Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells

Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (<6700 Da) exhibiting greatest stimulation of VIC proliferation. Low molecular weight HA degradation products added to VIC cultures also resulted in a four-fold increase in total matrix production and a two-fold increase in elastin production over untreated controls. VIC internalization of HA, as shown by cellular uptake of fluorescently labeled HA, likely activates signaling cascades resulting in the biological responses seen here. Lastly, VICs encapsulated within HA hydrogels remained viable, and significant elastin production was observed after 6 weeks of culture. This work shows promise for the creation of a tissue-engineered heart valve utilizing the synergistic relationship between hyaluronic acid and VICs.     

Preparation and characterisation of antibody modified gelatin nanoparticles as drug carrier system for uptake in lymphocytes

Established methods of protein chemistry can be used for the effective attachment of drug targeting ligands to the surface of protein-based nanoparticles. In the present work gelatin nanoparticles were used for the attachment of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. These antibody modified nanoparticles represent a promising carrier system for the specific drug targeting to T-lymphocytes. The objective of this work was the comprehensive quantification of every chemical reaction step during the preparation procedure of these cell specific nanoparticles. Gelatin nanoparticles were formed by a two-step desolvation process. After the first desolvation step the remaining sediment and the supernatant were analysed for molecular weight distribution by size exclusion chromatography (SEC). Nanoparticles then were formed using the high molecular gelatin fraction and subsequently were stabilised by glutaraldehyde crosslinking. A part of the detectable amino groups on the particle surface was reacted with 2-iminothiolane in order to introduce reactive sulfhydryl groups. The thiolated nanoparticles were coupled to NeutrAvidin (NAv) which previously was activated with the heterobifunctional crosslinker sulfo-MBS. All these reaction steps were quantified by photometry or gravimetry. The functionality of NAv after covalent conjugation was confirmed by a biotin-4-fluorescein assay. The NAv-modified nanoparticles then were used for the binding of biotinylated anti-CD3 antibodies by avidin-biotin-complex formation. A highly effective attachment of the ligand was ascertained by different, indirect methods: immunoblotting and fluorimetry. Therefore, a well-defined nanoparticle system with drug targeting ligand modification was established that holds promise for further effective preclinical testing.     

矿化胶原改性骨水泥的制备及生物力学测定

目的 基于矿化胶原（MC）对聚甲基丙烯酸甲酯（PMMA）进行改性,提高其在骨修复领域应用的可行性。方法 将不同粒径MC（＜200 μm、200 ～ 300 μm、300 ～ 400 μm、400 ～ 500 μm）按照5 wt%、10 wt%、15 wt%、20 wt%的含量加入到PMMA骨水泥,制备不同粒径及含量的复合骨水泥。通过工作特性、抗压强度、抗弯强度、压缩模量、弹性模量测试,评估MC/PMMA复合骨水泥的生物力学性能,确定最佳性能的复合骨水泥比例。结果 复合骨水泥混合时间30 s,等待时间2 ～ 5 min,工作时间5 ～ 12 min,固化时间10 ～ 20 min;不同粒径MC（＜200 μm、200 ～ 300 μm、300 ～ 400 μm、400 ～ 500 μm）复合骨水泥,在不同含量（5 wt%、10 wt%、15 wt%、20 wt%）下其抗压强度均可保持在70 MPa以上,对PMMA骨水泥的抗压强度影响不大。含量15 wt%、粒径300 ～ 400 μm MC/PMMA复合骨水泥的抗压强度90 MPa,抗弯强度50 MPa,压缩模量1.2 GPa,弹性模量较Osteopal V和Mendec Spine骨水泥明显降低,且与人体松质骨的弹性模量相匹配。结论 MC在保持PMMA机械强度的同时,可降低其弹性模量,含量15 wt%、粒径300 ～ 400 μm MC/PMMA骨水泥的工作特性、抗压和抗弯强度、弹性模量符合国际标准和临床使用要求。     

Preparation and characterization of hyaluronan/collagen II microspheres under an electrostatic field system with disc electrodes

