肾上皮细胞损伤使草酸钙晶体黏附增强的分子机制

研究了非洲绿猴肾上皮细胞(Vero)在损伤前后与一水合草酸钙(COM)和二水合草酸钙(COD)晶体的黏附作用及其引起的细胞反应,探讨了肾结石形成机理.COM和COD晶体与损伤细胞的黏附加重了细胞的过氧化损伤程度,导致损伤细胞的活力进一步降低,乳酸脱氢酶(LDH)释放量和活性氧(ROS)进一步增加,坏死细胞数量进一步增多,细胞体积缩小,并出现凋亡小体.COM晶体对细胞的损伤能力显著大于COD晶体.扫描电子显微镜(SEM)观测结果表明,损伤组Vero与COM微晶的黏附作用显著强于对照组,且能促进COM微晶的聚集.共聚焦显微镜观测结果表明,Vero损伤后,其表面表达的晶体黏附分子透明质酸(HA)显著增加,HA分子是促进微晶黏附的重要原因.细胞表面草酸钙的黏附量和晶体聚集程度与细胞的损伤程度成正相关.本文结果从分子和细胞水平上提示,细胞损伤是导致草酸钙肾结石形成的重要因素.     

Calcium-phosphorus interactions at a nano-structured silicate surface

Nano-structured calcium silicate (NCS), a highly porous material synthesized by controlled precipitation from geothermal fluids or sodium silicate solution, was developed as filler for use in paper manufacture. NCS has been shown to chemisorb orthophosphate from an aqueous solution probably obeying a Freundlich isotherm with high selectivity compared to other common environmental anions. Microanalysis of the products of chemisorption indicated there was significant change from the porous and nano-structured morphology of pristine NCS to fibrous and crystalline morphologies and non-porous detritus. X-ray diffraction analysis of the crystalline products showed it to be brushite, CaHPO42H2O, while the largely X-ray amorphous component was a mixture of calcium phosphates. A two-step mechanism was proposed for the chemisorption of phosphate from an aqueous solution by NCS. The first step, which was highly dependent on pH, was thought to be desorption of hydroxide ions from the NCS surface. This was kinetically favoured at lower initial pH, where the predominant form of phosphate present was H2PO(-)4, and led to decreased phosphorus uptake with increasing pH. The second step was thought to be a continuing chemisorption process after stabilization of the pH-value. The formation of brushite as the primary chemisorption product was found to be consistent with the proposed mechanism.     

草酸钾诱导的LB膜缺陷及对草酸钙堆积图形的影响

采用扫描电子显微镜(SEM)和原子力显微镜(AFM)研究了经不同浓度草酸钾(K2C2O4)处理后二棕榈酰磷酯酰胆碱(DPPC)的缺陷LB膜及其对一水草酸钙(COM)成核和生长的影响. K2C2O4的处理可进一步破坏LB膜中圆形畴区内的分子列阵, 尤其是使处在液态扩张相(LE)/液态凝集相(LC)边界的分子列阵无序程度增加, 从而促进了COM晶体在此处的成核和生长, 最终诱导圆形堆积的COM晶体图形出现. 随着损伤LB膜的K2C2O4浓度c(K2C2O4)从0.3 mmol/L增加到5.0 mmol/L, 其对LB膜畴区有序结构的破坏作用逐渐增强, 圈状堆积的晶体图形数量增多. 当c(K2C2O4)为0.3 mmol/L时, 主要诱导实心的圆形堆积的COM晶体图形, 而当c(K2C2O4)增加至5.0 mmol/L时, 生成圈状COM晶体图形, 且图形的半径减小. 这一研究结果将有助于从分子和超分子水平上了解肾小管上皮细胞膜损伤后的微结构变化及其与肾结石形成的关系.     

胆盐与磷酸钙的相互作用

胆汁的pH条件下(pH=6～8)，应该生成无定形磷酸钙(ACP)，而在胆结石中磷酸钙通常以羟基磷灰石的形式出现.利用谱学方法研究了ACP与胆盐的作用.结果表明，胆盐以胶团的形式与ACP作用，在溶液中形成复合胶团，使其溶解度增加.不同类型胆盐与ACP的作用能力不同：脱氧胆酸钠(NaDC) > 牛磺胆酸钠(NaTC) > 胆酸钠(NaC).胆盐与ACP中结合钙的亲和能力大于结合钙的亲和能力，使ACP在胆汁的环境下容易转化为羟基磷灰石.     

