Preparation,characterization and storage of human ferritin for use as a standard for the assay of serum ferritin

Summary. This paper describes studies on the source, preparation, characterization and storage of human ferritin for use as a standard for the immunoassay of serum ferritin. Ferritin was prepared from the liver or spleen by methods including either ultracentrifugation or cadmium sulphate crystallization. Preparations were characterized by polyacrylamide gel electrophoresis in dissociating and non-dissociating buffers, iso-electric focusing, analysis of amino acid composition and measurement of protein content. The protein content of solutions of liver or spleen ferritin may be determined by the method of Lowry with bovine serum albumin as standard. Lyophilization under carefully controlled conditions in buffer containing high concentrations of albumin provides a stable preparation of ferritin. Accelerated degradation and collaborative immunological studies of two lyophilized preparations of ferritin, one from liver and one from spleen, indicate that either is an acceptable reference material.     

泻白散及其主药体外抗氧化活性研究

目的 建立泻白散和方中3味主药甘草、地骨皮、桑白皮的体外抗氧化活性测定方法,并对31批药材和10批泻白散煎液的抗氧化活性进行测定。方法 采用紫外可见分光光度法检测一定浓度的药材提取液引起DPPH溶液吸光度（A）值降低,考察波长为517 nm,分别探索3味药材抗氧化活性成分的提取条件;并进行不同溶剂的吸收考察、专属性考察、DPPH线性考察、药材提取液线性考察、精密度试验、重复性试验、耐用性考察等方法学验证;以清除DPPH自由基的半抑制浓度（IC₅₀）作为评价指标,对泻白散和方中3味药材的体外抗氧化活性进行考察。结果 地骨皮、甘草、桑白皮和泻白散提取液的IC₅₀均值为0.31、1.24、1.49和0.91 g/L,泻白散提取工艺对方中药物抗氧化活性的保留均值为56%。结论 建立的抗氧化活性测定方法可用于泻白散及方中主药的抗氧化活性测定,为多维度评价中药和中药材质量提供新思路。     

Development of novel quality control material based on CRISPR/Cas9 editing and xenografts for MLH1 protein deficiency testing

BackgroundMismatch repair deficiency (dMMR) status induced by MLH1 protein deficiency plays a pivotal role in therapeutic decision‐making for cancer patients. Appropriate quality control (QC) materials are necessary for monitoring the accuracy of MLH1 protein deficiency assays used in clinical laboratories.MethodsCRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein‐deficient cell lines. The positive cell lines were screened and validated by Sanger sequencing, Western blot (WB), and next‐generation sequencing (NGS) and were then used to prepare formalin‐fixed, paraffin‐embedded (FFPE) samples through xenografting. These FFPE samples were tested by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for suitability as novel QC materials for MLH1 protein deficiency testing.ResultsWe successfully cultured 358 monoclonal cells, with a survival rate of 37.3% (358/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and named as GM12878Cas9_6 and GM12878Cas9_10. The NGS results further confirmed that the MLH1 gene mutation in these two cell lines would cause the formation of stop codons and terminate the expression of the MLH1 protein. The H&E staining and IHC results also verified the deficiency of the MLH1 protein, and FFPE samples from xenografts proved their similarity and consistency with clinical samples.ConclusionsWe successfully established MLH1 protein‐deficient cell lines. Followed by xenografting, we developed novel FFPE QC materials with homogenous, sustainable, and typical histological structures advantages that are suitable for the standardization of clinical IHC methods.     

Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture

Limbal epithelial stem cells are responsible for the maintenance of the human corneal epithelium and these cells reside in a specialised stem cell niche. They are located at the base of limbal crypts, in a physically protected microenvironment in close proximity to a variety of neighbouring niche cells. Design and recreation of elements of various stem cell niches have allowed researchers to simplify aspects of these complex microenvironments for further study in vitro. We have developed a method to rapidly and reproducibly create bioengineered limbal crypts (BLCs) in a collagen construct using a simple one-step method. Liquid is removed from collagen hydrogels using hydrophilic porous absorbers (HPAs) that have custom moulded micro-ridges on the base. The resulting topography on the surface of the thin collagen constructs resembles the dimensions of the stromal crypts of the human limbus. Human limbal epithelial cells seeded onto the surface of the constructs populate these BLCs and form numerous layers with a high proportion of the cells lining the crypts expressing putative stem cell marker, p63α. The HPAs are produced using a moulding process that is flexible and can be adapted depending on the requirements of the end user. Creation of defined topographical features using this process could be applicable to numerous tissue-engineering applications where varied 3-dimensional niche architectures are required.     

