组织工程研究的进展

组织工程从提出到现在不过10年的历史,但在世界范围内引起高度重视,许多国家已将它列为“经济的新增长点”,投入了相当的人力和财力进行研究,成果斐然.我国也有相关的研究课题开始运行.第二届组织工程学会于1998年在美国召开,会上发表了组织工程的最新研究成果.作为抛砖引玉,总结了有关组织工程研究的进展,并提出保证工程组织的功能化是值得深入研究的问题.     

Intestinal canine pythiosis in Venezuela confirmed by serological and sequencing analysis

An 11-months-old mixed Terrier male originally from Venezuela, was referred to a Veterinary Hospital with signs of depression, anorexia, vomiting and diarrhea. The illness had begun 1 month earlier. Despite antibiotic chemotherapy and vitamins, the disease progressed. Radiological exams showed involvement of the small intestine. Histopathological studies of tissue samples taken during surgical intervention revealed eosinophilic areas in the center of which, abundant eosinophils, histiocytes and giant cells were observed. Silver stained cross-sections of the small intestine showed slender sparsely septate hyphae within the necrotic areas. Attempts to isolate the etiologic agent in pure culture were fruitless. The dog died without a definitive diagnosis. Fixed tissue samples of the small intestine were later investigated using specific fluorescent antibodies for pythiosis and molecular tools. These exams indicated that the hyphae in the infected tissues belong to the straminipilan pathogen Pythium insidiosum. This is the first confirmed case of dog pythiosis in Venezuela.     

一种组织微阵列供体取样与受体蜡块制作的 新器具和新方法

发明一种组织微阵列供体取样与受体蜡块制作的新器具和新方法 . 利用这种新器具和新方法成功地制作了分别含 448 和 390 个供体组织点阵的组织微阵列受体蜡块和切片 . 这种新方法制作的组织微阵列切片经 H&E 染色,显微镜下观察证实,所有切片均无供体点阵组织脱落,切片厚度适中,组织结构无挤压变形,细胞形态均匀一致 . 免疫组化检测 P53 和 P16 蛋白在组织微阵列切片与其相应的常规组织切片中的表达结果完全一致 . 这种组织微阵列供体取样与受体蜡块制作新器具和新方法成本低廉,操作简便,具有在实验室推广应用的价值 .     

Fetal Tissue Banking for Transplantation: Characteristics of the Donor Population and Considerations for Donor and Tissue Screening

We initiated this study to evaluate the suitability for therapeutic use in transplantation of tissues obtained from human abortuses. We have developed protocols for the collection, handling and preservation of hepatic stem cells from electively aborted embryos and have developed methods for assessment of the cells so derived and processed. In this paper we present our findings regarding screening of potential donors, acquisition of fetal tissues, and assessment of the tissues for potentially infectious contaminants. We assess the suitability of the tissue donors according to current standards used for donors of commonly transplanted tissues (e.g., bone grafts, skin grafts and heart valves) and present data regarding the real availability of tissues from elective abortion procedures that would meet those standard tissue banking criteria.We specifically evaluated the donor's willingness to provide a blood sample for testing, conducted a detailed interview similar to those used for typical organ and tissue donors, and assessed the type and incidence of contamination in collected tissues. We find that although many women are willing to consent to use of the tissues for transplantation, attrition from the study for various reasons results in few fetal organs ultimately realistically available for transplantation. Typical reasons for attrition include: unwillingness to have a blood sample drawn or tested, positive serology results, social/medical high risk factors for acquisition of transmissible disease, no identifiable organs available, and unacceptable microbial contamination. Thus, although it might seem that due to the numbers of abortions performed annually, that there would be substantial numbers of suitable tissues available, only a small proportion are truly suitable for transplantation.     

Long-term storage of tissue samples for cell culture

Summary The establishment of cultured cell lines from skin biopsies stored at −196°C for periods up to 1 year has been investigated. Attempts to initiate cell cultures from the frozen tissue samples were uniformly successful. There was no alteration in chromosome constitution, morphological appearance, or specific activities of lysosomal enzymes in cells cultured from the stored samples. This process can safeguard against failure of the initial tissue culture and provide an alternate means of storing viable cells when it is impossible or impractical to initiate a cell culture immediately. This project was supported by the South Carolina Department of Mental Retardation and the Self Foundation.     