Induction of Ring‐Shaped Calcium Oxalate Patterns by Boundaries between Liquid Expanded Phase and Liquid Condensed Phase in Langmuir‐Blodgett Film

The formation of calcium oxalate kidney stones was related to injuries of renal epithelial membranes. The liquid condensed (LC) domains in Langmuir‐Blodgett (LB) film of dipalmitoylphosphatidylcholine (DPPC) were used as a model system to induce crystal growth of urinary mineral calcium oxalate monohydrate (COM). The circular defective boundaries between the LC and liquid expanded (LE) phases of the DPPC monolayer could provide much more nucleating sites for crystallization of COM crystals. It induced ring‐shaped or solid circular patterns of COM crystals on hydrophobic quartz substrates depending on the crystallization time.     

Influence of PSSS additive and temperature on morphology and phase structures of calcium oxalate

Calcium oxalate (CaOx) particles with different morphologies and phase structures were prepared by a facile precipitation reaction of sodium oxalate with calcium chloride in the absence and presence of poly(sodium 4-styrene-sulfonate) (PSSS) at different temperatures. The as-prepared products were characterized with scanning electron microscopy and X-ray diffraction. The influence of experimental conditions including pH, temperature, and concentration of PSSS and CaC2O4 on the morphologies and phase structures of the prepared calcium oxalate particles were investigated. It was found that variations in the concentration of PSSS and CaC2O4, temperature, and pH significantly influenced the crystal structure, morphology, and particle size of the samples. Various crystal morphologies of calcium oxalate, such as plate, leaf-shaped, bipyramid, and cylinder could be fabricated, depending on the experimental conditions. Higher PSSS concentration and reaction temperature favored the formation of metastable calcium oxalate dihydrate (COD) crystals and stable calcium oxalate monohydrate (COM), respectively. Especially, cylinder-shaped CaC2O4 particles were obtained at 80 degrees C in the presence of PSSS for the first time. This research may provide new insight into understanding and potentially regulating the formation of kidney stones and the control of morphology and phase structures of calcium oxalate particles.     

Effects of amino acids on crystal growth of CaC2O4 in reverse microemulsion

Crystal growth of calcium oxalate (CaC2O4) in bulk aqueous solution, reverse microemulsion of p-octyl polyethylene glycol phenylether (OP)/iso-octyl alcohol (IOA)/cydohexane/water and above microemulsions containing different kinds of amino acids, such as aspartic acid (Asp), tyrosine (Tyr) and tryptophan (Trp) were studied. The results indicated that different crystallization types of the crystals, which were calcium oxalate monohydrate (COM), calcium oxalate dihydrate (COD) and calcium oxalate trihydrate (COT), existed in bulk aqueous solution. But CaC2O4 growth mainly paralleled with (1 01) plane of COM in reverse microemulsion because of the induction of surfactant at water/oil interface. After adding amino acids into microemulsions, the growth of CaC2O4 crystals mainly influenced by the varieties of amino acids and the pH values of the amino acid aqueous solution. When pH values of the solutions was higher than isoelectric points of amino acids, CaC2O4 crystal paralleled with (1 01) plane of COM more easily with the addition of Trp, Tyr, Asp in turn; however, when pH of the solutions was lower than isoelectric points of Trp, CaC2O4 crystal growth paralleled with (020) face of COM. It is obviously that amino acids, pH values of the solutions and surfactant played important roles in the process of crystal growth of CaC2O4 in the microemulsions. The formation mechanism of CaC2O4 was also discussed in different microemulsions at last.     

硅凝胶体系中不同结构羧酸钾对草酸钙结晶的影响

采用扫描电子显微镜(SEM)和X射线衍射(XRD)方法研究了硅凝胶体系中不同种类羧酸钾对草酸钙(CaC₂O₄)晶体生长的调控作用. 加入一元醋酸钾(KOAc)只生成一水草酸钙(COM)晶体; 三元柠檬酸钾(K₃Cit)和四元乙二胺四乙酸二钾(K₂EDTA)可诱导二水草酸钙(COD)形成, 且随着其浓度增加, 对COD的诱导能力增加, 而二元酒石酸钾(K₂Tart)同时诱导了COM, COD和三水草酸钙(COT)生成. 随着结晶温度降低, 多元酸钾可以进一步减小COM晶体的比表面积, 增加COD的百分含量, 但K₂Tart诱导COT的能力减弱. 由于诱导COD和COT晶体形成、减小COM的比表面积均有利于防止草酸钙尿石的形成, 因此, 多元羧酸钾可用于草酸钙结石的预防和治疗.     

