PET imaging of a collagen matrix reveals its effective injection and targeted retention in a mouse model of myocardial infarction.

Injectable biomaterials have shown promise for cardiac regeneration therapy. However, little is known regarding their retention and distribution upon application in vivo. Matrix imaging would be useful for evaluating these important properties. Herein, hexadecyl-4-[(18)F]fluorobenzoate ((18)F-HFB) and Qdot labeling was used to evaluate collagen matrix delivery in a mouse model of myocardial infarction (MI). At 1 wk post-MI, mice received myocardial injections of (18)F-HFB- or Qdot-labeled matrix to assess its early retention and distribution (at 10 min and 2h) by positron emission tomography (PET), or fluorescence imaging, respectively. PET imaging showed that the bolus of matrix at 10 min redistributed evenly within the ischemic territory by 2h. Ex vivo biodistribution revealed myocardial matrix retention of ∼ 65%, which correlated with PET results, but may be an underestimate since (18)F-HFB matrix labeling efficiency was ∼ 82%. For covalently linked Qdots, labeling efficiency was ∼ 96%. Ex vivo Qdot quantification showed that ∼ 84% of the injected matrix was retained in the myocardium. Serial non-invasive PET imaging and validation by fluorescence imaging confirmed the effectiveness of the collagen matrix to be retained and redistributed within the infarcted myocardium. This study identifies matrix-targeted imaging as a promising modality for assessing the biodistribution of injectable biomaterials for application in the heart.

Micro- and nano-CT for the study of bone ultrastructure.

Micro-computed tomography (micro-CT)-a version of X-ray CT operating at high spatial resolution-has had a considerable success for the investigation of trabecular bone micro-architecture. Currently, there is a lot of interest in exploiting CT techniques at even higher spatial resolutions to assess bone tissue at the cellular scale. After recalling the basic principles of micro-CT, we review the different existing system, based on either standard X-ray tubes or synchrotron sources. Then, we present recent applications of micro- and nano-CT for the analysis of osteocyte lacunae and the lacunar-canalicular network. We also address the question of the quantification of bone ultrastructure to go beyond the sole visualization.

A novel poly(amido amine)-dendrimer-based hydrogel as a mimic for the extracellular matrix.

The extracellular matrix is mimicked by a novel dendrimer-based hydrogel, which exhibits a highly interconnected porous network, enhanced mechanical stiffness, and a low swelling ratio. The hydrogel system supports the proliferation and differentiation of mesenchymal stem cells without any cytotoxic effects. This dendrimer-based hydrogel may serve as a model for developing new advanced materials with applications in tissue engineering.

Visualizing the three-dimensional mesoscopic structure of dermal tissues.

Thorough knowledge of dermal tissue structure in three dimensions is not only a prerequisite for understanding the relationship between cells and their extracellular matrix, but also provides a basis for understanding of wound healing and scar formation for designing the ideal scaffold for skin tissue engineering. Here, we show for the first time the visualization of the three dimensional (3D) structure of dermal tissue by phase-contrast microtomography (µCT) with third-generation synchrotron radiation (SR). Compared with irregular dermal tissue (such as scar tissue), the normal dermal tissues were found to consist of a network of elliptically shaped regions containing a web of fibre bundles. The bundles, composed of fibres, were found to be orientated along specific directions, indicative of helical weaving. A regular array of dentate structure was shown on the fibres. The results showed that phase-contrast µCT with SR had a distinct advantage in accurately viewing the 3D microstructure of dermal tissues.

Functional ultrasound imaging for assessment of extracellular matrix scaffolds used for liver organoid formation.

A method of 3D functional ultrasound imaging has been developed to enable non-destructive assessment of extracellular matrix scaffolds that have been prepared by decellularization protocols and are intended for recellularization to create organoids. A major challenge in organ decellularization is retaining patent micro-vascular structures crucial for nutrient access and functionality of organoids. The imaging method described here provides statistical distributions of flow rates throughout the tissue volumes, 3D vessel network architecture visualization, characterization of microvessel volumes and sizes, and delineation of matrix from vascular circuits. The imaging protocol was tested on matrix scaffolds that are tissue-specific, but not species-specific, matrix extracts, prepared by a process that preserved >98% of the collagens, collagen-associated matrix components, and matrix-bound growth factors and cytokines. Image-derived data are discussed with respect to assessment of scaffolds followed by proof-of-concept studies in organoid establishment using Hep3B, a human hepatoblast-like cell line. Histology showed that the cells attached to scaffolds with patent vasculature within minutes, achieved engraftment at near 100%, expressed liver-specific functions within 24 h, and yielded evidence of proliferation and increasing differentiation of cells throughout the two weeks of culture studies. This imaging method should prove valuable in analyses of such matrix scaffolds.

