Skin regeneration stimulation: the role of PCL‐platelet gel nanofibrous scaffold

Skin is the largest organ of the human body. Thus far, tissue engineering of skin has developed rapidly and has used many types of growth factors and nanofibrous scaffolds. In this study, we differentiated neonate keratinocytes for epithelialization on the polycaprolactone‐Platelet gel (PCL‐PG) scaffold. Fabricated PCL nanofibers prepared by electrospinning technology and coated by platelet gel. Subsequently, the structure of the scaffold was evaluated by SEM, FTIR‐ATR, contact angle and tensile test assays. After seeding the neonate keratinocytes on neat PCL and PCL‐PG scaffolds, the epidermal maturation was tested by detecting cytokeratin 10 and loricrin determinants by immunocytochemistry; moreover, keratinocyte genes such as keratin 14, keratin 10, and Involucrin were investigated by real‐time PCR. The results of MTT assay indicated an increase in cell viability and cell proliferation of neonate keratinocytes on PCL‐PG nanofiber scaffolds compared with PCL. RT‐PCR and immunocytochemical analysis showed better cell differentiation on the PCL‐PG scaffolds than neat PCL. Furthermore, SEM microscopy images demonstrated that neo‐keratinocytes enhance adhesion and proliferation on PCL‐PG nanofiber scaffolds. We found that PG increases biocompatibility and wettability of scaffold, cell adhesion, and expression of keratinocyte markers. Overall, this procedure is recommended to be employed in skin tissue engineering and wounds healing.     

Fabrication of combinatorial polymer scaffold libraries

We have designed a novel combinatorial research platform to help accelerate tissue engineering research. Combinatorial methods combine many samples into a single specimen to enable accelerated experimentation and discovery. The platform for fabricating combinatorial polymer scaffold libraries can be used to rapidly identify scaffold formulations that maximize tissue formation. Many approaches for screening cell-biomaterial interactions utilize a two-dimensional format such as a film or surface to present test substrates to cells. However, cells in vivo exist in a three-dimensional milieu of extracellular matrix and cells in vitro behave more naturally when cultured in a three-dimensional environment than when cultured on a two-dimensional surface. Thus, we have designed a method for fabricating combinatorial biomaterial libraries where the materials are presented to cells in the form of three-dimensional, porous, salt-leached, polymer scaffolds. Many scaffold variations and compositions can be screened in a single experiment so that optimal scaffold formulations for tissue formation can be rapidly identified. In summary, we have developed a platform technology for fabricating combinatorial polymer scaffold libraries that can be used to screen cell response to materials in a three-dimensional, scaffold format.     

Ultrafast optical pulse delivery with fibers for nonlinear microscopy

Nonlinear microscopies including multiphoton excitation fluorescence microscopy and multiple-harmonic generation microscopy have recently gained popularity for cellular and tissue imaging. The optimization of these imaging methods for minimally invasive use requires optical fibers to conduct light into tight space, where free-space delivery is difficult. The delivery of high-peak power laser pulses with optical fibers is limited by dispersion resulting from nonlinear refractive index responses. In this article, we characterize a variety of commonly used optical fibers in terms of how they affect pulse profile and imaging performance of nonlinear microscopy; the following parameters are quantified: spectral bandwidth and temporal pulse width, two-photon excitation efficiency, and optical resolution. A theoretical explanation for the measured performance of these fibers is also provided.     

Improvement of the representative volume element method for 3-D scaffold simulation

Predicting the mechanical properties of the 3-D scaffold using finite element method (FEM) simulation is important to the practical application of tissue engineering. However, the porous structure of the scaffold complicates computer simulations, and calculating scaffold models at the pore level is time-consuming. In some cases, the demands of the procedure are too high for a computer to run the standard code. To address this problem, the representative volume element (RVE) theory was introduced, but studies on RVE modeling applied to the 3-D scaffold model have not been focused. In this paper, we propose an improved FEM-based RVE modeling strategy to better predict the mechanical properties of the scaffold prior to fabrication. To improve the precision of RVE modeling, we evaluated various RVE models of newly designed 3-D scaffolds using FEM simulation. The scaffolds were then constructed using microstereolithography technology, and their mechanical properties were measured for comparison.     

