低温保护剂和降温速率对猪关节软骨压缩杨氏模量的影响

利用动态热机械分析仪(DMA),研究了3种低温保护剂(DMSO、甘油、丙二醇)、4种不同低温保护剂浓度(10%V/V、30%V/V、60%V/V、80%V/V)以及两种降温速率(1℃/min和20℃/min)对猪关节软骨杨氏模量的影响。研究结果表明:在相同的低温保护剂浓度条件下,样品经DMSO处理过的杨氏模量最大,甘油的次之,丙二醇的最小,这是由3种保护剂官能团的水合作用所决定的;随着低温保护剂浓度的增加,杨氏模量也增加,如降温速率为20℃/min时,10%甘油的杨氏模量为1.68 MPa,30%甘油的杨氏模量为2.15 MPa,60%甘油的杨氏模量为2.97 MPa,其它两种低温保护剂也是呈同样的趋势;较慢的降温速率更有利于关节软骨生物力学特性的保存,如新鲜关节软骨的杨氏模量为2.53 MPa,当丙二醇浓度为60%、降温速率为1℃/min时的杨氏模量为2.38 MPa,20℃/min时的杨氏模量为2.25 MPa,其他两种低温保护剂也呈同样的趋势。     

保存方法对同种异体软骨移植物免疫原性的影响

背景：异体软骨移植是治疗关节软骨缺损主要手段,寻找软骨组织保存方法来降低移植软骨的免疫原性已成为临床上关键性的技术问题。 目的：比较慢速梯度降温法、玻璃化法与60Co射线照射+梯度降温法对同种异体骨软骨移植物免疫原性的影响。 方法：将关节软骨块随机分为4组：新鲜组不采取任何措施;慢速梯度降温组给予程序降温法保存关节软骨;玻璃化组利用玻璃化溶液处理后保存;60Co射线照射+梯度降温组给予60Co射线照射后,再按程序降温法保存关节软骨。保存8,15,30,60 d后关节软骨块经细胞培养及扩增后制备成细胞悬液。流式细胞仪检测关节软骨细胞表面主要组织相容性复合体Ⅰ,Ⅱ抗原表达率。 结果与结论：经过保存后,慢速梯度降温组、玻璃化组和60Co射线照射+梯度降温组细胞表面的主要组织相容性复合体Ⅰ和Ⅱ抗原表达率有大幅下降,与新鲜组相比有显著性差异,但前3组间没有显著性差异。各保存组主要组织相容性复合体Ⅰ类抗原和主要组织相容性复合体Ⅱ类抗原表达率均随着时间的推移下降,并且在30 d时3种不同保存方法保存的软骨细胞表面主要组织相容性复合体表达率就降低到最低。预示着经保存处理的关节软骨可能会提高同种异体骨软骨移植手术的成功率。     

玻璃化冷冻法保存关节软骨

背景：同种异体骨软骨移植技术是治疗关节软骨缺损有效的方法之一,但由于移植物保存方法不理想,明显制约着该技术的临床应用。 目的：探讨玻璃化冷冻法保存关节软骨组织的可行性和优越性。 方法：切取成年猪骨软骨,制成约5 mm×6 mm(直径×长度)大小的圆柱形骨软骨块。以新鲜软骨组为对照,分别采用0.5 mol/L甘油 、1 mol/L二甲基亚砜、1 mol/L玻璃化溶液3种方法预处理软骨块,再行冷冻法保存软骨块8周,采用组织化学染色、免疫荧光染色观察并比较软骨细胞活性的变化。 结果与结论：玻璃化溶液预处理组的关节软骨细胞存活率达到74.5%,明显高于甘油和二甲基亚砜预处理组,软骨基质成分仅少量丢失。3种方法相比较,玻璃化溶液预处理后慢速梯度降温冷冻保存法可以明显提高冻存关节软骨组织的活性。     

关节软骨冻结过程热膨胀行为的实验研究

采用热机械分析仪(TMA)研究了不同低温保护剂及其不同浓度、降温速率对猪关节软骨降温过程热膨胀行为的影响。温度范围取0℃～-100℃,低温保护剂选用二甲基亚砜、甘油和1,2丙二醇,每种保护剂设置3种浓度,分别为10%(v/v),30%(v/v),60%(v/v)。以热应变表征热膨胀行为。研究结果表明:关节软骨表面不同区域的含水量有较大差异,这会导致完整关节软骨冻结时的非均匀热膨胀现象;5℃/min时的冻结相变过程最大热应变变化值为5.39%±0.05%,是1℃/min(最大热应变变化值为3.73%±0.06%)的1.45倍,但后者达到最大热应变变化值所需时间是前者的3倍;低温保护剂能显著抑制关节软骨冻结膨胀行为,但在低浓度时(如10%(v/v)),二甲基亚砜(最大热应变变化值为3.22%±0.07%)比甘油(最大热应变变化值为4.20%±0.20%)和丙二醇(最大热应变变化值为0.045 0±0.001 0)的热膨胀要小一些;而当浓度足够高时(如60%(v/v)),3种低温保护剂均能完全抑制关节软骨冻结膨胀现象,有利于保护关节软骨的生物力学性能。     

