Evaluation of hepatic histology by near-infrared confocal microscopy: a pilot study

Liver biopsy is a necessary procedure in establishing the tissue diagnosis of many liver conditions and often guides therapeutic strategies. Current histopathologic techniques are either time-consuming or tissue-destroying; hence the potential need for a fast and nondestructive imaging technique of unfixed human liver. This pilot study evaluates the use of near-infrared reflectance confocal microscopy (CM) in the study of human liver histopathology. Without cutting or staining the tissue, CM provides images of bulk parenchyma showing cellular and subcellular detail and depicting morphologic features of hepatic parenchyma in both diseased and nondiseased states. This article presents a series of 12 human liver biopsy samples, providing an overview on the potential of this technique in assessing common findings from light microscopy.     

Use of polarization-sensitive optical coherence tomography to determine the directional polarization sensitivity of articular cartilage and meniscus

The directional polarization sensitivity of articular cartilage and meniscus is investigated by use of polarization-sensitive optical coherence tomography (PS-OCT) by varying the angle of incident illumination. Experimental results show that when the incident light is perpendicular to the tissue surface, normal articular cartilage demonstrates little polarization sensitivity, while meniscus demonstrates strong polarization sensitivity. Differences in optical phase retardation produced by articular cartilage and meniscus are observed when the incident angle of the scanning light beam is adjusted between 0 and 90 deg relative to the tissue surface. Directional polarization sensitivity of articular cartilage and meniscus as obtained by PS-OCT imaging using variations in the angle of incident illumination can be used to assess the orientation and organization of the collagen matrix of these tissues. The polarization sensitivity as evidenced by the Stokes vector and optical phase retardation images can be explained by the orientation of the angle of illumination relative to the unique structural organization of the collagen fibrils and fibers of articular cartilage and meniscus.     

Quantitative characterization of articular cartilage using Mueller matrix imaging and multiphoton microscopy

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.     

Low Temperature Backscattered Electron Imaging in the Study of Human Tissues

A scanning electron microscopic technique for the examination of bulk, fresh, hydrated human tissue is described. Samples of fresh tissue are frozen in liquid nitrogen against a mirror-finished copper block and planed in a cryoultramicrotome before transfer to a low temperature scanning electron microscope. After sublimation of water from the specimen surface, the tissue is examined in secondary electron and back-scattered electron modes. Adjacent pieces of tissue, and those retrieved after backscattered electron observation, can be readily prepared for and examined by light and by conventional transmission electron microscopy.

The method has been tested with multiple blocks taken from 6 cases of human breast carcinoma. In the backscattered electron mode, the infiltrating columns of neoplastic cells can be distinguished from mammary adipose and fibrous tissue. Within a carcinoma, the collagenous stroma, carcinoma cells, and perivascular and perineural infiltrates can be identified. These features have been contrasted with those obtained by light microscopy, by low temperature scanning, and by transmission electron microscopy. This use of back-scattered electron imaging for the investigation of unfixed hydrated tissue offers the possibility that the technique could be of considerable value in the microscopy of very small samples in which, because of a need for subsequent biochemical, histochemical, and immunologic investigation, fixation and dehydration are to be avoided.     

Low temperature backscattered electron imaging in the study of human tissues

A scanning electron microscopic technique for the examination of bulk, fresh, hydrated human tissue is described. Samples of fresh tissue are frozen in liquid nitrogen against a mirror-finished copper block and planed in a cryoultramicrotome before transfer to a low temperature scanning electron microscope. After sublimation of water from the specimen surface, the tissue is examined in secondary electron and backscattered electron modes. Adjacent pieces of tissue, and those retrieved after backscattered electron observation, can be readily prepared for and examined by light and by conventional transmission electron microscopy. The method has been tested with multiple blocks taken from 6 cases of human breast carcinoma. In the backscattered electron mode, the infiltrating columns of neoplastic cells can be distinguished from mammary adipose and fibrous tissue. Within a carcinoma, the collagenous stroma, carcinoma cells, and perivascular and perineural infiltrates can be identified. These features have been contrasted with those obtained by light microscopy, by low temperature scanning, and by transmission electron microscopy. This use of backscattered electron imaging for the investigation of unfixed hydrated tissue offers the possibility that the technique could be of considerable value in the microscopy of very small samples in which, because of a need for subsequent biochemical, histochemical, and immunologic investigation, fixation and dehydration are to be avoided.     