Constant composition dissolution of mixed phases. II. Selective dissolution of calcium phosphates

Characterization of the dissolution kinetics of individual synthetic and biological calcium phosphates is of considerable importance since these phases often coexist in biological minerals. The constant composition method has been used to study the dissolution kinetics of a series of synthetic calcium phosphates, brushite (DCPD), beta-tricalcium phosphate (TCP), octacalcium phosphate (OCP), hydroxyapatite (HAP), and carbonated apatite (CAP) in the presence and absence of citric acid, as a function of pH and thermodynamic driving force. While citric acid markedly accelerates the dissolution of TCP, HAP dissolution is significantly inhibited. Moreover, this additive has almost no influence on the dissolution of DCPD, OCP, and CAP. Dual constant composition dissolution studies of mixed calcium phosphates in the presence of citric acid have also been made. Another factor, pH, also plays an important role in the dissolution of these calcium phosphates. In suspensions of calcium phosphate mixtures, specific phases can be selectively dissolved by changing experimental parameters such as pH and the presence of rate modifiers. This result has important applications for the dissolution control of dental hard tissues such as dentin, enamel, and calculus.     

二水草酸钙的热力学转化及海藻多糖的稳定作用

草酸钙结石的形成与尿液中草酸钙的存在形式密切相关，一水草酸钙(COM)促进尿石症形成，而二水草酸钙(COD)易随尿液排出体外。本文采用体外模拟方法，比较研究了COD晶体在水溶液、正常人尿液和结石患者尿液3个不同体系中的稳定性及海藻龙须菜多糖(SPS)对COD的稳定作用。在水溶液和患者尿液中，不但COD转化率高，而且得到的转化产物COM晶体聚集程度大；而在正常人尿液中，COD转化率低，转化产物聚集程度较小。COD在不同体系中转化的速度依次为：水溶液＞患者尿液＞正常人尿液。从海藻龙须菜中提取的硫酸多糖可以稳定COD的存在并减小COM的聚集，这有利于阻止草酸钙结石的形成，因此，海藻龙须菜多糖有可能用于防止草酸钙结石形成。     

Effect of Concentration of Structurally-Different Carboxylic Acids on Growth and Aggregation of Calcium Oxalate in Gel Systems

The effect of concentration of structurally-different carboxylic acids such as ethylene diamine tetraacetic acid （H4edta）, citric acid （H3cit）, tartaric acid （H2tart）, and acetic acid （HOAc） on growth and aggregation of calcium oxalate （CaOxa） in gel systems was comparatively investigated. H2tart and H3cit could change the morphology of cal- cium oxalate monohydrate （COM） and induce the formation of calcium oxalate dihydrate （COD）. H4edta could induce the formation of COD at a lower concentration of 0.33 mmol/L and have the strongest ability to inhibit aggregation of COM. HOAc inhibited COM aggregation only at a higher concentration than 500 mmol/L. With increasing the number of carboxylic groups in an acid or increasing the concentration of carboxylic acid, the capacity of this acid to induce COD formation and to inhibit growth and aggregation of COM crystals increased. That is, this capacity followed the order： H4edta〉H3cit〉H2tart〉 〉HOAc. The result in this work suggested that the presence of H3cit and H2tart in urine played a role in the natural defense against stone formation.     