基于快速原型的组织工程支架成形技术

组织工程是生物制造的重要发展阶段,通过先成形生物材料的支架,然后在上面复合细胞的方式,来实现组织器官的人工制造。支架的性能在组织工程的应用中非常重要。相比传统的支架成形方法,基于离散—堆积原理的快速原型技术,所制造的支架个性化程度较高:支架的孔隙率、力学性能、生物相容性和降解特性等方面,可以通过参数设计和材料选择的方法进行人工设定;因此非常适于构建结构性的组织和器官。介绍了现有的基于快速原型的组织工程支架的成形技术,总结了其特点及应用现状,并分析讨论了其优缺点。     

Bioactive composites for bone tissue engineering

One of the major challenges for bone tissue engineering is the production of a suitable scaffold material. In this review the currently available composite material options are considered and the methods of production and assessing the scaffolds are also discussed. The production routes range from the use of porogens to produce the porosity through to controlled deposition methods. The testing regimes include mechanical testing of the produced materials through to in vivo testing of the scaffolds. While the ideal scaffold material has not yet been produced, progress is being made.     

Structural evaluation of scaffolds prototypes produced by three-dimensional printing

The fabrication of porous scaffolds with complex architectures represents a challenge in tissue engineering. Recent studies have shown that it is possible to construct tissue-engineered bone repair scaffolds with tight pore size distributions and controlled geometries using 3D printing techniques. In this context, this work aims to evaluate the 3D printing process in order to study its potential for scaffold fabrication. Despite the wide use of porous scaffolds, its design, geometry optimization and mechanical assessment, for successful integration in tissue engineering, require further developments and studies to help in its optimal design. In the present work, cubical scaffolds prototypes with different architectures were produced by 3D printing technology. Scaffolds dimensional accuracy, porosity and mechanical stiffness were comprehensive analysed by means of an experimental investigation. The microporosity, inherent to the fabrication process, and the mechanical characterization of the bulk material were also considered. This paper addresses methodologies to overcome some limitations of 3D printing technique to produce scaffolds for tissue engineering and proposes procedures for their suitable mechanical characterization. Results of this work indicate that 3D printing process has great potential for scaffold fabrication.     

Imaging cellular responses to mechanical stimuli within three-dimensional tissue constructs

The cellular response to environmental cues is complex, involving both structural and functional changes within the cell. Our understanding of this response is facilitated by microscopy techniques, but has been limited by our ability to image cell structure and function deep in highly-scattering tissues or 3D constructs. A novel multimodal microscopy technique that combines coherent and incoherent imaging for simultaneous visualization of structural and functional properties of cells and engineered tissues is demonstrated. This microscopic technique allows for the simultaneous acquisition of optical coherence microscopy and multiphoton microscopy data with particular emphasis for applications in cell biology and tissue engineering. The capability of this technique is shown using representative 3D cell and tissue engineering cultures consisting of primary fibroblasts from transgenic green fluorescent protein (GFP) mice and GFP-vinculin transfected fibroblasts. Imaging is performed following static and dynamic mechanically-stimulating culture conditions. The microscopy technique presented here reveals unique complementary data on the structure and function of cells and their adhesions and interactions with the surrounding microenvironment.     

骨组织工程细胞载体框架的两种快速成形新工艺

采用纳米羟基磷石灰石／胶原自组装材料，同时在成形过程中加入聚左旋乳酸以改善的成形性能，开发了两种新的制备骨组织工程细腻载体框架结构的快速成形工艺：T（组织工程材料）＋F（框架成形材料）成形工艺和低温喷射成形工艺。这两种工艺实现了材料的精确堆积，能成形200－500μm的可控大孔隙和孔隙梯度结构，获得理想的形态参数。     

基于RP的骨组织工程支架构造及生物学特性分析

应用三维CAD软件设计支架和相应的模具结构,通过光固化快速成形技术制造出树脂模具,并在模具中填充磷酸钙(CPC)生物材料,然后通过热分解方法去掉树脂模具,得到具有可控微管道结构的骨组织工程支架。该方法克服了传统构造方法中支架内部微管道结构不可控的缺点,为制造出更有利于细胞/组织长入和成活的支架三维空间结构提供了一个更理想的方案。INSTRONMicrotester试验设备上测得支架的最大抗压强度为7.12MPa;测得其表面粗糙度Ra=2.16μm。扫描电镜观察支架表面微结构特征,能谱分析测出支架中所含钙元素和磷元素的摩尔比是1.59∶1。结果表明所构造的支架具有良好的生物学特性。     

Fabrication and characterization of three-dimensional poly(ether- ether- ketone)/-hydroxyapatite biocomposite scaffolds using laser sintering