Combining multispectral polarized light imaging and confocal microscopy for localization of nonmelanoma skin cancer

Multispectral polarized light imaging (MSPLI) enables rapid inspection of a superficial tissue layer over large surfaces, but does not provide information on cellular microstructure. Confocal microscopy (CM) allows imaging within turbid media with resolution comparable to that of histology, but suffers from a small field of view. In practice, pathologists use microscopes at low and high power to view tumor margins and cell features, respectively. Therefore, we study the combination of CM and MSPLI for demarcation of nonmelanoma skin cancers. Freshly excised thick skin samples with nonmelanoma cancers are rapidly stained with either toluidine or methylene blue dyes, rinsed in acetic acid, and imaged using MSPLI and CM. MSPLI is performed at 630, 660, and 750 nm. The same specimens are imaged by reflectance CM at 630, 660, and 830 nm. Results indicate that CM and MSPLI images are in good correlation with histopathology. Cytological features are identified by CM, and tumor margins are delineated by MSPLI. A combination of MSPLI and CM appears to be complementary. This combined in situ technique has potential to guide cancer surgery more rapidly and at lower cost than conventional histopathology.     

Low Temperature Backscattered Electron Imaging in the Study of Human Tissues

A scanning electron microscopic technique for the examination of bulk, fresh, hydrated human tissue is described. Samples of fresh tissue are frozen in liquid nitrogen against a mirror-finished copper block and planed in a cryoultramicrotome before transfer to a low temperature scanning electron microscope. After sublimation of water from the specimen surface, the tissue is examined in secondary electron and back-scattered electron modes. Adjacent pieces of tissue, and those retrieved after backscattered electron observation, can be readily prepared for and examined by light and by conventional transmission electron microscopy.

The method has been tested with multiple blocks taken from 6 cases of human breast carcinoma. In the backscattered electron mode, the infiltrating columns of neoplastic cells can be distinguished from mammary adipose and fibrous tissue. Within a carcinoma, the collagenous stroma, carcinoma cells, and perivascular and perineural infiltrates can be identified. These features have been contrasted with those obtained by light microscopy, by low temperature scanning, and by transmission electron microscopy. This use of back-scattered electron imaging for the investigation of unfixed hydrated tissue offers the possibility that the technique could be of considerable value in the microscopy of very small samples in which, because of a need for subsequent biochemical, histochemical, and immunologic investigation, fixation and dehydration are to be avoided.     

Design Characteristics for the Tissue Engineering of Cartilaginous Tissues

Tissues like the temporomandibular joint (TMJ) disc and the knee meniscus are often mistakenly viewed as a tantamount to hyaline cartilage, largely due to the absence of a comprehensive understanding of the distinguishing properties of cartilaginous tissues. Because of this confusion, fibrocartilaginous tissue engineering attempts may not be based on suitable experimental designs. Fibrocartilaginous tissues are markedly different than hyaline cartilage; however, the dearth of knowledge related to their cellular and biochemical composition, as well as their biomechanical characteristics, is stunning. Hyaline articular cartilage is exclusively composed of chondrocytes that produce primarily type II collagen, whereas the TMJ disc and the knee meniscus have a mixed cell population of fibroblasts and cells similar to chondrocytes, which predominantly secrete type I collagen. Additionally, fibrocartilaginous tissues have a low glycosaminoglycan content, a low compressive modulus, and a high tensile modulus when compared to hyaline cartilage. Therefore, it is crucial for fibrocartilaginous tissue engineering attempts to be tissue-specific, utilizing the knowledge of the distinct and unique properties of these tissues. At the same time, advances and insights related to the science and engineering aspect of hyaline cartilage regeneration must be carefully considered for the in vitro engineering of fibrocartilaginous tissues.     