异体软骨组织移植物的体外液体保存

背景：由于关节软骨无神经和血管,其营养主要来源于滑液和滑膜血管的渗透作用,自身修复能力有限,因而如何更好的修复关节软骨损伤成为亟待解决的医学难题。 目的：回顾近年来关于软骨损伤修复方法以及异体软骨体外保存方法的文献研究,找到适合异体软骨组织体外保存的最佳保存条件以及培养液介质成分,从而提高异体软骨组织体外保存效果。 方法：计算机检索PubMed数据库和中国期刊全文数据库(CNKI)于1990年1月至2013年2月有关异体软骨组织移植物体外液体保存方法的研究,检索关键词分别为“Osteochondral allograft; tissue culture; chondrocyte survival rate;in vitro”和“关节软骨;异体移植;液体保存”,排除发表时间较早或重复研究。 结果与结论：目前主要有2种方法体外保存异体骨软骨。低温冷冻保存法保存后的软骨细胞存活率降低明显,影响移植效果,因此临床应用较少。液体保存法能够提高细胞成活率,保持组织活性,但保存时间不长,不能广泛应用。学者们又进一步改良液体培养环境以及培养液成分,延长了软骨组织体外保存时间,提高了软骨组织保存效果。     

生殖细胞的细胞膜渗透特性研究进展

生殖细胞的低温保存在多个领域具有重要应用价值。为提高保存后细胞的存活率与活性,研究细胞膜对水和低温保护剂的渗透特性十分必要。本文综述了近年来动物生殖细胞的细胞膜渗透特性研究概况,列出了典型的精子细胞和卵母细胞的细胞膜对水和常见低温保护剂的渗透系数,分析了这些细胞的渗透特性对制定其低温保存方案的影响。论文还介绍了细胞膜渗透特性的最新测量方法。     

低温保护剂载入关节软骨过程的传质建模

掌握加载过程中软骨组织内部低温保护剂浓度的时空分布特性,这对合理设计加载程序进而成功保存关节软骨至关重要,为此构建描述低温保护剂载入关节软骨过程的扩散传质模型。根据二元扩散热力学模型,将有效扩散系数与无限稀释扩散系数进行关联,活度系数采用UNIFAC模型估算,扩散系数的浓度依赖性采用Vignes公式表示,无限稀释扩散系数采用Siddiqi-Lucas公式计算。对于二甲亚砜(Me₂SO)、甘油(GLY)、乙二醇(EG)和丙二醇(PG)这4种典型的低温保护剂(CPA),由模型计算得到的软骨组织内CPA的平均浓度与文献报道的实验值之间的平均相对偏差(MRE)和决定系数(R²)分别为1.90%～36.29%和0.959～0.998(Me₂SO),13.56%～19.19%和0.990～0.995(GLY),8.89%～22.09%和0.969～0.988(EG),5.35%～23.76%和0.971～0.992(PG)。结果表明,该模型能较好地适用于这4种CPA,可用于直接预测加载过程中软骨组织内部CPA浓度的时空分布,从而指导加载程序的设计与优化。     

玻璃化保存肌腱的生物力学观察

目的探讨玻璃化保存法对兔肌腱力学性能的影响。方法玻璃化保存组，6条新鲜胫前肌肌腱，以18．64％二甲基亚砜＋13.37％乙酰胺＋9．17％1．2丙二醇＋浓度0．10mmol／L海藻糖＋10％小牛血清为玻璃化冷冻保护剂．将新西兰纯种大白兔肌腱采用三步法预处理．-196℃液氮保存14d；“两步法”深低温冷冻保存组，6条新鲜胫前肌肌腱，以15％二甲基亚砜＋10％小牛血清作为冷冻保护剂，“两步法”处理后-196℃液氮冷冻保存14d；对照组．6条新鲜新西兰纯种大白兔胫前肌肌腱。分别进行肌腱拉伸实验．检测肌腱破坏载荷峰值、最大载荷拉伸位移及杨氏弹性模量。结果肌腱破坏荷载峰值：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组间差异均有统计学意义（P=0．002），玻璃化保存组与“两步法”深低温冷冻保存组无统计学意义（P=0．256）；最大载荷拉伸位移：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组3组间均无统计学意义（P=0．065）；杨氏弹性模量：新鲜肌腱组与玻璃化保存组及“两步法”深低温冷冻保存组间差异均有统计学意义（P=0．006）．玻璃化保存组与“两步法”深低温冷冻保存组差异无统计学意义（P=0．577）。结论玻璃化保存法保存肌腱具有良好的发展前景。     