A scanning electron microscope study of healthy pecan tissues showing the presence or absence of internal fungal contamination

Summary Healthy pecan, Caryaillinoensis (Wang) Koch, tissue was obtained from an 8-year-old grafted Cherokee tree. Dormant buds were gathered and stored until spring growth. After rigorous surface sterilization, halves of both stored and freshly harvested dormant buds and of actively growing shoots were plated onto sterile PDA. Corresponding halves wre fixed in FAA and processed for scanning electron microscopy (SEM). All plated dormant buds (both stored and freshly harvested) showed presence internally of a fungus, and SEM studies revealed hyphalike strands similar to those of the isolated fungi within cells of those buds. The spring flush was sterile in culture, and strands were absent in SEM studies.     

组织工程的一般考虑

组织工程的最终目标是通过体外增减活细胞与其胞外环境相互作用而发育成具胡生物活性的组织或器官替代物,替换、修复组织或器官,或强化其生物学功能。本文主要讨论细胞移植和组织重建的影响因素和可能的方法。     

Design and characterization of a modified T‐flask bioreactor for continuous monitoring of engineered tissue stiffness

Controlling environmental conditions, such as mechanical stimuli, is critical for directing cells into functional tissue. This study reports on the development of a bioreactor capable of controlling the mechanical environment and continuously measuring force‐displacement in engineered tissue. The bioreactor was built from off the shelf components, modified off the shelf components, and easily reproducible custom built parts to facilitate ease of setup, reproducibility and experimental flexibility. A T‐flask was modified to allow for four tissue samples, mechanical actuation via a LabView controlled stepper motor and transduction of force from inside the T‐flask to an external sensor. In vitro bench top testing with instrumentation springs and tissue culture experiments were performed to validate system performance. Force sensors were highly linear (R² > 0.998) and able to maintain force readings for extended periods of time. Tissue culture experiments involved cyclic loading of polyurethane scaffolds seeded with and without (control) human foreskin fibroblasts for 8 h/day for 14 days. After supplementation with TGF‐β, tissue constructs showed an increase in stiffness between consecutive days and from the acellular controls. These experiments confirmed the ability of the bioreactor to distinguish experimental groups and monitor tissue stiffness during tissue development. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

UCP1 mRNA does not produce heat

Because of the possible role of brown adipose tissue and UCP1 in metabolic regulation, even in adult humans, there is presently considerable interest in quantifying, from in-vitro data, the thermogenic capacities of brown and brite/beige adipose tissues. An important issue is therefore to establish which parameters are the most adequate for this. A particularly important issue is the relevance of UCP1 mRNA levels as estimates of the degree of recruitment and of the thermogenic capacity resulting from differences in physiological conditions and from experimental manipulations. By solely following UCP1 mRNA levels in brown adipose tissue, the conclusion would be made that the tissue's highest activation occurs after only 6 h in the cold and then successively decreases to being only some 50% elevated after 1 month in the cold. However, measurement of total UCP1 protein levels per depot ("mouse") reveals that the maximal thermogenic capacity estimated in this way is reached first after 1 month but represents an approx. 10-fold increase in thermogenic capacity. Since this in-vitro measure correlates quantitatively and temporally with the acquisition of nonshivering thermogenesis, this must be considered the most physiologically relevant parameter. Similarly, observations that cold acclimation barely increases UCP1 mRNA levels in classical brown adipose tissue but leads to a 200-fold increase in UCP1 mRNA levels in brite/beige adipose tissue depots may overemphasise the physiological significance of these depots, as the high fold-increases are due to very low initial levels, and the UCP1 mRNA levels reached are at least an order of magnitude lower than in brown adipose tissue; furthermore, based on total UCP1 protein amounts, the brite/beige depots attain only about 10% of the thermogenic capacity of the classical brown adipose tissue depots. Consequently, inadequate conclusions may be reached if UCP1 mRNA levels are used as a proxy for the metabolic significance of recruited versus non-recruited brown adipose tissue and for estimating the metabolic significance of brown versus brite/beige adipose tissues. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