运动性半月板损伤及组织工程半月板的应用

背景：半月板损伤是常见的膝关节运动损伤之一,常伴有关节软骨的损伤,其原因是膝关节内环境的紊乱。 目的：评价组织工程半月板在半月板构建中的生物支架材料的性能,以寻求合理的半月板替代物。 方法：以“组织工程;半月板修复;生物材料支架;运动性半月板损伤”为中文关键词,以“tissue engineering, the meniscus repair,biological material scaffold; athletic meniscal injury”为英文关键词,应用计算机检索维普数据库(1994-01/2009-12)和Pubmed数据库(1994-01/2009-12),纳入30篇运动性半月板损伤和组织工程半月板相关的文献。对半月板组织的特征、半月板组织工程种子细胞的来源、组织工程半月板支架材料生物相容性和可降解性以及细胞因子在组织工程半月板构建过程中的作用进行综合评价。 结果与结论：与传统半月板修复相比,组织工程化半月板具有无抗原性,来源不受限制,可按预先设计塑型,具有生命力等许多优点。但是组织工程化半月板仍有许多问题有待研究和解决,如何模拟体内环境,在体外成功构建半月板组织,如何提高支架材料的应用性,研制具有与正常人体半月板组织相接近的力学性能的支架材料是一个半月板修复的关键性问题。     

MRI after unicondylar knee arthroplasty: The preserved compartments

PurposeThe aim of this study was to assess the reproducibility of using magnetic resonance imaging (MRI) to analyze the preserved anatomic knee compartments following unicondylar knee arthroplasty (UKA) with zirconium femoral components. It was hypothesized that evaluation of the cartilage, ligaments, meniscus, and tendons would result in a high rate of inter-observer reliability.ScopeTen patients underwent MRI of the knee tailored to reduce metallic artifact following medial UKA with zirconium femoral implants. Cartilage, external meniscus, collateral and cruciate ligaments, the quadriceps and patellar tendons, and the presence of joint effusion were evaluated by two independent investigators. The reviewers provided degrees of confidence with their evaluation of each parameter through the use of a five-point scale. Inter-observer agreement was calculated and inter-observer reliability was determined by use of Cohen's Kappa.Artifacts originating from the implants were rarely observed. There was excellent inter-observer reliability (i.e., high Cohen's Kappa) for all assessed structures, and a high level of observer confidence for the evaluation of the cartilage, meniscus, tendons, ligaments, and joint effusion.ConclusionIn this study tailored MRI allows for reproducible analysis of the preserved knee joint compartment after UKA for zirconium implants. This technique might prove helpful in the assessment of painful knee joints after UKA with other metallic materials as new MRI software programs, which suppress metal artifacts, are developed.     

Tissue engineering of the meniscus

Meniscus lesions are among the most frequent injuries in orthopaedic practice and they will inevitably lead to degeneration of the knee articular cartilage. The fibro-cartilage-like tissue of the meniscus is notorious for its limited regenerative capacity. Tissue engineering could offer new treatment modalities for repair of meniscus tears and eventually will enable the replacement of a whole meniscus by a tissue-engineered construct.Many questions remain to be answered before the final goal, a tissue-engineered meniscus is available for clinical implementation. These questions are related to the selection of an optimal cell type, the source of the cells, the need to use growth factor(s) and the type of scaffold that can be used for stimulation of differentiation of cells into tissues with optimal phenotypes. Particularly in a loaded, highly complex environment of the knee, optimal mechanical properties of such a scaffold seem to be of utmost importance.With respect to cells, autologous meniscus cells seems the optimal cell source for tissue engineering of meniscus tissue, but their availability is limited. Therefore research should be stimulated to investigate the suitability of other cell sources for the creation of meniscus tissue. Bone marrow stroma cells could be useful since it is well known that they can differentiate into bone and cartilage cells. With respect to growth factors, TGF-beta could be a suitable growth factor to stimulate cells into a fibroblastic phenotype but the problems of TGF-beta introduced into a joint environment should then be solved. Polyurethane scaffolds with optimal mechanical properties and with optimal interconnective macro-porosity have been shown to facilitate ingrowth and differentiation of tissue into fibro-cartilage. However, even these materials cannot prevent cartilage degeneration in animal models. Surface modification and/or seeding of cells into the scaffolds before implantation may offer a solution for this problem in the future.This review focuses on a number of specific questions; what is the status of the development of procedures for lesion healing and how far are we from replacing the entire meniscus by a (tissue-engineered) prosthesis. Subquestions related to the type of scaffold used are: is the degree of tissue ingrowth and differentiation related to the initial mechanical properties and if so, what is the influence of those properties on the subsequent remodelling of the tissue into fibro-cartilage; what is the ideal pore geometry and what is the optimal degradation period to allow biological remodelling of the tissue in the scaffold. Finally, we will finish with our latest results of the effect of tear reconstruction and the insertion of prostheses on articular cartilage degradation.     