不同保存方法对同种异体软骨移植修复软骨缺损的免疫原性影响

背景：同种异体骨软骨移植在修复软骨缺损中仍存在免疫排斥等问题。 目的：评价程序深低温冷冻保存法、低温保护剂程序降温保存法、组织培养法、化学处理法对软骨移植物免疫原性的影响。 方法：以“软骨、保存、移植”为中文关键词,采用电子检索的方式,在万方数据库、PubMed数据库中检索1998-01/2011-11关于不同保存方法对软骨移植物免疫原性影响的文章。排除重复研究、普通综述或Meta分析类文章,筛选纳入36篇文献进行评价。 结果与结论：软骨细胞膜上带有组织相容性抗原,软骨细胞能够表达主要相容性复合物且有特异性分化抗原。基质完整时,软骨细胞不会与受体淋巴细胞及浆细胞接触,表现为低免疫性,但软骨下骨组织无基质包绕,所以带有软骨下骨的移植物虽可增加固定的牢固性,但也会增加免疫原性。从不同保存方法对同种异体软骨移植修复软骨缺损的免疫原性影响来看,程序深低温冷冻保存法、低温保护剂程序降温保存法、组织培养法3种方法均有较好的应用效果。     

玻璃化深低温冷冻保存气管的实验研究

玻璃化深低温冷冻在降温过程中大大减少冰晶的形成，极大的减轻了冷冻保存过程中对组织、细胞的损伤，前景广阔。本文旨在研究玻璃化快速降温对气管组织结构的影响，实验分为玻璃化快速降温组、程控缓慢降温组、新鲜未冷冻组3组，通过HE染色、TUNEL染色、透射电镜及扫描电镜等检查，评价不同冷冻方法对气管软骨和纤毛结构影响。结果显示，玻璃化快速降温组的气管软骨板结构完整，黏膜上皮及其纤毛结构、软骨细胞形态保持良好，与传统程控缓慢降温相比，快速降温冷冻对软骨细胞损伤轻，有更高的软骨细胞存活率和纤毛上皮的覆盖率，获得较高的软骨复苏率，较好地保持形态结构完整，其保存效果优于传统缓慢降温的保存效果。     

Sample size requirement for digital image analysis of collagen proportionate area in cirrhotic livers

Hall A R, Tsochatzis E, Morris R, Burroughs A K & Dhillon A P
(2012) Histopathology
Sample size requirement for digital image analysis of collagen proportionate area in cirrhotic livers Aims: The requirements for adequate cirrhotic liver biopsy size have not been established for quantitative fibrosis measurements (collagen proportionate area: CPA). We evaluated the CPA of virtual biopsies in cirrhosis to elucidate (i) the amount of tissue required to achieve reliable CPA measurements and (ii) the effect of aetiology on sample size requirements. Method and results: A total of 120 cirrhotic tissue blocks (six aetiologies) were studied. A representative 100 mm² region was selected from each block and a reference CPA measured. Each image (n = 120) was divided into 100 × 1 mm² images; CPA was measured for each 1 mm² and virtual biopsies of different sizes were created from the 1 mm² components. For each virtual biopsy size the probability that the virtual biopsy CPA would be within 5% of the reference CPA was calculated. There were 441 000 virtual biopsies. Biopsy size versus probability plots indicated that, for 90% probability that the virtual biopsy CPA can be expected to be within 5% of the reference CPA, 22–28 mm² of analysable tissue is required depending on liver disease aetiology; and that a 75% probability level requires a biopsy with 12–15 mm² of analysable tissue. Conclusion: The sample size required for a given probability level depends on the aetiology of cirrhosis, and this should be taken into account when judging the reliability of cirrhotic liver biopsy CPA.     