The ability to have precise control over porosity, scaffold shape, and internal pore architecture is critical in tissue engineering. For anchorage-dependent cells, the presence of three-dimensional scaffolds with interconnected pore networks is crucial to aid in the proliferation and reorganization of cells. This research explored the potential of rapid prototyping techniques such as selective laser sintering to fabricate solvent-free porous composite polymeric scaffolds comprising of different blends of poly(ether-ether-ketone) (PEEK) and hydroxyapatite (HA). The architecture of the scaffolds was created with a scaffold library of cellular units and a corresponding algorithm to generate the structure. Test specimens were produced and characterized by varying the weight percentage, starting with 10 wt% HA to 40 wt% HA, of physically mixed PEEK-HA powder blends. Characterization analyses including porosity, microstructure, composition of the scaffolds, bioactivity, and in vitro cell viability of the scaffolds were conducted. The results obtained showed a promising approach in fabricating scaffolds which can produce controlled microarchitecture and higher consistency.     

Fabrication and evaluation of calcium phosphate cement scaffold with controlled internal channel architecture and complex shape

The ability to have precise control over internal channel architecture, porosity, and external shape is essential for tissue engineering. The feasibility of using indirect stereo-lithography (SL) to produce scaffolds from calcium phosphate cement materials for bone tissue engineering has been investigated. The internal channel architecture of the scaffolds was created by removal of the negative resin moulds made with SL. Scanning electron microscopy (SEM) showed highly open, well-interconnected channel architecture. The X-ray diffraction examination revealed that the hydroxyapatite phase formed at room temperature in the cement was basically stable up to 850 degrees C. There was no phase decomposition of hydroxyapatite, although the crystallinity and grain size were different. The ability of resulting structure to support osteoblastic cells culture was tested in vitro. Cells were evenly distributed on exterior surfaces and grew into the internal channels of scaffolds. To exploit the ability of this technique, anatomically shaped femoral supracondylar scaffolds with 300-800 microm interconnected channels were produced and characterized.     

面向骨组织工程的三维仿生支架的微观结构研究

骨缺损修复是当今国际组织工程领域的一个难题，而细胞载体支架的制取是骨缺损问题研究的重点。探讨了支架结构制取的几种方法并进行了比较，采用数模相结合的方法进行微观结构的设计，获得了参数(孔的形状、尺寸，孔隙率等)可控的骨支架模型。     

Fibre-optic nonlinear optical microscopy and endoscopy

Nonlinear optical microscopy has been an indispensable laboratory tool of high‐resolution imaging in thick tissue and live animals. Rapid developments of fibre‐optic components in terms of growing functionality and decreasing size provide enormous opportunities for innovations in nonlinear optical microscopy. Fibre‐based nonlinear optical endoscopy is the sole instrumentation to permit the cellular imaging within hollow tissue tracts or solid organs that are inaccessible to a conventional optical microscope. This article reviews the current development of fibre‐optic nonlinear optical microscopy and endoscopy, which includes crucial technologies for miniaturized nonlinear optical microscopy and their embodiments of endoscopic systems. A particular attention is given to several classes of photonic crystal fibres that have been applied to nonlinear optical microscopy due to their unique properties for ultrashort pulse delivery and signal collection. Furthermore, fibre‐optic nonlinear optical imaging systems can be classified into portable microscopes suitable for imaging behaving animals, rigid endoscopes that allow for deep tissue imaging with minimally invasive manners, and flexible endoscopes enabling imaging of internal organs. Fibre‐optic nonlinear optical endoscopy is coming of age and a paradigm shift leading to optical microscope tools for early cancer detection and minimally invasive surgery.     

Differentiating the two main histologic categories of fibroadenoma tissue from normal breast tissue by using multiphoton microscopy

Multiphoton microscopy has become a novel biological imaging technique that allows cellular and subcellular microstructure imaging based on two‐photon excited fluorescence and second harmonic generation. In this work, we used multiphoton microscopy to obtain the high‐contrast images of human normal breast tissue and two main histologic types of fibroadenoma (intracanalicular, pericanalicular). Moreover, quantitative image analysis was performed to characterize the changes of collagen morphology (collagen content, collagen orientation). The results show that multiphoton microscopy combined with quantitative method has the ability to identify the characteristics of fibroadenoma including changes of the duct architecture and collagen morphology in stroma. With the advancement of multiphoton microscopy, we believe that the technique has great potential to be a real‐time histopathological diagnostic tool for intraoperative detection of fibroadenoma in the future.     