Replacement of the rabbit medial meniscus with a polyester-carbon fibre bioprosthesis

Eighteen New Zealand white rabbits underwent prosthetic replacement of the meniscus which was attached to the intercondylar area of the tibia through a predrilled hole and around the internal surface of the capsule by sutures, in one knee, and meniscectomy alone in the contralateral knee. The animals were killed 3 and 6 month after implantation. Fragmentation of carbon fibres was noted in six knee joints. Histological sections demonstrated more progressive articular cartilage degeneration of the tibial surface which had the meniscus removed alone (P = 0.05). The implant was incorporated into the capsule with minimal invasion of fibrous tissue into the interstices of the prosthesis. Scanning electron microscopy revealed the extent of articular cartilage fibrillation which was more evident in the area where the meniscus covered the tibia.     

生长因子在组织工程半月板构建中的作用

背景：生长因子诱导细胞向纤维软骨分化是半月板组织工程研究热点。半月板的体外构建和体内重塑与生长因子的作用关系密切。 目的：概述近年来生长因子半月板组织工程研究进展,并对其机制进行探讨。 方法：应用计算机检索 2008年1月至2013年3月维普数据库(http://lib.cqvip.com)、中国知网数据库(www.cnki.net)及Pubmed数据库(http://www.ncbi.nlm.nih.gov/pubmed)相关文章,以“半月板组织工程;软骨;生长因子”为中文检索词;以“meniscus tissue engineering, cartilage, growth factors”为英文检索词。纳入53篇关于半月板组织工程中生长因子研究的文章。 结果与结论：软骨组织工程研究中生长因子的种类繁多,新的生长因子亦在不断的被发现。生长因子对软骨调节作用的研究,从以前的单一生长因子模式开始向多生长因子间相互作用研究模式转变;生长因子对软骨调节作用的分子机制也得到了广泛的研究。生长因子在组织工程中具有良好的运用前景,但还存在着许多尚待解决的问题,如在半月板愈合过程中,不同时间阶段、不同生长因子的表达与作用均不相同,因此如何适时、适量以及怎样发挥生长因子之间的相互作用来更好的模拟体内微环境,探究生长因子对软骨调节作用的分子机制以及发现新的生长因子等都将是半月板组织工程中的研究重点。     

Effect of mechanical load on articular cartilage collagen structure: a scanning electron-microscopic study

Little is known about the morphological effect of a mechanical load upon articular cartilage. The objective of this study was to describe and quantify the deformation of the articular cartilage collagen structure of the tibial plateau under static loading. Whole intact rabbit knee joints were loaded in vitro by simulating a quadriceps force of 3x, 1x or 0.5x body weight (high, medium, low) over durations of 30 or 5 min (long, short). Specimens were cryopreserved while under load and prepared for morphological evaluation by field emission scanning electron microscopy. Under high force and long duration loading the collagen fibers exhibited high deformation with an increased thickness of the layer of collagen fibers oriented almost parallel to the surface and a cartilage thickness reduced to 54%. Collagen fiber deformation occurred mostly in the transitional and upper radial zone. The area of tibial indentation and the cartilage thickness reduction increased with magnitude and duration of load. The collagen matrix did show a bulging edge at the border of the meniscus and exhibited remarkable deformation under the meniscus.     