Mechanisms of ion transport across the choroid plexus

1. Mechanisms of ion transport across the choroidal epithelium were investigated using an in vitro preparation of the frog choroid plexus.2. Sodium was actively transported across the plexus from the vascular to the ventricular surface by an ouabain sensitive electrically silent pump. As in other epithelial membranes the rate of sodium transport was stimulated by the presence of bicarbonate ions in the Ringer solutions. Chloride and bicarbonate ions accompany the net flux of sodium across this tissue.3. Some experiments suggest that potassium is actively transported from the ventricular to the serosal surface, and that the rate of transport is a function of the extracellular potassium concentration.4. No evidence was obtained to suggest that calcium is actively transported across this tissue in either direction.5. Diamox, ethoxyzolamide, pitocin, pitressin, hydrocortisone, amiloride, spironolactone and anoxia all failed to influence sodium transport.6. The sequence of passive ion permeation across the plexus was P(Rb) approximately P(K) > P(Cs) approximately P(Na) approximately P(Cl) approximately P(HCO3) > P(Li) as deduced from diffusion potential measurements. At least for Na, K and Cl there was a good correlation between the permeability coefficients derived from unidirectional flux measurements and from electrical parameters. This indicates that exchange diffusion is unimportant as a mechanism for passive ion transport.7. The instantaneous current-voltage curves were linear in both symmetrical and asymmetrical salt solutions and the choroid plexus conductance was found to be directly proportional to the external salt concentration. These and other lines of evidence suggest that the major route of passive ion permeation across this epithelium is via the tight junction route and not through the cell interior.8. These results are discussed in relation to the in vivo studies of c.s.f. secretion and the mechanisms of active and passive ion transport across other epithelial membranes such as the gall-bladder, intestine and renal proximal tubule.     

大鼠软脑膜微血管通透性的计算机分析

本文应用微循环活体观察和荧光示踪技术 ,通过计算机数字图象分析对荧光素钠 (FiNa)在软脑膜微血管的通透过程进行了定量研究 ,以互成角度的两根血管为研究对象 ,建立了正常大鼠软脑膜和缺血大鼠软脑膜微血管对荧光素钠的通透方程 ,得到了不同缺血条件下的微血管通透速度方程及对通透速度评价的定量方法。通过对所得结果进一步分析 ,计算出两血管夹角与微血管通透性之间的关系 ,并代入不同缺血条件下大鼠软脑膜的通透曲线进行了验证。结果 :大鼠软脑膜微血管的通透方程成对数分布 ,通透速度成幂函数下降 ,一段时间后趋于稳定。以缺血 1h再灌注大鼠通透最为迅速 ,缺血 12h再灌注大鼠通透速度略快于正常大鼠。对于互成角度的两根血管 ,荧光物质的扩散速度与两血管间夹角存在一定关系。结论 :本方法能够较好地反映软脑膜微血管通透的实际情况 ,为进一步建立复杂结构血管的通透方程 ,以及定量评价微血管物质交换参数 ,提供了数学基础。     

Effect of cyproterone acetate on structure and function of rhesus monkey reproductive organs

A low dose of Cyproterone acetate (CPA; 1 mg/kg body weight/day for 70 days) was administered to adult male rhesus monkeys to assess its effects on testicular and epididymal structure and function in a nonhuman primate species. CPA caused extensive degenerative changes in morphology of seminiferous, efferent duct, and epididymal epithelia, including decrease in diameter of seminiferous and epididymal tubules and their lumen, height of epididymal epithelium, and an increase in intertubular connective tissue. The protein profile of spermatozoa showed alterations during their epididymal transit in control and CPA-treated monkeys. In CPA-treated animals, 19 polypeptides were acquired and nine were eliminated during epididymal transit in contrast to acquisition of 12 and loss of 14 polypeptides in control animals. Treatment with CPA also resulted in the appearance of 14 new polypeptides in epididymal cytosol and luminal fluid, probably of lysosomal origin. The protein pattern of caput and cauda epididymal tubule cytosol, maintained in organ culture and exposed to 100 μM CPA for 3 days, showed absence of eight polypeptides. These results indicate that even at the low dose used in this study, CPA has caused spermatogenic arrest, degenerative changes in the epididymal structure, and alterations in epididymal and sperm protein profile. Suppression of serum testosterone levels indicates the need for androgen supplementation if CPA is to be used for male contraception. © 1992 Wiley-Liss, Inc.     

