Real-time thickness measurement of biological tissues using a microfabricated magnetically-driven lens actuator

A fiber optic confocal catheter with a micro scanning lens was developed for real-time and non-contact thickness measurement of biological tissue. The catheter has an outer diameter and rigid length of 4.75 mm and 30 mm respectively and is suitable for endoscopic applications. The catheter incorporates a lens actuator that is fabricated using microelectromechanical systems (MEMS) technology. The lens is mounted on a folded flexure made of nickel and is actuated by magnetic field. Thickness measurements are performed by positioning the catheter in front of the tissue and actuating the lens scanner in the out-of-plane direction. A single-mode optical fiber (SMF) is used to deliver a 785 nm laser beam to the tissue and relay back the reflected light from the tissue to a photomultiplier tube (PMT). When the focal point of the scanning lens passes tissue boundaries, intensity peaks are detected in the reflecting signal. Tissue thickness is calculated using its index of refraction and the lens displacement between intensity peaks. The utility of the confocal catheter was demonstrated by measuring the cornea and skin thicknesses of a mouse. Measurement uncertainty of 8.86 μm within 95% confidence interval has been achieved.     

京尼平交联大鼠肾去细胞生物支架提高支架生物学性能的实验研究

目的　通过京尼平交联大鼠肾去细胞生物支架,提高支架的生物学性能。 方法　取250 g左右的健康SD大鼠80只,分为正常组、未交联支架组、戊二醛交联支架组和京尼平交联支架组。游离大鼠肾、肾动脉,连接蠕动泵,经PBS灌注去血后得到的肾作为正常组。其余大鼠肾依次灌入肝素化PBS溶液、1% TritonX-100、1%十二烷基硫酸钠（SDS）、去离子水,完成大鼠肾去细胞生物支架制备。戊二醛交联支架组继续灌入0.625%戊二醛（GA）1500 mL;京尼平交联支架组浸入0.5%的京尼平溶液于37 ℃恒温箱中行化学交联24 h;未经戊二醛或京尼平交联的肾去细胞支架作为未交联支架组。对4组肾分别作HE、Masson、免疫荧光染色及电子显微镜扫描,观察支架组织形态学及超微结构改变;力学拉伸试验检测机械力学性能。 结果　SD大鼠肾支架经京尼平交联后,HE、Masson染色显示胶原纤维排列更加紧密有序,肾小球处的纤维呈聚集状,Collagen I和 Collagen IV荧光染色增强,电镜扫描可见交联后的去细胞支架内蜂窝状孔洞结构更加立体,并可见典型的肾小球龛样结构轮廓更加清晰;交联组肾弹性模量较支架组明显增强。 结论　京尼平交联大鼠肾支架能提高支架的生物学性能有助于为后期细胞植入和器官再生。     

Stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin)

The study was undertaken to investigate the stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin) at distinct elapsed storage durations. The glutaraldehyde-fixed counterpart was used as a control. Porcine pericardia procured from a slaughterhouse were used as raw materials. After fixation, the fixed tissues were sterilized in a graded series of ethanol solutions and thoroughly rinsed in phosphate buffered saline for 1 day, and then stored in a jar containing sterilized water. The samples were taken out and tested for their stability during the durations of 1day through 6 months after storage. The stability of each study group was tested by measuring its tensile strength, free-amino-group content, and denaturation temperature. Additionally, the cytotoxicity of each test sample and its corresponding storage solution were investigated in vitro using 3T3 fibroblasts. The results were examined using a microscope and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the stability of the genipin-fixed tissue during storage was superior to its glutaraldehyde-fixed counterpart. The differences in stability between the genipin- and glutaraldehyde-fixed tissues during storage may be caused by their differences in crosslinking structure. There was no apparent cytotoxicity for both the genipin-fixed tissue and its corresponding storage solution throughout the entire course of the study, whereas significant cytotoxicity was observed for both the glutaraldehyde-fixed tissue and its storage solution. However, the cytotoxicity of the glutaraldehyde-fixed tissue decreased with increasing elapsed storage duration, whereas that of its corresponding storage solution increased. This suggested that the toxic residues remaining in the glutaraldehyde-fixed tissue leached out slowly into its corresponding storage solution during the course of storage.     

Biomechanical and angiogenic properties of tissue-engineered rat trachea using genipin cross-linked decellularized tissue

In this study, the obtainment and characterization of decellularized rat tracheal grafts are described. The detergent-enzymatic method, already used to develop bioengineered pig and human trachea scaffolds, has been applied to rat tracheae in order to obtain airway grafts suitable to be used to improve our knowledge on the process of tissue-engineered airway transplantation and regeneration. The results demonstrated that, after 9 detergent-enzymatic cycles, almost complete decellularized tracheae, retaining the hierarchical and mechanical properties of the native tissues with strong in vivo angiogenic characteristics, could be obtained. Moreover, to improve the mechanical properties of decellularized tracheae, genipin is here considered as a naturally derived cross-linking agent. The results demonstrated that the treatment increased mechanical properties, in term of secant modulus, without neither altering the pro-angiogenic properties of decellularized airway matrices or eliciting an in vivo inflammatory response.     