Measurement of myocardial extracellular volume fraction by using equilibrium contrast-enhanced CT: validation against histologic findings.

PURPOSE: To develop and validate equilibrium contrast material-enhanced computed tomography (CT) to measure myocardial extracellular volume (ECV) fraction by using a histologic reference standard and to compare equilibrium CT with equilibrium contrast-enhanced magnetic resonance (MR) imaging. MATERIALS AND METHODS: A local ethics committee approved the study, and all subjects gave fully informed written consent. An equilibrium CT protocol was developed using iohexol at 300 mg of iodine per milliliter (bolus of 1 mg per kilogram of body weight administered at a rate of 3 mL/sec, followed immediately by an infusion of 1.88 mL/kg per hour with CT imaging before and at 25 minutes after injection of bolus of contrast agent) and ECV within the myocardial septum measured using both equilibrium CT and equilibrium MR imaging in patients with severe aortic stenosis. Biopsy samples of the myocardial septum collected during valve replacement surgery were used for histologic quantification of extracellular fibrosis with picrosirius red staining. Equilibrium CT- and equilibrium MR imaging-derived ECV measurements were compared with histologically quantified fibrosis by using Pearson correlation. Agreement between equilibrium CT and equilibrium MR imaging was assessed by using Bland-Altman comparison. RESULTS: Twenty-three patients (16 male, seven female; mean age, 70.8 years; standard deviation, 8.3) were recruited. The mean percentage of histologic fibrosis was 18% (intersubject range, 5%-40%). There was a significant correlation between both equilibrium CT- and equilibrium MR imaging-derived ECV and percentage of histologic fibrosis (r = 0.71 [P < .001] and r = 0.84 [P < .0001], respectively). Equilibrium CT-derived ECV was significantly correlated to equilibrium MR imaging-derived ECV (r = 0.73). CONCLUSION: ECV measured by using equilibrium CT in patients with aortic stenosis correlates with histologic quantification of myocardial fibrosis and with ECV derived by using equilibrium MR imaging.

Mammographic density: intersection of science, the law, and clinical practice.

High mammographic density is associated with a two- to sixfold increased risk of breast cancer. Mammographic density can be altered by endogenous and exogenous hormonal factors and generally declines with age. Mammographic density is affected by confounding factors such as age, parity, menopausal status, and body mass index (BMI), thus making interpretation of mammographic density challenging. None of the established means of measuring mammographic density are entirely satisfactory because they are time consuming and/or subjective. Although mammographic density has been shown to predict breast cancer risk, the role of mammographic density in precisely assessing a woman's breast cancer risk over her lifetime and evaluating response to risk-reduction strategies cannot be fully realized until we have a better understanding of the biology that links mammographic density to breast cancer risk.

Noninvasive imaging of myocardial extracellular matrix for assessment of fibrosis.

PURPOSE OF REVIEW: Myocardial fibrosis is a common feature of many cardiomyopathies, including hypertrophic cardiomyopathy. Myocardial fibrosis has been shown to be reversible and treatable with timely intervention. Although early detection and assessment of fibrosis is crucial, adequate diagnostics are still in development. Recent studies have shown progress on noninvasive imaging methods of fibrosis using cardiovascular magnetic resonance (CMR) and nuclear imaging modalities. RECENT FINDINGS: T1 mapping and extracellular volume mapping (ECV) combined with CMR imaging are cutting edge methods that have the potential to assess interstitial myocardial fibrosis. Recent findings show that ECV measurement can be correlated to the extent of diffuse fibrosis. Comparatively, molecular imaging targets specific biomarkers in the fibrosis formation pathway and provides enhanced sensitivity for imaging early disease. Biomarkers include molecules involved in angiogenesis, ventricular remodeling, and fibrotic tissue formation, whereas collagen targeted agents can directly identify fibrotic tissue in the heart. SUMMARY: This review introduces novel methods of fibrosis imaging that utilize properties of extracellular matrix and its biomarkers. Changes in characteristics and cellular biomarkers of the extracellular space can provide significant information regarding fibrosis formation and its role in cardiomyopathy. Ultimately, these findings may improve detection and monitoring of disease and improve efficiency and effectiveness of the treatment.