A Direct Compression Stimulator for Articular Cartilage and Meniscal Explants

This paper describes the development and use of a direct compression stimulator for culturing explants from the meniscus of the knee and articular cartilage. Following design and fabrication of the instrument along with its data acquisition system, the function of the machine was verified by both mechanical means and tissue effect. The loading chamber can hold up to 45 5 mm diameter samples. While designed to stimulate samples up to 4 mm thick, axial displacements as little as 0.127 μm are within the theoretical capacity of the stimulator. In gene expression studies, collagen II and aggrecan expression were examined in explants from articular cartilage as well as medial and lateral menisci subjected to dynamic stimulation and static compression. These results were then compared to free swelling samples. It was found that static compression to cut thickness down-regulated aggrecan and collagen II expression in articular cartilage explants compared to free swelling controls by 94% and 90%, respectively. The application of a dynamic, intermittent, 2% oscillation around the cut thickness returned expression levels to those of free swelling controls at 4 h but not at 76 h. In medial meniscus samples, dynamic compression up-regulated aggrecan expression by 108%, but not collagen II expression, at 4 and 76 h compared to static controls. No difference in gene expression was observed for lateral meniscal explants. Thus, effects of direct compression seen in articular cartilage may not necessarily translate to the knee meniscus. The design of this stimulator will allow a variety of tissues and loading regimens to be examined. It is hoped that regimens can be found that not only return samples to the production levels of free swelling controls, but also surpass them in terms of gene expression, protein synthesis, and functional properties.     

Application of X-ray grating interferometry for the imaging of joint structures

Conventional X-ray absorption contrast imaging does not depict soft tissues, such as cartilage, in sufficient detail. For visualization of the soft tissues, X-ray phase-contrast imaging is more sensitive than absorption-contrast imaging. The basic concept of the X-ray phase-contrast imaging used in this study is similar to that of differential interference contrast (Nomarski) microscopy. We applied Talbot–Lau X-ray interferometry to visualize the joint structures in the right hand and knee of a donated cadaver. This imaging system simultaneously produced three different types of images: an absorption image, a differential phase image, and a visibility image. The interface between the articular cartilage of the metacarpo-phalangeal joint and fluid or the bony cortex was clearly demonstrated on the differential phase image, whereas this interface was unclear on the absorption image. Within the knee joint, the surface of the articular cartilage was demonstrated both on the differential phase and visibility images; the medial collateral ligament and medial meniscus were also visualized successfully. These results are clinically significant for the diagnosis and therapeutic estimation of rheumatoid arthritis and related joint diseases. This feasibility study on the clinical application of this imaging tool was a collaborative effort of researchers in the fields of physics, radiology, and gross anatomy.     

Correlative evaluation of variable pressure-scanning electron microscopy to histology methods for vascular tissues

Abstract

For pathological investigation, vascular tissues implanted with biomaterials or medical devices require microscopic histology and scanning electron microscopy (SEM) assessment to evaluate biocompatibility with the vascular luminal surface. However, these valuable specimens are often susceptible to SEM and histology tissue preparation artifacts. Correlative variable pressure-scanning electron microscopy (VP-SEM) and histology methods were developed and evaluated, using current SEM technology, allowing direct imaging of hydrated samples without lengthy tissue processing, labor, equipment, and their sequela artifacts. Unstained and silver-stained, fixed swine carotid arteries were assessed using a cooling stage and an environmental secondary electron detector. After VP-SEM imaging, arteries were processed for histology evaluation. Assessment of luminal endothelial surface SEM images and tissue histology of the underlying mural wall was made to determine if low vacuum, cryo-freezing, electron beam exposure, and/or staining affected tissue morphology. Conventional SEM requires lengthy tissue processing with dedicated equipment for solvent dehydration, critical point drying, and metallic sputter coating in order to image under high vacuum for secondary electron imaging. This process causes notable artifacts when samples are later processed for microscopic histology. In contrast, silver staining of swine carotid arteries, followed by aldehyde fixation, facilitated VP-SEM imaging and histology preparation with results that were comparable to conventional SEM and had acceptable tissue morphology. VP-SEM with a cooling stage and an environmental secondary electron detector allows efficient SEM imaging and correlative histological assessment of vascular tissue samples. This correlative method may be further developed and implemented for research applications with biomaterial and medical device implanted tissues.     