Polyethylene terephthalate membrane grafted with peptidomimetics: endothelial cell compatibility and retention under shear stress

The present work aimed to treat a polyethylene terephthalate (PET) surface to make the biomaterial more ‘attractive’ in terms of attachment and shear stress response to endothelial cells with a view to possible applications in vascular grafting. A surface wet-chemistry protocol was applied to graft track-etched PET membranes with RGD peptidomimetics based on the tyrosine template and active at the nano-level vs. isolated human α_vβ₃ receptor, which was monitored by X-ray photoelectron spectroscopy, contact angle measurement and atomic force microscopy for characterization. A primary culture of human saphenous vein endothelial cells was used before and after sterilization of the membranes (heat treatment or γ-ray irradiation) to test the benefit of grafting. The optimal surface concentrations of grafted molecules were around 50?pmol/cm². Compared to GRGDS, the peptidomimetics promoted cell attachment with similar or slightly better performances. Endothelialized grafted supports were further exposed to 2?h of shear stress mimicking arterial conditions. Cells were lost on non-grafted PET whereas cells on grafted polymers sterilized by γ-ray irradiation withstood forces with no significant difference in focal contacts. At the mRNA level, cells on functionalized PET were able to respond to shear stress with NFkB upregulation. Thus, grafting of peptidomimetics as ligands of the α_vβ₃ integrin could be a relevant strategy to improve the adhesion of human endothelial cells and to obtain an efficient endothelialized PET for the surgery of small-diameter vascular prostheses.     

Effect of coating thickness and its material on the stress distribution for dental implants

Dental implants have been increasingly used to recover the masticatory function of lost teeth. It has been well known that the success of a dental implant is heavily dependent on initial stability and long-term osseointegration due to optimal stress distribution in the surrounding bones by the concept implant surface coating. Hydroxyapatite (HAP), as a coating material, has been widely used in dentistry due to its biocompatibility. Some investigations show a benefit of coating dental implants with HAP, and others concluded that HAP coating reduces the long-term implant survival. Therefore, the aim of this investigation is to design a new functionally graded dental implant coating, as well as studying the effect of coating thickness on the maximum von Mises stresses in bone adjacent to the coating layer. The gradation of the elastic modulus is changed along the longitudinal direction. Stress analysis using a finite element method showed that using a coating thickness of 150 µm, functionally graded from titanium at the apex to the collagen at the root, will successfully reduce the maximum von Mises stress in bone by 19% and 17% compared to collagen and HAP coating respectively.     

显微血管减压术中单纯应用涤纶垫棉治疗基底动脉压迫所致三叉神经痛的临床疗效

目的初步探讨显微血管减压术中单纯应用涤纶垫棉治疗基底动脉压迫所致三叉神经痛的临床疗效。方法回顾性分析2012年1月至2019年12月陆军军医大学大坪医院神经外科收治的31例基底动脉压迫引发三叉神经痛患者的临床资料。31例患者在显微血管减压术中均单纯使用涤纶垫棉作为减压材料,且未使用其他减压方式。手术采用经乙状窦后入路,于脑干和责任动脉之间放置涤纶垫棉实现减压。采用巴罗神经学研究所(BNI)提出的疼痛分级评估手术效果。结果31例三叉神经痛患者中,27例(87.1%)术后疼痛即刻完全缓解(BNI分级Ⅰ级),3例在术后3个月内完全缓解(BNI分级Ⅰ级),1例疼痛部分缓解(BNI分级Ⅲ级)。5例(16.1%)患者术后出现面部感觉减退,其中3例自愈;1例患者出现渐进性听力下降。31例患者的随访时间为6~85个月(中位时间为40个月),随访期间有4例(12.9%)复发(BNI分级Ⅳ~Ⅴ级),其中2例再次接受手术治疗,另外2例采用立体定向放射治疗配合药物治疗可部分控制面部疼痛。结论显微血管减压术中单纯应用涤纶垫棉治疗基底动脉所致三叉神经痛的术后即刻效果显著,但其复发率及并发症的发生率较高。     

Cytocompatibility study of NHLBI primary reference materials using human endothelial cells

Four different materials, low density polyethylene (LDPE), polydimethylsiloxane (PDMS), polyvinylcholoride (PVC) and cellulose, were selected by the Devices and Technology Branch of the National Heart Lung and Blood Institute (NHLBI) as primary reference materials for blood contacting. Among the wide variety of tests proposed to assess hemocompatibility of short-term blood contacting catheters, it was desirable to rule out whether these materials could release toxics for vascular cells of the physiological environment. Thus, the cytocompatibility of these materials have been checked towards human umbilical vein endothelial cells: the method used avoids direct contact between cells and materials but evaluates the effect of possible toxic substances leached from materials. These substances were obtained under defined conditions according to a standard. The results show that the extracts of cellulose and LDPE provoke an important cytotoxic effect on the endothelial cell cultures, while the extracts of PDMS and PVC allow the obtention of endothelial cell lining of the reference surface, with a correct global metabolic activity and the intracellular presence of von Willebrand factor.     

变应变率下骨骼肌连续介质本构模型

目的 针对不同结构耦合肌肉主被动行为无法考虑肌肉组织连续介质力学特性的问题,提出运用被动与主动耦合在同一个本构方程的方法,构建骨骼肌连续介质超弹性主被动本构模型。方法 为标定被动本构模型参数,给出单轴拉伸实验方法 及条件,并通过理论推导,介绍利用试验数据求解被动模型参数的具体方法。为验证主动模型的有效性,以实例对模型进行验证。结果 模型预测曲线与实验输出应力拉伸比曲线具有较好的一致性,在相同应变下的情况下,被动应力和总应力最大误差仅为20、40 kPa。结论 该连续介质超弹性本构模型能较好模拟骨骼肌的主被动行为,从而有利于下一步人体肌肉的建模与仿真。