Adhesion between molecules and calcium oxalate crystals: critical interactions in kidney stone formation

Kidney stones are crystal aggregates, most commonly containing calcium oxalate monohydrate (COM) crystals as the primary constituent. Notably, in vitro studies have suggested that anionic molecules or macromolecules with substantial anionic functionality (e.g., carboxylate) play an important role in crystal aggregation and crystal attachment to renal epithelial cells. Furthermore, kidney stones contain measurable amounts of carboxylate-rich proteins that may serve as adhesives and promote aggregation of COM crystals. Atomic force microscopy (AFM) measurements of adhesion forces between tip-immobilized molecules and the COM (100) surface in aqueous media, described herein, reveal the effect of functional groups on adhesion and support an important role for the carboxylate group in processes responsible for kidney stone formation, specifically macromolecule-mediated adhesion of COM crystals to cells and crystal aggregation. The presence of poly(aspartic acid) during force measurements results in a reduction in the adhesion force measured for carboxylate-modified tips, consistent with the blocking of binding sites on the COM (100) surface by the carboxylate-rich polymer. This competitive binding behavior mimics the known reduction in attachment of COM crystals to renal epithelial cells in the presence of carboxylate-rich urinary macromolecules. These results suggest a feasible methodology for identifying the most important crystal surface-macromolecule combinations related to stone formation.     

二维琼脂凝胶体系中过渡金属离子对草酸钙晶体生长和周期性沉淀的影响

在二维凝胶圆盘体系中,研究过渡金属离子对草酸钙晶体生长和周期性环状沉淀的影响,并对其形成机理进行探讨.结果表明:在一定浓度的Fe3 存在下,体系中出现了周期性环状沉淀现象.同时发现在此条件下抑制了一水合草酸钙(COM)晶型生成.此外,加入Zn2 、Co2 、Ni2 体系中的晶体都以二水合草酸钙(COD)为主;而加Cu2 离子的圆盘中,三水合草酸钙(COT)占据了主要的成分,这说明不同的过渡金属离子对三种草酸钙晶型的生成影响不同.另外,还对出现的与尿结石有关的特殊现象(白斑和放射状的条纹)进行了探讨.     

Brushite (Ca,M)HPO4, 2H2O doping with bioactive ions (M = Mg2+, Sr2+, Zn2+, Cu2+, and Ag+): a new path to functional biomaterials?

Dicalcium phosphate dihydrate, DCPD (CaHPO₄·2H₂O), brushite, is an important calcium phosphate compound encountered in mineralized tissues and used in medicine, especially in bone cement formulations. However, the use of DCPD as direct implantable biomaterial has not received dedicated attention. In addition, the possibility to dope DCPD with biologically active ions to modulate its performances was not systematically explored. We have investigated in depth the doping of DCPD with Mg²⁺, Sr²⁺, Zn²⁺, Cu²⁺, and Ag⁺ ions. Clear modifications in terms of chemical composition, particle size, pore distribution, crystal morphology, and affinity for water were pointed out. Then, the samples were cultured with human adipose-derived stem cells to explore cytotoxicity and proliferation. Various behaviors were noticed dependent on the incorporated metal ions. Such DCPD compounds associated with bioactive metal ions, and particularly Ag⁺ and Zn²⁺, appear promising as a new family of reactive materials for use, as such or in combination, in bone-related applications.     

玉米须提取液对尿液中草酸钙晶体形成的影响

本文采用X射线衍射(XRD)、红外光谱(FTIR)、扫描电子显微镜(SEM)等方法分析了玉米须提取液对正常人尿液中草酸钙晶体形成的影响,通过电导率法研究了草酸钙晶体生长的动力学过程,以及从生物矿化的角度对玉米须提取液影响尿液中草酸钙晶体的可能机理进行了探讨。由于玉米须提取液中有机酸或多糖的羟基、羰基等通过配位作用与Ca²⁺结合形成可溶性配位化合物,减少了Ca²⁺与Oxa^2-的结合能力,从而抑制了CaOxa的成核和生长。同时,可能由于玉米须提取液中有效成分与二水草酸钙(COD)的吸附点键合,增强了COD晶体在溶液中的热力学稳定性,进而抑制了COD晶体向热力学更稳定态的一水草酸钙(COM)晶体转变。结果显示,这种抑制作用随玉米须浓度增大而增大,且COD晶体尺寸随着玉米须浓度的增大而减小。玉米须抑制COD晶体向COM晶体转变的作用为开发预防和治疗尿结石的药物提供了启示。     

Intercalation of n-alkyltrimethylammonium ions into layered calcium octyl phosphates thermally treated in vacuo