脂肪干细胞在再生医学中的应用

再生医学是一门研究如何促进创伤与组织再生及功能重建的新兴学科,主要通过研究干细胞分化、机体等正常组织创伤修复与再生等机制来维持、修复、再生或改善损伤组织和器官功能。脂肪干细胞（adipose-derived stem cells,ASCs）是近年来从脂肪组织中分离得到的一种具有多向分化潜能的干细胞,是一种足量的、可用于实际的、有一定吸引力的自体细胞代替的供体资源,并能够广泛的用于组织修复、再生、发育的可塑性及细胞治疗等研究中。阐述了脂肪干细胞在旁分泌、软组织重建及损伤修复、骨骼肌重建、心血管重建、神经系统重建及癌症转移与入侵方面的作用模式,概括总结了目前利用脂肪干细胞参与的临床治疗方法,以期对脂肪干细胞在再生医学中应用研究提供参考。     

Individual Limb Muscle Bundles Are Formed through Progressive Steps Orchestrated by Adjacent Connective Tissue Cells during Primary Myogenesis

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Peridermal and interthecal tissue in Silurian retiolitid graptolites: with examples from Sweden and Arctic Canada

Peridermal tissue, and thecal 'floors' are shown to be present in some specimens of Pseudoretiolites, Retiolites and Stomatograptus. The development of a dense reticulum and thecal hoods is also recognized as being a progressive developmental feature in Gothograptus. It is suggested that all these structures, whether internal or external, are the product of mature or old-age development. It appears questionable whether the Rhabdopleura model of rhabdosomal development adequately explains late-stage membranes and other adventitious tissues such as discussed herein.     

Quantification of Mucosa oxygenation using three discrete spectral bands of visible light

Quantification of the mucosa oxygenation levels during Endoscopic imaging provides useful physiological/diagnostic information. In this work a method for non‐contact quantification of the oxygen saturation index during Endoscopic imaging using three discrete spectral‐band in the blue, the green, and the red parts of the spectrum (RGB bands) has been investigated. The oxygen saturation index (TOI_rgb) was calculated from the three discrete RGB spectral bands using diffusion approximation modeling and least‐square analysis. A parametric study performed to identify the optimum band width for each of the three spectral bands. The quantification algorithm was applied to in vivo images of the endobronchial mucosa to calculate (TOI_rgb) from selected areas within the image view. The results were compared to that obtained from the full visible spectral (470–700 nm, 10 nm) measurements. The analysis showed that a band width of at least 20 nm in the blue and the green is required to obtain best results. The results showed that the method provides accurate estimation of the oxygenation levels with about 90% accuracy compared to that obtained using the full spectra. The results suggest the potential of quantifying the oxygen saturation levels from the three narrow RGB spectral bands/images. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Functional near infrared spectroscopy using spatially resolved data to account for tissue scattering: A numerical study and arm‐cuff experiment

Functional Near‐Infrared Spectroscopy (fNIRS) aims to recover changes in tissue optical parameters relating to tissue hemodynamics, to infer functional information in biological tissue. A widely‐used application of fNIRS relies on continuous wave (CW) methodology that utilizes multiple distance measurements on human head for study of brain health. The typical method used is spatially resolved spectroscopy (SRS), which is shown to recover tissue oxygenation index (TOI) based on gradient of light intensity measured between two detectors. However, this methodology does not account for tissue scattering which is often assumed. A new parameter recovery algorithm is developed, which directly recovers both the scattering parameter and scaled chromophore concentrations and hence TOI from the measured gradient of light‐attenuation at multiple wavelengths. It is shown through simulations that in comparison to conventional SRS which estimates cerebral TOI values with an error of ±12.3%, the proposed method provides more accurate estimate of TOI exhibiting an error of ±5.7% without any prior assumptions of tissue scatter, and can be easily implemented within CW fNIRS systems. Using an arm‐cuff experiment, the obtained TOI using the proposed method is shown to provide a higher and more realistic value as compared to utilizing any prior assumptions of tissue scatter.     

