached confluence on the scaffolds after 7 weeks in static culture. The formation of bone in PEMA scaffolds was investigated with water proton MRM. Quantitative MRM maps of the magnetization transfer ratio (MTR) yielded maps of protein deposition, and magnetic resonance (MR) relaxation times (T1 and T2) yielded maps of mineral deposition. The location of newly formed bone and local mineral concentrations were confirmed by XMT. By comparing MRM and XMT data from selected regions-of-interest in one sample, the inverse relationship between the MR relaxation times and mineral concentration was validated, and calibration curves for estimating the mineral content of cell-seeded PEMA scaffolds from quantitative MRM images were developed.
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Regeneración Ósea/fisiología , Metilmetacrilatos , Osteoblastos/fisiología , Ingeniería de Tejidos , Animales , División Celular/fisiología , Embrión de Pollo , Imagen por Resonancia Magnética , Osteoblastos/citologíaRESUMEN
Magnetic resonance imaging (MRI) studies of tissue engineered constructs prior to implantation clearly demonstrate the utility of the MRI technique for studying the bone formation process. To test the utility of our MRI protocols for explant studies, we present a novel test platform in which osteoblast-seeded scaffolds were implanted on the chorioallantoic membrane of a chick embryo. Scaffolds from the following experimental groups were examined by high-resolution MRI: (a) cell-seeded implanted scaffolds (CIM), (b) unseeded implanted scaffolds (UCIM), (c) cell-seeded scaffolds in static culture (CIV) and (d) unseeded scaffolds in static culture (UCIV). The reduction in water proton transverse relaxation times and the concomitant increase in water proton magnetization transfer ratios for CIM and CIV scaffolds, compared to UCIV scaffolds, were consistent with the formation of a bone-like tissue within the polymer scaffold, which was confirmed by immunohistochemistry and fluorescence microscopy. However, the presence of angiogenic vessels and fibrotic adhesions around UCIM scaffolds can confound MRI findings of bone deposition. Consequently, to improve the specificity of the MRI technique for detecting mineralized deposits within explanted tissue engineered bone constructs, we introduce a novel contrast agent that uses alendronate to target a Food and Drug Administration-approved MRI contrast agent (Gd-DOTA) to bone mineral. Our contrast agent termed GdALN was used to uniquely identify mineralized deposits in representative samples from our four experimental groups. After GdALN treatment, both CIM and CIV scaffolds, containing mineralized deposits, showed marked signal enhancement on longitudinal relaxation time-weighted (T1W) images compared to UCIV scaffolds. Relative to UCIV scaffolds, some enhancement was observed in T1W images of GdALN-treated UCIM scaffolds, subjacent to the dark adhesions at the scaffold surface, possibly from dystrophic mineral formed in the fibrotic adhesions. Notably, residual dark areas on T1W images of CIM and UCIM scaffolds were attributable to blood inside infiltrating vessels. In summary, we present the efficacy of GdALN for sensitizing the MRI technique to the deposition of mineralized deposits in explanted polymeric scaffolds.
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Sustitutos de Huesos/química , Membrana Corioalantoides/metabolismo , Medios de Contraste/farmacología , Imagen por Resonancia Magnética/métodos , Alendronato/farmacología , Animales , Huesos/patología , Pollos , Fibrosis/patología , Compuestos Heterocíclicos/farmacología , Inmunohistoquímica/métodos , Microscopía Fluorescente/métodos , Compuestos Organometálicos/farmacología , Osteoblastos/citología , Óvulo/patología , Polímeros/química , Prótesis e Implantes , Ingeniería de Tejidos/métodos , Andamios del TejidoRESUMEN
Paramagnetic manganese can be employed as a calcium surrogate to sensitize the magnetic resonance imaging (MRI) technique to the processing of calcium during the bone formation process. At low doses, after just 48h of exposure, osteoblasts take up sufficient quantities of manganese to cause marked reductions in the water proton T1 values compared with untreated cells. After just 24h of exposure, 25µM MnCl(2) had no significant effect on cell viability. However, for mineralization studies 100µM MnCl(2) was used to avoid issues of manganese depletion in calvarial organ cultures and a post-treatment delay of 48h was implemented to ensure that manganese ions taken up by osteoblasts is deposited as mineral. All specimens were identified by their days in vitro (DIV). Using inductively coupled plasma optical emission spectroscopy (ICP-OES), we confirmed that Mn-treated calvariae continued to deposit mineral in culture and that the mineral composition was similar to that of age-matched controls. Notably there was a significant decrease in the manganese content of DIV18 compared with DIV11 specimens, possibly relating to less manganese sequestration as a result of mineral maturation. More importantly, quantitative T1 maps of Mn-treated calvariae showed localized reductions in T1 values over the calvarial surface, indicative of local variations in the surface manganese content. This result was verified with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). We also found that ΔR1 values, calculated by subtracting the relaxation rate of Mn-treated specimens from the relaxation rate of age-matched controls, were proportional to the surface manganese content and thus mineralizing activity. From this analysis, we established that mineralization of DIV4 and DIV11 specimens occurred in all tissue zones, but was reduced for DIV18 specimens because of mineral maturation with less manganese sequestration. In DIV25 specimens, active mineralization was observed for the expanding superficial surface and ΔR1 values were increased due to the mineralization of small, previously unmineralized areas. Our findings support the use of manganese-enhanced MRI (MEMRI) to study well-orchestrated mineralizing events that occur during embryonic development. In conclusion, MEMRI is more sensitive to the study of mineralization than traditional imaging approaches.