In vitro study of enzymatic degradation of biological tissues fixed by glutaraldehyde or epoxy compound

The study, using bacterial collagenase, was to investigate the changes in characteristics of a collagen-rich tissue, porcine pericardium, fixed by glutaraldehyde or epoxy compound (ethylene glycol diglycidyl ether) during the course of degradation. Fresh porcine pericardium was used as a control. During degradation, the heat released by the reaction of collagenase with a test sample was monitored by a highly sensitive microcalorimeter. Also, the degree of degradation of each test sample was determined by measuring its increment in free amino group content and changes in denaturation temperature and tensile strength. Microcalorimetric analysis of collagenase degradation of fresh, epoxy-fixed, and glutaraldehyde-fixed tissues revealed that the heat released during degradation correlates well with the degree of tissue degraded. The cleaving of peptide bonds in biological tissue by collagenase degradation may increase its free amino group content and reduce its denaturation temperature and tensile strength. It was noted that the fresh tissue cannot resist bacterial collagenase degradation, while the glutaraldehyde-fixed tissue had a relatively better resistance to degradation than its epoxy-fixed counterpart.     

Preparation of gelatin/genipin nanofibrous membrane for tympanic member repair

Abstract

The wound healing for tympanic membrane (TM) perforations is one of the most common clinical treatment in otomicrosurgery. Recently, tissue-engineered composites and grafts, also new designs of biomaterials are applied to the management of TM perforation. In this work, a series of gelatin/genipin nanofibrous films were prepared as a patch for repairing TM perforation. Gelatin, a type of biomaterial with excellent electrospinning performance, has been used for preparing the nanofibers. The genipin, as a crosslinking agent, has been blended into the gelatin nanofibers. The reaction between gelatin and genipin engender suitable tensile strength and water-resistance for TM patch. The survival rate of human umbilical vein endothelial cells and fibroblasts demonstrated that the gelatin/genipin nanofiber scaffolds had good biocompatibility, which indicated the genipin was a kind of effective and nontoxic crosslinking agent for improving the mechanical property and water-resistance of gelatin films. In short, our work provides a novel macromolecular material with good mechanical properties, water-tolerance and excellent biocompatibility which could be used as a potential patch for TM repair.     

Polarization singularities of biological tissues images

The ways to the polarization singularities of the biological tissues images of various morphological structures have been theoretically analyzed. The coordinate distributions of singly (linear states of polarization) and doubly degenerated (circular states of polarization) polarization singularities of the physiologically normal and pathologically changed biological tissues have been experimentally examined. The statistical criteria of diagnostics of the kidney tissue collagenous disease (the third and the fourth statistical moments of the linear density singularity points) have been determined. It was discovered that the process of the pathological change of the kidney tissue morphology leads to the formation of the self-similar (fractal) coordinate distribution of the polarization singularities of its image.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 degrees C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

Numerical study of water diffusion in biological tissues using an improved finite difference method

An improved finite difference (FD) method has been developed in order to calculate the behaviour of the nuclear magnetic resonance signal variations caused by water diffusion in biological tissues more accurately and efficiently. The algorithm converts the conventional image-based finite difference method into a convenient matrix-based approach and includes a revised periodic boundary condition which eliminates the edge effects caused by artificial boundaries in conventional FD methods. Simulated results for some modelled tissues are consistent with analytical solutions for commonly used diffusion-weighted pulse sequences, whereas the improved FD method shows improved efficiency and accuracy. A tightly coupled parallel computing approach was also developed to implement the FD methods to enable large-scale simulations of realistic biological tissues. The potential applications of the improved FD method for understanding diffusion in tissues are also discussed.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3- dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 °C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

京尼平对缺氧/复氧损伤后大鼠心肌细胞凋亡及自噬的影响

目的 探讨京尼平对缺氧/复氧(H/R)损伤后大鼠心肌细胞凋亡及自噬的影响。方法 建立H/R损伤模型,体外培养的大鼠H9c2心肌细胞行缺氧12 h、复氧4 h。实验分为对照组（Con）、京尼平组(GE)、缺氧/复氧组(H/R)、缺氧/复氧+京尼平组(H/R+GE)。细胞计数试剂盒-8（CCK-8）检测细胞存活率,流式细胞仪检测细胞凋亡,透射电子显微镜观察自噬体,Western blotting检测Bax、Bcl-2、P62、Beclin1、LC3-Ⅱ、蛋白激酶B(Akt)、p-Akt、哺乳动物雷帕霉素靶蛋白（mTOR）和p-mTOR蛋白的表达。结果 京尼平预处理增强了H/R损伤后的H9c2心肌细胞活力,抑制细胞凋亡及自噬体累积,降低自噬结构断面积与细胞质断面积的比值。Western blotting结果显示,京尼平预处理减少了H/R损伤后Bax、LC3-Ⅱ和Beclin1蛋白表达,增加Bcl-2、P62、p-Akt和p-mTOR蛋白表达。结论 京尼平可以抑制H/R损伤后心肌细胞凋亡及自噬,其机制可能与上调Akt/mTOR信号通路有关。     