Computerized Determination of Adipocyte Size

Objective: Fat cell size is a fundamental parameter in the study of adipose tissue metabolism, because it markedly influences the cellular rates of metabolism. Previous techniques for the sizing of adipocytes are often complicated or time‐consuming. The aim of this study was to develop a new, computerized method for rapid and accurate determination of adipocyte size in a cell suspension obtained by incubating human or rat adipose tissue biopsies with collagenase. Research Methods and Procedures: The cell suspension was placed between a siliconized glass slide and a cover slip. Using the reference method [designated as (R)], the cell diameters were determined manually using a microscope with a calibrated ocular. The new method presented here [designated as (C)] was based on computerized image analysis. Results: After two well‐defined corrective adjustments, measurements with (R) and (C) agreed very well. The small remaining differences seemed, in fact, to depend on inconsistencies in (R). Discussion: We propose that (C) constitutes a valuable tool to study fat cell size, because this method is fast and allows the assessment of a sufficient number of cells to get reliable data on size distribution. Furthermore, images of cell preparations may be stored for future reference.     

Omental and subcutaneous adipose tissue steroid levels in obese men

We examined plasma and fat tissue sex steroid levels in a sample of 28 men aged 24.8-62.2 years (average BMI value of 46.3 +/- 12.7 kg/m(2)). Abdominal adipose tissue biopsies were obtained during general or obesity surgery. Omental and subcutaneous adipose tissue steroid levels were measured by gas chromatography and chemical ionization mass spectrometry after appropriate extraction procedures. BMI and waist circumference were negatively correlated with plasma testosterone (r = -0.49 and -0.50, respectively, p < 0.01) and dihydrotestosterone (r = -0.58 and -0.56, respectively, p < 0.01), and positively associated with estrone levels (r = 0.64 and 0.62, respectively, p < 0.001). Regional differences in adipose tissue steroid levels were observed for dihydrotestosterone (p < 0.005), androstenedione (p < 0.0001) and dehydroepiandrosterone levels (p < 0.05), which were all significantly more concentrated in omental versus subcutaneous fat. Positive significant associations were found between circulating level of a steroid and its concentration in omental and subcutaneous adipose tissue, for estrone (r = 0.72 and 0.57, respectively, p < 0.01), testosterone (r = 0.66 and 0.58, respectively, p < 0.01) and dihydrotestosterone (r = 0.58 and 0.45, respectively, p < 0.05). Positive correlations were observed between plasma dehydroepiandrosterone-sulfate and omental (r = 0.56, p < 0.01) as well as subcutaneous adipose tissue dehydroepiandrosterone level (r = 0.38, p = 0.05). Positive significant associations were found between omental adipocyte responsiveness to positive lipolytic stimuli (isoproterenol, dibutyryl cyclic AMP and forskolin) and plasma or omental fat tissue androgen levels. In conclusion, although plasma androgen or estrogen levels are strong correlates of adipose tissue steroid content both in the omental and subcutaneous fat depots, regional differences may be observed. Androgen concentration differences in omental versus subcutaneous adipose tissue suggest a depot-specific impact of these hormones on adipocyte function and metabolism.     

Near‐infrared human finger measurements based on self‐calibration point: Simulation and in vivo experiments

Near‐infrared light allows measuring tissue oxygenation. These measurements relay on oxygenation‐dependent absorption spectral changes. However, the tissue scattering, which is also spectral dependent, introduces an intrinsic error. Most methods focus on the volume reflectance from a semi‐infinite sample. We have proposed examining the full scattering profile (FSP), which is the angular intensity distribution. A point was found, that is, the iso‐path length (IPL) point, which is not dependent on the tissue scattering, and can serve for self‐calibration. This point is geometric dependent, hence in cylindrical tissues depends solely on the diameter. In this work, we examine an elliptic tissue cross section via Monte Carlo simulation. We have found that the IPL point of an elliptic tissue cross section is indifferent to the input illumination orientation. Furthermore, the IPL point is the same as in a circular cross section with a radius equal to the effective ellipse radius. This is despite the fact that the FSPs of the circular and elliptical cross sections are different. Hence, changing the orientation of the input illumination reveals the IPL point. In order to demonstrate this experimentally, the FSPs of a few female fingers were measured at 2 perpendicular orientations. The crossing point between these FSPs was found equivalent to the IPL point of a cylindrical phantom with a radius similar to the effective radius. The findings of this work will allow accurate pulse oximetry assessment of blood saturation.