Ectopic cortisol-producing adrenocortical adenoma in the renal hilum: histopathological features and steroidogenic enzyme profile

Ectopic cortisol-producing adrenocortical adenomas (CPA) are extremely rare, and only four cases have previously been reported so far but the tumors were not ultrastructurally studied. Presented in this paper is the fifth case with ectopic CPA which was extensively examined to gain deeper insights in terms of the histopathological features and steroidogenic enzyme profile of the tumor. A 53-year-old woman complained of accidental discovery of left renal mass. She had a 5-year history of hypertension, weight gain, moon face, thin skin and systemic edema. These symptoms completely relieved after the tumor removal. Two years later, the above symptoms recurred, and a recurrent tumor was revealed in left renal hilum. The tumor was removed completely with relief of her symptoms of Cushing’s syndrome. Histologically and ultrastructurally, the tumor was composed of compact cells and clear cells, and the former was prominent, suggesting an active secretory function of the tumor. The adenoma tissue showed a strong immunostaining for Melan-A, 3beta-hydroxysteroid dehydrogenase (HSD3B2) and 17alpha-hydroxylase1 (CYP17A1). Expression pattern for 11beta-hydroxylase 1 (CYP11B1), 11beta-hydroxylase 2 (CYP11B2), CYP17 and HSD3B2 mRNA in ectopic CPA was similar to that in the adrenal CPA. In conclusion, in terms of histopathological characteristic and steroidogenic enzyme profile, ectopic CPA is similar to adrenal CPA, suggesting that they are of identical cell origin.     

Delivery of large heterologous polypeptides across the cytoplasmic membrane of antigen-presenting cells by the Bordetella RTX hemolysin moiety lacking the adenylyl cyclase domain

The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC(-) toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8(+) T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8(+) CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b(+) target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines.     

Use of X-ray Tomography to Map Crystalline and Amorphous Phases in Frozen Biomaterials

The outcome of both cryopreservation and cryosurgical freezing applications is influenced by the concentration and type of the cryoprotective agent (CPA) or the cryodestructive agent (i.e., the chemical adjuvants referred to here as CDA) added prior to freezing. It also depends on the amount and type of crystalline, amorphous and/or eutectic phases formed during freezing which can differentially affect viability. This work describes the use of X-ray computer tomography (CT) for non-invasive, indirect determination of the phase, solute concentration and temperature within biomaterials (CPA, CDA loaded solutions and tissues) by X-ray attenuation before and after freezing. Specifically, this work focuses on establishing the feasibility of CT (100–420 kV acceleration voltage) to accurately measure the concentration of glycerol or salt as model CPA and CDAs in unfrozen solutions and tissues at 20°C, or the phase in frozen solutions and tissue systems at −78.5 and −196°C. The solutions are composed of water with physiological concentrations of NaCl (0.88% wt/wt) and DMEM (Dulbecco’s Modified Eagle’s Medium) with added glycerol (0–8 M). The tissue system is chosen as 3 mm thick porcine liver slices as well as 2 cm diameter cores which were either imaged fresh (3–4 h cold ischemia) or after loading with DMEM based glycerol solutions (0–8 M) for times ranging from hours to 7 days at 4°C. The X-ray attenuation is reported in Hounsfield units (HU), a clinical measurement which normalizes X-ray attenuation values by the difference between those of water and air. NaCl solutions from 0 to 23.3% wt/wt (i.e. water to eutectic concentration) were found to linearly correspond to HU in a range from 0 to 155. At −196°C the variation was from −80 to 95 HU while at −78.5°C all readings were roughly 10 HU lower. At 20°C NaCl and DMEM solutions with 0–8 M glycerol loading show a linear variation from 0 to 145 HU. After freezing to −78.5°C the variation of the NaCl and DMEM solutions is more than twice as large between −90 and +190 HU and was distinctly non-linear above 6 M. After freezing to −196°C the variation of the NaCl and DMEM solutions increased even further to −80 to +225 HU and was distinctly non-linear above 4 M, which after modeling the phase change and crystallization process is shown to correlate with an amorphous phase. In all tissue systems the HU readings were similar to solutions but higher by roughly 30 HU, as well as showing some deviations at 0 M after storage, probably due to tissue swelling. The standard deviations in all measurements were roughly 5 HU or below in all samples. In addition, two practical examples for CT use were demonstrated including: (1) glycerol loading and freezing of tissue cores and, (2) a mock cryosurgical procedure. In the loading experiment CT was able to measure the permeation of the glycerol into the sample at 20°C, as well as the evolution of distinct amorphous vs. crystalline phases after freezing to −196°C. In the mock cryosurgery example, the iceball edge was clearly visualized, and attempts to determine the temperature within the iceball are discussed. An added benefit of this work is that the density of these frozen samples, an essential property in measurement and modeling of thermal processes, was obtained in comparison to ice.