Extracellular matrix heterogeneity regulates three-dimensional morphologies of breast adenocarcinoma cell invasion.

Plasticity and reciprocity of breast cancer cells to various extracellular matrice (ECMs) are three-dimensionally analyzed in quantitative way in a novel and powerful microfluidic in vitro platform. This successfully demonstrates the metastatic potential of cancer cells and their effective strategies of ECM proteolytic remodeling and morphological change, while interacting with other cells and invading into heterogeneous ECMs.

Laser-treated Nucleus pulposus as an innovative model of intervertebral disc degeneration.

This study describes an innovative experimentally induced model of intervertebral disc degeneration. This innovative approach is based on the induction of extracellular matrix disorders in the intervertebral disc (IVD) using a diode laser. For this study, 15 one-year-old and five 30-month-old New Zealand White rabbits were used. Two procedures were tested to trigger IVD degeneration: needle aspiration (reference technique) and a laser approach. The IVD degeneration process was assessed 20, 40, 60, 90 and 120 days after surgery by X-ray radiography (IVD height), magnetic resonance imaging (MRI) (T2 intensity of IVD signal) and histological analysis using modified Boos' scoring. Our data indicate that a marked IVD degeneration was found compared with sham-operated animals regardless of the procedure tested. A significant decrease in disc height on X-ray radiographs was first demonstrated. In addition, MRI disc signals were significantly reduced in both groups. Finally, a statistically significant increase in Boos' scoring was found in both laser and aspiration-induced IVD degeneration. Interestingly, IVD degeneration induced by laser treatment was more progressive compared with aspiration. Moreover, the histological results indicated that laser-induced disc degeneration was quite similar to that obtained during the natural aging process as observed in 30-month-old rabbits. Our study describes the consistency of this innovative experimentally-induced animal model of IVD degeneration. The radiological, MRI and histological data confirm its relevance. The histological examination indicates that IVD degeneration induced by laser treatment is comparable to the degenerative process observed during the onset of spontaneous IVD degeneration. This model could be a useful tool to help us validate biomaterial-assisted, cell-based, regenerative medicine strategies for the prevention and treatment of IVD degeneration.

Ultrasonic assessment of extracellular matrix content in healing Achilles tendon.

Although several imaging modalities have been utilized to observe tendons, assessing injured tendons by tracking the healing response over time with ultrasound is a desirable method which is yet to be realized. This study examines the use of ultrasound for non-invasive monitoring of the healing process of Achilles tendons after surgical transection. The overall extracellular matrix content of the transection site is monitored and quantified as a function of time. B-mode images (built from successive A-scan signatures) of the injury site were obtained and compared to biomechanical properties. A quantitative measure of tendon healing using the extracellular matrix (ECM) content of the injury site was analyzed using linear regression with all biomechanical measures. Contralateral tendons were used as controls. The trend in the degree of ECM regrowth in the 4 weeks following complete transection of excised tendons was found to be most closely paralleled with that of linear stiffness (R(2) = 0.987, p < .05) obtained with post-ultrasound biomechanical tests. Results suggest that ultrasound can be an effective imaging technique in assessing the degree of tendon healing, and can be used to correlate structural properties of Achilles tendons.

Clinically applied CT arthrography to measure the sulphated glycosaminoglycan content of cartilage.

OBJECTIVE: Similar to delayed gadolinium enhanced MRI of cartilage, it might be possible to image cartilage quality using CT arthrography (CTa). This study assessed the potential of CTa as a clinically applicable tool to evaluate cartilage quality in terms of sulphated glycosaminoglycan content (sGAG) and structural composition of the extra-cellular matrix (ECM). METHODS: Eleven human cadaveric knee joints were scanned on a clinical CT scanner. Of each knee joint, a regular non-contrast CT (ncCT) and an ioxaglate injected CTa scan were performed. Mean X-ray attenuation of both scans was compared to identify contrast influx in seven anatomical regions of interest (ROIs). All ROIs were rescanned with contrast-enhanced µCT, which served as the reference standard for sGAG content. Mean X-ray attenuation from both ncCT and CTa were correlated with µCT results and analyzed with linear regression. Additionally, residual values from the linear fit between ncCT and µCT were used as a covariate measure to identify the influence of structural composition of cartilage ECM on contrast diffusion into cartilage in CTa scans. RESULTS: CTa resulted in higher X-ray attenuation in cartilage compared to ncCT scans for all anatomical regions. Furthermore, CTa correlated excellent with reference µCT values (sGAG) (R=0.86; R(2)=0.73; P<0.0001). When corrected for structural composition of cartilage ECM, this correlation improved substantially (R=0.95; R(2)=0.90; P<0.0001). CONCLUSIONS: Contrast diffusion into articular cartilage detected with CTa correlates with sGAG content and to a lesser extent with structural composition of cartilage ECM. CTa may be clinically applicable to quantitatively measure the quality of articular cartilage.