Non-invasive imaging of optical parameters of biological tissues

The normalised back-scattered intensity (NBI) profiles at various locations on the forearms of ten human subjects were obtained by moving the multi-probe of a laser reflectometer. The statistical analysis of the NBI data showed that the variation in the NBI was significantly higher at the ulnar region compared with that at other regions. For determination of the scattering (μ _s ) and absorption (μ _a ) coefficients and the anisotropy parameter g at each location on the forearm, these profiles were matched with the NBI profiles simulated by a Monte Carlo procedure (χ _0.99 ² ). For the reconstruction of images of variation of these parameters, the averaged values ofμ _a ,μ _s and g at all locations on the forearms of the subjects were determined. The absorption coefficient had a minimum (1.92 cm⁻¹) and maximum (2.21 cm⁻¹) at the wrist and the lateral region of the forearm, respectively. The scattering coefficient had a maximum (194 cm⁻¹) at the medial side and near the elbow, and a minimum (186 cm⁻¹) at the lateral side of the forearm. Similar changes in the anisotropy parameter were also observed. By interpolation of the data of each parameter on a 100×100 image matrix and after median filtering, colour-coded images of the variation in the optical parameters were constructed. These images could be useful for diagnostic and therapeutic applications of lasers.     

Measurement of thermal and ultrasonic properties of some biological tissues

The measurement of thermal and ultrasonic properties of biological tissues is essential for the assessment of the temperature rise induced in vivo by diagnostic ultrasound. In this paper, we present measurements of thermal conductivity, thermal diffusivity, speed of sound and ultrasonic attenuation of fresh ex vivo porcine tissue, namely ‘muscle’ (from abdomen and leg), ‘skin with subcutaneous fat’ (from abdomen and leg), ‘abdominal fat’ and ‘bone’. The measurements of the thermal properties of biological tissue samples are based on a transient method. Thermal property measurements show that subcutaneous fat has the lowest thermal conductivity (0.23 W m^?1 K^?1), while muscle gives the highest values (0.46 W m^?1 K^?1). Thermal diffusivity of muscle tissue recorded the highest value among the studied tissues (0.16 mm² s^?1) while that of skin with subcutaneous fat gave the lowest value (0.11 mm² s^?1). A scanning acoustic macroscope was used to measure attenuation coefficient and speed of sound for the tissue samples. The results for the speed of sound are broadly similar to those reported in the literature. The power law dependence of the attenuation coefficient of the form η = a f ^b as a function of frequency was found to be more appropriate than the linear fit in this study.     

Applications of digital image correlation to biological tissues

Optical methods are becoming commonplace in investigations of the physical and mechanical behavior of biological tissues. Digital image correlation (DIC) is a versatile optical method that shows tremendous promise for applications involving biological tissues and biomaterials. We present the fundamentals of DIC with an emphasis on the application to biological materials. An approach for surface preparation is described that facilitates its application to hydrated substrates. Three examples are presented that highlight the use of DIC for biomedical research. The first example describes the use of DIC to study the mechanical behavior of arterial tissues up to 40% elongation. The second example describes an evaluation of the mechanical properties of bovine hoof horn in the dehydrated and fully hydrated states. Uniaxial tension experiments are performed to determine the elastic modulus (E) and Poisson's ratio (nu) of both the arterial and dermal tissues. Spatial variations in the mechanical properties are evident from the full-field characterization of both tissues. Finally, an application of DIC to study the evolution of loosening in cemented total hip replacements is described. The noncontact analysis enables measurement of the relative displacement between the bone/bone cement and bone cement/prosthesis interfaces. Based on the elementary optical arrangement, the simple surface preparation, and the ability to acquire displacement/strain measurements over a large range of deformation, DIC should serve as a valuable tool for biomedical research. Further developments will enable the use of DIC for in vivo applications.     

Dielectric properties of tissues and biological materials: a critical review

We critically review bulk electrical properties of tissues and other biological materials, from DC to 20 GHz, with emphasis on the underlying mechanisms responsible for the properties. We summarize the classical principles behind dielectric relaxation and critically review recent developments in this field. Special topics include a summary of the significant recent advances in theories of counterion polarization effects, dielectric properties of cancer vs. normal tissues, properties of low-water-content tissues, and macroscopic field-coupling considerations. Finally, the dielectric properties of tissues are summarized as empirical correlations with tissue water content in other compositional variables; in addition, a comprehensive table is presented of dielectric properties. The bulk electrical properties of tissues are needed for many bioengineering applications of electric fields or currents, and they provide insight into the basic mechanisms that govern the interaction of electric fields with tissue.     

The identification of particulate matter in biological tissues and fluids.

Sections, 1-2 micron thick, of Araldite embedded tissue were prepared to provide a link between optical and electron microscopy for the identification of crystals. This technique permits examination of individual particles by means of polarising light microscopy, electron microscopy, X-ray energy spectroscopy and electron diffraction. It provides a reliable method for routine and research studies. It has led to the positive identification of individual crystals of calcium pyrophosphate dihydrate and hydroxyapatite in synovial fluids. The method can be applied to the identification of other particulate matter in a variety of biological specimens.