Collagen II and hyaluronan are the two major components of the native extracellular matrix (ECM). Both biopolymers are responsible for providing the associated tissues with tensile strength, and also serve as a structural scaffold for cell adhesion and growth. Over the years, many researchers have focused on the preparation and evaluation of man-made ECM comprising the two polymers in the form of a membrane for chondrocyte culture applications. Here, a simple and in situ method, involving the injection of the hyaluronan/collagen II (HA/Col II) mixture solution through a pair of hollow-centered parallel disc electrodes (HCPDEs) of a high-voltage electrostatic field system, was developed and employed to prepare HA/Col II microspheres in watery phase. The HA/Col II microspheres were firmed up by a two-step cross-linking treatment (first by FeCl(3) and then by 1-ethyl-3-(3-dimethyl aminopropyl) carbodimide, EDC) to secure the spherical structure shape. Then, at 37 degrees C, reconstitution treatment of the Col II molecules was conducted to further strengthen the microspheres. Depending on treatment conditions, the resulting series of HA/Col II microspheres all exhibited good sphericity in the range of 486+/-43 to 679+/-24microm in diameter. Furthermore, the ratio and amount of HA/Col II in the mixture solutions would affect the morphological structure and basic characteristics, including mechanical strength, thermal properties and water content. In the preliminary study, the HA/Col II microspheres have shown to provide favorable ECM characteristics, with appropriate mechanical strength, and exhibited a 3D inclination.     

Effects of cyclosporin on serum hyaluronan levels in collagen arthritis

Serum hyaluronan (HA) levels were measured in a rat model of collagen arthritis using a sandwich enzyme-linked immunosorbent assay. Values became elevated as the arthritis developed, correlating with its severity. Daily subcutaneous treatment with cyclosporin at the dose of 25 mg/kg per day for fourteen days completely prevented anti-type II collagen antibody production and the serum HA increase as well as development of collagen arthritis. HA in the blood may thus provide a good quantitative marker for joint disease in rat collagen arthritis with potential as a tool for evaluation of drug efficacy in this experimental model.     

功能化Fe3O4的制备及在基因转染中的应用

目的探讨修饰了多聚赖氨酸的超顺磁性葡聚糖Fe3O4纳米粒子（简称功能化Fe3O4,DMNP）的制备及其作为基因载体的可行性。方法采用碱沉淀法一步合成了外包葡聚糖的磁性纳米粒子,并用多聚赖氨酸对其表面进行修饰,使之通过静电作用吸附连接DNA。同时应用扫描电镜、红外分光光度计对该纳米复合物的结构及成分进行表征,并用琼脂糖凝胶电泳和紫外分光光度计对其结合质粒DNA的能力进行测量．该复合纳米粒子作为基因载体将绿色荧光蛋白质粒pEGFP—C1转入人肺癌细胞系A549中,并进一步将载肺癌耐药基因ABCG2-PCDNA3．1质粒转入人肺癌细胞系A549中。结果该复合纳米粒子分散性好,大小较均一,与质粒DNA有较好的结合能力,用荧光显微镜观察到了绿色荧光蛋白的表达,并用PCR技术检测到了耐药基因ABCG2的表达。结论该复合纳米粒子能作为一种新型有效的基因载体在体外将载肺癌耐药基因ABCG2-PCDNA3．1质粒转入人肺癌细胞系A549中并表达。     

Fibroblast contraction of collagen matrices with and without covalently bound hyaluronan

Hyaluronan, which is found in high concentrations in fetal tissues, has been suggested to play a major role in preventing scar formation in fetal wounds. We have developed a floating collagen fibrillar matrix (CFM) made out of reconstituted type I collagen for the purpose of evaluating the ability of hyaluronan to inhibit the fibroblast induced contraction of the matrix. When hyaluronan is covalently bound to collagen it appears to better support fibroblast proliferation and matrices are less contractible by these cells than when hyaluronan interacts only ionically. When hyaluronan is bridged between collagen fibrils by a network of extensive covalent crosslinks, contractibility by fibroblasts is abolished. These modified collagen matrices may prove to be very useful in the development of bioprostheses and implants.     

Preparation and characterization of hyaluronan oligosaccharides for angiogenesis study