Electron microscopy of wet tissues: a case study in renal pathology

In this report we introduce wet-tissue scanning electron microscopy, a novel technique for direct imaging of wet tissue samples using backscattered electrons. Samples placed in sealed capsules are imaged through a resilient, electron-transparent membrane. The contrast of the imaged samples may be enhanced by chemical staining. The samples several millimeters thick and imaged without sectioning, makes this technique suitable for rapid analysis of tissue specimens. We applied this technique to D-limonene-induced nephropathy where accumulation of hyaline protein droplets is induced in proximal and distal convoluted tubules of the kidney. Images obtained by scanning electron microscopy of hydrated kidney specimens exhibited superior resolution, contrast, and magnification compared with those obtained by conventional light microscopy of paraffin sections. The electron micrographs can be obtained within an hour of tissue removal, whereas preparation for light microscopy requires at least 1 day. These advantages of the wet scanning electron microscopy technique indicate its potential utility in a wide range of applications in histopathology and toxicology.     

A porous polymer scaffold for meniscal lesion repair--a study in dogs

Meniscal lesions often occur in the avascular area of the meniscus with little chance of spontaneous repair. An access channel in the meniscal tissue can function as an entrance for ingrowing repair tissue from the vascular periphery of the meniscus to the lesion in the avascular zone which again induced healing of the lesion. Implantation of a porous polymer in a full-thickness access channel induced healing. However, a better integration between meniscal tissue and the implant might be achieved with the combination of the newly developed porous polymers and a modified surgical technique. This might improve meniscal lesion healing and the repair of the access channel with neo-meniscal tissue. Longitudinal lesions were created in the avascular part of 24 canine lateral menisci and a partial-thickness access channel was formed to connect the lesion with the meniscal periphery. In 12 menisci, the access channel was left empty (control group), while in the remaining 12 menisci the polymer implant was sutured into the access channel. Repair of the longitudinal lesions was achieved with and without polymer implantation in the partial-thickness access channel. Polymer implants induced fibrous ingrowth with cartilaginous areas, which resembled neo-meniscal tissue. Implantation did not prevent articular cartilage degeneration.     

Conditioned media from human osteoarthritic synovium induces inflammation in a synoviocyte cell line

Aim: Osteoarthritis (OA) is a whole joint pathology involving cartilage, synovial membrane, meniscus, subchondral bone, and infrapatellar fat pad (IFP). Synovitis has been widely documented in OA suggesting its important role in pathogenesis. The aim of this study was to investigate the role of different joint tissues in promoting synovitis. Materials and methods: Conditioned media (CM) from cartilage, synovial membrane, meniscus, and IFP were generated from tissues of five patients undergoing total knee replacement and used to stimulate a human fibroblast-like synoviocytes cell line (K4IM). Cytokines, chemokines, and metalloproteases release was analyzed in all CM by Bio-Plex Assay and sulfated glycosaminoglycan (GAG) content by dimethylmethylene blue assay. Gene expression of several markers was evaluated by real-time PCR in K4IM cells stimulated with the CM obtained from joint tissues. Results: CM from all tissues produced high levels of IL-6, IL-8, and CCL2. CCL21, MMP-3, and ?13 levels were detected in all CM except IFP. MMP-10 was present only in CM of cartilage and synovial tissues. IL-1β, IL-15, TNF-α, CCL5, and CCL19 were undetectable. However, only K4IM cells stimulated by the CM from OA synovium showed an increase of IL-6, CXCL-8, CCL21, MMP10, and IL-1β expression. Conclusion: Our study showed that K4IM might be a suitable in vitro model for evaluating different cellular pathways in OA studies. Importantly, we demonstrated that in OA, all joint tissues might be involved in the progression of synovitis with a predominant role of synovial membrane itself compared to the other joint tissues.