Biomimetic approach to perforation repair using dental pulp stem cells and dentin matrix protein 1.

INTRODUCTION: Dentin regeneration could be an ideal treatment option to restore tissue function. This study was conducted to evaluate the ability of dental pulp stem cells (DPSCs) and dentin matrix protein 1 (DMP1) impregnated within a collagen scaffold to regenerate dentin. METHODS: Simulated perforations were created in 18 dentin wafers made from freshly extracted human molars. Six groups were established. They were (1) empty wafers, (2) mineral trioxide aggregate, (3) collagen scaffold, (4) scaffold with DMP1, (5) scaffold with DPSCs, and (6) scaffold with DPSCs and DMP1. One sample was placed subcutaneously in each mouse with three mice in each group. After 12 weeks, the samples were subjected to radiographic, histological, and immunohistochemical evaluations. RESULTS: DPSCs impregnated within a collagen scaffold differentiated into odontoblast-like cells forming a highly cellular, vascular, and mineralized matrix in the presence of DMP1. CONCLUSIONS: A triad consisting of DPSCs, DMP1, and a collagen scaffold promotes dentin regeneration in a simulated perforation repair model.

Ultrastructural localization of cochlin in the rat cochlear duct.

Cochlin, a product of the COCH gene, is a major constituent of the inner ear extracellular matrix. Type II collagen, a protein that contributes to structural stability, is also a component of this extracellular matrix. In this study, using the postembedding immunogold method, we demonstrate the localization of cochlin and type II collagen in the cochlear duct at the ultrastructural level. The immunolabeling of cochlin was observed in the fibrillar substance in the spiral limbus, beneath the inner sulcus cells, and in the basilar membrane, the spiral prominence and the spiral ligament. Immunolabeling of type II collagen was observed in the same fibrillar substance in the extracellular matrix of the cochlear duct. This localization of cochlin is consistent with the expected localization of type II collagen. The localization of cochlin and type II collagen indicates the important roles played by these proteins in the hearing process.

Micro-computed tomographical imaging of soft biological materials using contrast techniques.

The aim of this work was to introduce high-resolution computed tomography (micro-CT) for scaffolds made from soft natural biomaterials, and to compare these data with the conventional techniques scanning electron microscopy and light microscopy. Collagen-based scaffolds were used as examples. Unlike mineralized tissues, collagen scaffolds do not provide enough X-ray attenuation for micro-CT imaging. Therefore, various metal-based contrast agents were applied and evaluated using two structurally distinct scaffolds, one with round pores and one with unidirectional lamellae. The optimal contrast techniques for obtaining high-resolution three-dimensional images were either a combination of osmium tetroxide and uranyl acetate, or a combination of uranyl acetate and lead citrate. The data obtained by micro-CT analysis were in line with data obtained by light and electron microscopy. However, small structures (less than a few mum) could not be visualized due to limitation of the spot size of the micro-CT apparatus. In conclusion, reliable three-dimensional images of scaffolds prepared from soft natural biomaterials can be obtained using appropriate contrast protocols. This extends the use of micro-CT analysis to soft materials, such as protein-based biomaterials.

Ultrasound anatomy of normal nails unit with 18 mhz linear transducer.