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Calcificación Fisiológica/fisiología , Imagen por Resonancia Magnética/métodos , Manganeso/farmacología , Animales , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Pollos , Medios de Cultivo/farmacología , Terapia por Láser , Espectrometría de Masas , Técnicas de Cultivo de Órganos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Fósforo/metabolismo , Cráneo/citología , Espectrofotometría Atómica , Fracciones Subcelulares/efectos de los fármacosRESUMEN
Several years ago, manganese-enhanced magnetic resonance imaging (MEMRI) was introduced as a new powerful tool to image active brain areas and to identify neural connections in living, non-human animals. Primarily restricted to studies in rodents and later adapted for bird species, MEMRI has recently been discovered as a useful technique for neuroimaging of invertebrate animals. Using crayfish as a model system, we highlight the advantages of MEMRI over conventional techniques for imaging of small nervous systems. MEMRI can be applied to image invertebrate nervous systems at relatively high spatial resolution, and permits identification of stimulus-evoked neural activation non-invasively. Since the selection of specific imaging parameters is critical for successful in vivo micro-imaging, we present an overview of different experimental conditions that are best suited for invertebrates. We also compare the effects of hardware and software specifications on image quality, and provide detailed descriptions of the steps necessary to prepare animals for successful imaging sessions. Careful consideration of hardware, software, experiments, and specimen preparation will promote a better understanding of this novel technique and facilitate future MEMRI studies in other laboratories.
RESUMEN
PURPOSE: To determine if magnetic resonance (MR) microscopy can yield images sufficient for discriminating early progressive atherosclerotic lesions from nonprogressive atherosclerotic lesions in human coronary arteries. MATERIALS AND METHODS: Institutional review board approval and informed consent were not required. Seventeen coronary artery segments (mean diameter, 2.8 mm +/- 1.0 [standard deviation]) were collected within 36 hours after death from 11 cadavers (six men, five women; age range at death, 33-65 years). Quantitative T1, T2, intensity-weighted (IW), and magnetization transfer (MT) maps were acquired with a 9.4-T vertical-bore magnet. Coronary artery lesions were classified as adaptive intimal thickening (AIT), pathologic intimal thickening (PIT), or intimal xanthoma (IXA). Internal anatomic fiducial landmarks and stains were applied to proximal and epicardial vessel surfaces and used to register histologic sections with MR images and thus enable comparison of MR images and Movat pentachrome-stained histologic specimens. Unique 0.0012-0.0287-cm(2) regions of interest were visually identified on quantitative T1, T2, MT, and IW maps of AIT, IXA, and PIT lesions. Distributions of T1, T2, MT, and IW values were compared with Student t and Wilcoxon two-sample tests. RESULTS: MR microscopic images of nonprogressive AIT and IXA lesions revealed two intimal layers. The luminal intima had higher T1 and T2 values and lower MT values than did the medial intima; these findings were consistent with compositional differences observed in histologic sections. In the IXA lesion, T2 values of both intimal layers were markedly reduced when compared with T2 values of AIT lesions because of the accumulation of lipid-laden macrophages in both layers. Progressive PIT lesions had a typical multilayered appearance or foci with a short T2 relaxation time and low IW values; these features were not observed in AIT or IXA lesions. CONCLUSION: MR microscopy enabled identification of morphologic arterial wall features that enable discrimination of progressive PIT lesions from nonprogressive AIT or IXA lesions.
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Enfermedad de la Arteria Coronaria/patología , Vasos Coronarios/patología , Imagen por Resonancia Magnética/métodos , Microscopía/métodos , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Túnica Íntima/patologíaRESUMEN
Magnetic resonance imaging (MRI) has become a staple of diagnostic radiology. Despite its diagnostic utility the resolving power of typical clinical MRI instruments is only on the order of 1 mm. This has led to the development of magnetic resonance microscopy (MRM), which employs the same physical imaging principals used in MRI, but with instrumentation designed to resolve structural details down to the level of 10-100 microns in samples ranging from less than 1mm to several centimeters in size. Until recently, major advancements in MRM have focused on hardware and software developments allowing the detection of radio-frequency signals originating from very small volume elements within the sample. Such high-resolution images have facilitated the early detection of diseased tissue by focusing on sub-millimeter structural changes induced in the tissue. To sensitize the MRM technique to pathologic tissue changes, investigators have developed techniques, such as chemical shift imaging to detect pre-cancerous changes in tissue metabolism and MR relaxometry to detect changes in tissue composition during the earliest stages of degeneration for diseases such as osteoarthritis or multiple sclerosis. However, such non-specific measurements can only serve as surrogate measures of disease progression and potential measures of treatment efficacy. As disease diagnosis moves from the anatomic to the molecular stage, scientists will require imaging techniques that can detect molecular events deep inside the human body. To meet this goal, MR scientists are working to improve imaging resolutions in vivo and they are developing molecular probes that can dramatically amplify the MR signal in response to specific and highly localized molecular events. This article will identify current trends in the MRM field aimed at meeting the challenges imposed by molecular imaging and areas for future development in this highly promising imaging field.