Layered calcium octyl phosphate (CH3(CH2)7OPO3Ca.1.6H2O: CaOP), which is composed of a multilayer alternating bilayer of octyl phosphates and a dicalcium phosphate dihydrate (DCPD)-like phase, was thermally treated in vacuo and the intercalation of n-alkyltrimethylammonium ions into the materials was examined. The octyl groups in the layer were eliminated by outgassing above 250 degrees C to give the amorphous calcium phosphates. Further, the specific surface area was steeply increased and mesopores with a diameter of ca. 2.0 nm were formed. IR results indicated that the surface P-OH groups were generated by outgassing at 250 degrees C. When the CaOP outgassed at 250 degrees C was treated with n-alkyltrimethylammonium ion solutions (carbon number of alkyl group, n=14-18), three XRD peaks reappeared below 2theta=15 degrees and the d-spacing ratio of these peaks was 1:1/2:1/3. These facts indicate that the n-alkyltrimethylammonium ions were intercalated into the amorphous calcium phosphate phases.     

不同种类羧酸钠对草酸钙结晶过程中晶相的影响

采用X射线衍射法研究了水溶液中不同种类羧酸钠对尿结石主要成份草酸钙结晶的影响，这些羧酸钠分别为含有一、二、三和四个羧基的羟基乙酸钠（NaGly）、酒石酸钠（Na2Tart）、柠檬酸钠（Na3Cit）和EDTA二钠盐（Na2EDTA）.结果表明，随着羧酸钠浓度的增加，草酸钙的晶相均发生规律性的变化：从最稳定的一水草酸钙（COM），到不稳定的二水草酸钙（COD），最后转化为次稳定的三水草酸钙（COT）.不同结构羧酸钠抑制COM生长、促进COD生成的顺序为:Na3Cit >Na2Tart >Na2EDTA >NaGly.该结果将为临床上选择防结石药物提供新的思路.     

海藻硫酸多糖抑制草酸钙结石形成的化学模拟

用体外模拟方法研究了从海藻异枝麒麟菜中提取的硫酸多糖(ESPS)对尿结石患者尿液中草酸钙晶体生长的影响. ESPS不但诱导与尿路细胞膜粘附力较弱的二水草酸钙晶体形成, 而且抑制一水草酸钙的生长和聚集, 归因于一水草酸钙的富钙(101)晶面与聚阴离子ESPS之间的静电相互作用. 上述结果表明, ESPS是一种抑制草酸钙结石的潜在绿色药物.     

Stability and transformation mechanism of weddellite nanocrystals studied by X-ray diffraction and infrared spectroscopy

This study is focused on the stability of weddellite, the dihydrate phase of calcium oxalate [CaC(2)O(4)·(2 + x)H(2)O], mainly detected in kidney stones and in oxalate films found on the surfaces of several ancient monuments. Its occurrence is a critical issue since, at environmental conditions, weddellite is unstable and quickly changes into whewellite, the monohydrate phase of calcium oxalate (CaC(2)O(4)·H(2)O). New single crystal X-ray diffraction experiments have been carried out, which confirm the structural model of weddellite previously published. Synthesised nanocrystals of weddellite have been kept under different hygrometric conditions in order to study, by X-ray powder diffraction, the influence of humidity on their stability. Moreover, the mechanism of transformation of weddellite nanocrystals has been investigated by infrared spectroscopy using D(2)O as a structural probe.     

Aggregation and dispersion characteristics of calcium oxalate monohydrate: effect of urinary species

In this research, screening and central composite experimental designs are used to determine the effect of various factors on the aggregation and dispersion characteristics of previously grown calcium oxalate monohydrate (COM) crystals in artificial urinary environments of controlled variables. The variables examined are pH and calcium, oxalate, pyrophosphate, citrate, and protein concentrations in ultrapure water and artificial urine. Optical density measurements, particle size analysis, optical microscopy, AFM force measurements, and protein adsorption have been used to assess the state of aggregation and dispersion of the COM crystals and to elucidate the mechanisms involved in such a complex system. The data indicate that our model protein, mucin, acts as a dispersant. This is attributed to steric hindrance resulting from the adsorbed mucoprotein. Oxalate, however, promotes aggregation. Interesting interactions between protein and oxalate along with protein and citrate are observed. Such interactions (synergistic or antagonistic) are found to depend on the concentrations of these species. Surface responses for these interactions are presented and discussed in this paper. In summary, solution, surface, and interface chemistries interact in a complex manner in the physiological environment to either inhibit or promote aggregation, and an understanding of such interactions may help determine and control the factors affecting kidney stone formation.