Interest is growing in non-invasive diagnostic methods for nails in dermatological pathology. Currently, nail disease diagnosis is based mostly on clinical evaluation; instrumental examination, traditionally, has been performed by magnetic resonance. Ultrasound (US) can be proposed as an easier and more available method for the study of the nail apparatus. In this study, the nail unit normal ultrasound anatomy was investigated to obtain data on adult normal parameters. On 35 healthy volunteers (20 women and 15 men--average age of 27 years) we performed an ultrasonographic study on the nail plate (dorsal and ventral), nail matrix and nail bed of all fingers of the hands using a 18 MHz linear transducer with Esaote Mylab 50. A thick gel layer allowed for appropriate transmission of ultrasound without any additional device. Macroscopic nail features were studied by clinical examination and photographic analysis. The following ultrasound parameters were investigated: nail thickness; nail bed thickness; matrix lenght; matrix-bone distance. Blood flow was studied with the use of colour and power colour Doppler. The nail apparatus echographic anatomy consists in: (a) nail plate, represented by two hyperechoic bands (dorsal and ventral) with an hypoechoic or anechoic space between them; (b) nail bed, represented by an area of dys-homogeneous hypo-echogeneity; (c) nail matrix, represented by a markedly hypoechoic area corresponding to the region under the nail sulcus; (d) ligaments, sometimes well detectable and formed by a specialized connective tissue; and (e) vessels, well evaluable through doppler examination.

Incomplete digestion preserves chondrocytes from dedifferentiating in long-termed culture on plastic substrate.

Epiphyseal pieces from young rat's costal cartilage were predigested for 30min by hyaluronidase then digested by collagenase for 1h with gentle beating applied. Resulted grape-like chondrocytes connecting with the residual cartilage matrix were seeded in plastic culture dishes and 4 passages at about 12-days interval were carried out. Morphological observations were performed daily. Compared with completely isolated chondrocytes at the same passage, detection for collagen II, integrin-beta(1) and focal adhesion kinase by immunochemistry staining, Western Blot and RT-PCR were performed to evaluate the preservation of chondrocytic phenotype and cellular functions. Primary chondrocytes isolated by complete enzymatic digestion served as control. Completely isolated chondrocytes in the monolayer culture were ready to lose the chondrocytic phenotype marked by the down-regulation of collagen II secretion and specific morphological alterations which were characterized as the cells gradually became long and spindle-like from their originally rounded shape. In case of the incompletely digested chondrocytes, the expression of collagen II was stable during the whole experiment while extensive cell-cell contacts and matrix-cell connections were observed. Transcription and expression of integrin-beta(1) and FAK were active and paracrine of BMP-7 was positive. These results suggested stable chondrocytic phenotype. Conclusionly, by the incomplete digestion method, the requisite time for enzymatic isolation was reduced and chondrocytes with residual matrix were harvested instead of mono-cell suspension. Compared with the novel techniques, the incomplete digestion shortened the enzymatic procedure greatly and simplified the subculturing operations with less financial cost. Especially, as extracellular matrix was preserved, chondrocytes expressed stable phenotype in a rather long-termed culture.

Quantifying the 3D macrostructure of tissue scaffolds.

The need to shift from tissue replacement to tissue regeneration has led to the development of tissue engineering and in situ tissue regeneration. Both of these strategies often employ the use of scaffolds--templates that allow cells to attach and then guide the new tissue growth. There are many design criteria for an ideal scaffold. These criteria vary depending on the tissue type and location in the body. In any application of a scaffold it is vital to be able to characterise the scaffold before it goes into in vitro testing. In vitro testing allows the cell response to be investigated before its in vivo performance is assessed. A full characterisation of events in vitro and in vivo, in three dimensions (3D), is necessary if a scaffold's performance and effectiveness is to be fully quantified. This paper focuses on porous scaffolds for bone regeneration, suggests appropriate design criteria for a bone regenerating scaffold and then reviews techniques for obtaining the vitally important quantification of its pore structure. The techniques discussed will include newly developed methods of quantifying X-ray microtomography (microCT) images in 3D and for predicting the scaffolds mechanical properties and the likely paths of fluid flow (and hence potential cell migration). The complications in investigating scaffold performance in vitro are then discussed. Finally, the use of microCT for imaging scaffolds for in vivo tests is reviewed.

[Chondromas (enchondroma, periosteal chondroma, enchondromatosis)].

Chondromas combine a group of benign cartilaginous tissue tumors with common histological manifestations. The tumors (enchondroma, periosteal chondroma, and enchondromatosis) differ in sites and clinical manifestations. Chondromas are generally hypocellular, avascular tumors with an abundance of hyaline cartilage matrix and chondrocytes located diffusely, in clones or lobules. Small bone chondromas, Ollier's disease, and Mafucci's syndrome are characterized by the tumor tissue containing a large number of cells and by greater cytological atypia. Enchondroma is successfully treated by surgical methods.