Mechanical and histological properties of an electrospun scaffold with a modified surface by plasma polymerization implanted in an <i>in vivo</i> model

This article presents the construction of scaffolds composed of polylactic acid (PLA) with different concentrations of hydroxyapatite (HA) by electrospinning, which were superficially modified with polypyrrole (PPy/I) by plasma polymerization. A preliminary study was conducted of the biological and mechanical behavior of the scaffolds when they were implanted in the back of rabbits for 30 days; bone cells differentiated from mesenchymal stem cells (MSCs) were used. The bone cell and scaffold structures were characterized by histological, immunohistochemical, and mechanical stress tests. Hematoxylin–eosin staining showed good tissue conformation. The immunohistochemical tests highlighted the presence of the main bone tissue proteins, such as collagen, osteocalcin, and osteopontin. The PLA/HA scaffolds were observed to exhibit cell adhesion and proliferation properties; however, the response was much better in the scaffolds that had a higher concentration of HA and that were coated with PPy/I. The results of the mechanical tests of the scaffolds indicated that the plasma treatment improved the adhesion and cell proliferation properties and contributed to the mechanical support, allowing the formation of neotissues with good viability of cell growth.     

Chitosan-based hyaluronan hybrid polymer fibre scaffold for ligament and tendon tissue engineering

To establish medical use of tissue engineering technology for ligament and tendon injuries, a scaffold was developed which has sufficient ability for cell growth, cell differentiation, and mechanical properties. The scaffold made from chitosan and 0.1 per cent hyaluronic acid has adequate biodegradability and biocompatibility. An animal experiment showed that the scaffold has less toxicity and less inflammation induction. Furthermore, in-vivo animal experiments showed that the mechanical properties of the engineered ligament or tendon had the possibility to stabilize the joint. It was shown that newly developed hybrid-polymer fibre scaffold has feasibility for joint tissue engineering.     

Multiphoton fluorescent images with a spatially varying background signal: a ML deconvolution method

By means of multiphoton laser scanning microscopy, neuroscientists can look inside the brain deeper than has ever been possible before. Multiphoton fluorescent images, as all optical images, suffer from degradation caused by a variety of sources (e.g. light dispersion and absorption in the tissue, laser fluctuations, spurious photodetection and staining deficiency). From a modelling perspective, such degradations can be considered the sum of stochastic noise and a background signal. Among the methods proposed in the literature to perform image deconvolution in either confocal or multiphoton fluorescent microscopy, Vicidomini et al. (2009) were the first to incorporate models for noise (a Poisson process) and background signal (spatially constant) in the context of regularized inverse problems. Unfortunately, the so-called split-gradient deconvolution method (SGM) they used did not consider possible spatial variations in the background signal. In this paper, we extend the SGM by adding a maximum-likelihood estimation step for the determination of a spatially varying background signal. We demonstrate that the assumption of a constant background is not always valid in multiphoton laser microscopy and by using synthetic and actual multiphoton fluorescent images, we evaluate the face of validity of the proposed method, and compare its accuracy with the previously introduced SGM algorithm.     

Use of confocal and multiphoton microscopy for the evaluation of micro-optical components and emitters

We report on the application of confocal and multiphoton microscopic techniques for the evaluation of the latest generation of micro optical components. The optical emitting characteristics of arrays of matrix addressable GaN micrometer-sized light emitting diodes (micro-LEDs) have been measured using a commercial confocal microscope utilising the LEDs' own emission along with reflection confocal microscopy to determine the surface structure. Multiphoton induced luminescence from the 10-20-micron diameter emitters has also been used to examine the structure of the device and we compare this with electrically induced emission. In related work, the optical properties of micro lens arrays (10-100-micron diameter) fabricated in SiC, Sapphire, and Diamond have been determined using transmission confocal microscopy. Such optical microscopy techniques offer a simple, non-destructive method to determine the structure and performance of such novel devices.     

Development of composite porous scaffolds based on poly(lactide-co-glycolide)/nano-hydroxyapatite via selective laser sintering

Poly(lactide-co-glycolide) (PLGA)/nano-hydroxyapatite (nano-HAP) composite porous scaffolds with well-controlled pore architectures as well as high exposure of the bioactive ceramics to the scaffold surface were fabricated via selective laser sintering. Neat PLGA and the composite of PLGA/nano-HAP were used to obtain suitable process parameters. The effects of nano-HAP content on the microstructure and mechanical properties were investigated. The testing results showed that the compressive strength and modulus of the scaffolds were highly enhanced when the nano-HAP content reached from 0 to 20 wt%, while the mechanical properties experienced a sharp dropped with the nano-HAP content further increased. This might be due to the large reduction in polymer which decreased the interface bond strength between particles. It suggests that the introduction of nano-HAP as a reinforcing phase can improve the mechanical properties of the polymer porous scaffolds. The novel developed scaffolds may serve as a three-dimensional bone substrate in tissue engineering.