Low molecular weight hyaluronan (HA) is proved to be active in stimulating endothelial cell (EC) proliferation. The study is to obtain biological active HA fragments of defined length from high molecular weight HA for angiogenesis research. A novel method, by a high-voltage polyacrylamide-gel electrophoresis (PAGE) that uses specific fluorophore labeling, was developed to identify the degraded products digested from native high molecular weight HA. The polymer HA was digested by hyaluornidase partially, and the enzymatic fragments of HA or HA oligosaccharides (o-HAs) were analyzed for molecular sizes by a combination of PAGE, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The purified HA oligomers were pooled and the biological effects of the mixtures on EC proliferation were studied, by separating the digests of HA fragments by size exclusion chromatography on a Bio-Gel P-10 column. The prepared o-HAs were collected and analyzed by a series of methods, PAGE, HPLC, and MS, which indicated a mixture of oligosaccharides covering a range of different sizes from 4 to 20-mer. The minimal molecular size determined by the special PAGE was 2 disaccharide, and the method can determine o-HA mass as high as 10 disaccharide (seeing the least clear band as much as possible on gel). Fractions containing HA fragments with sizes from 4 to 20-mer were pooled by distinct oligosaccharide species and added to the culture of pig endothelial cell (PIEC) for biological activities study. A significant increase in EC proliferation was found in comparison with nondigested high molecular weight HA. Our results suggested that o-HA can be obtained by a method of fluorophore-labeled olyacrylamide gel electrophoresis for identifying as small as 4-mer oligomer HA from high molecular weight HA-digested products. This method was proved to be sensitive, simple, and rapid in characterizing saccharides, which may be used by replacing the complicated ways of HPLC and MS. The prepared o-HA was found to be active in stimulating EC proliferation.     

普鲁兰多糖微球制备及影响因素考察

目的 采用水／油（W／O）乳化交联法制备普鲁兰多糖微球并观察多种因素对微球形态及粒径的影响。方法采用W／O乳化交联法，司班20作为表面活性剂，分别以蓖麻油和正己烷作为油相，不同浓度的普鲁兰多糖水溶液为水相，形成均一乳液后加入交联剂戊二醛，普鲁兰多糖被固化形成微球。观察普鲁兰多糖水溶液浓度、水／油比例、交联剂浓度、转速等多种因素对微球形态及大小的影响。通过扫描电镜观察微球形态，Mastersizer S激光粒度仪进行粒径分析。结果油的成分、水／油比例、多糖水溶液浓度、交联剂浓度及搅拌速度均不同程度影响微球形态和大小。蓖麻油为油相形成微球条件：水／油＜1：5，普鲁兰多糖水溶液浓度1％，交联剂浓度5％～50％。这些条件下形成微球表面光滑、粒径分布均一。正己烷为油相形成微球条件：1：5＜水／油＜1：2，普鲁兰多糖水溶液浓度1％，交联剂浓度＜25％，形成微球表面光滑，但粒径分布不均一。0．2％或5％普鲁兰多糖水溶液、转速＜200r／min均不能形成微球。结论采用W／O乳化交联法能够制备普鲁兰多糖微球，这种制备方法简单，多种因素控制微球形态与大小。     

低相对分子质量透明质酸脂质体的制备及透皮性能

背景：透明质酸是一种用途广泛的高分子材料,在实际应用中,由于其相对分子质量较大,透皮吸收效果不理想,利用脂质体作为其载体可以改善其传输效果,具有较好的应用前景。 目的：观察包裹低相对分子质量透明质酸脂质体的透皮性能。 方法：采用薄膜法制备低相对分子质量透明质酸脂质体,设计正交实验,采用蛇蜕研究其透皮吸收性能。 结果与结论:温度35 ℃、胆固醇与卵磷脂的比例为0.15∶1,透明质酸与卵磷脂的比例为0.03∶1、水合介质PBS的pH值为7.5,可制备包封率较高的低相对分子质量透明质酸脂质体;脂质体包裹低相对分子质量透明质酸,能有效得提高透明质酸透过蛇蜕的能力,达到了提高透明质酸透皮吸收率的作用。     

Chemically modified hemoglobin as an artificial oxygen carrier in dogs with experimental hemorrhagic shock

Leningrad Research Institute of Hematology and Blood Transfusion. All-Union Hematologic Scientific Center, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. I. Vorob'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 8, pp. 142–144, August, 1991.     

Differential effects of exogenous and endogenous hyaluronan on contraction and strength of collagen gels

The addition of exogenous hyaluronan to biomaterial scaffolds has been an important area of investigation for many decades. The ability to manipulate endogenous production of hyaluronan via the hyaluronan syntheses has offered another mechanism to study the effect of hyaluronan. While the literature suggests that exogenously added hyaluronan and endogenously produced hyaluronan will have varying impacts on extracellular matrix organization and function, no studies have directly shown this phenomenon. In this investigation, we demonstrate that the addition of exogenous high molecular weight (～1 MDa) hyaluronan and hyaluronan oligosaccharides have a distinct impact on both contraction and strength of smooth muscle cell-seeded collagen gels when compared to the effects of hyaluronan that is endogenously produced by the hyaluronan synthases. More specifically, the addition of exogenous high molecular weight hyaluronan resulted in more compact collagen gels with a higher ultimate tensile strength, whereas the endogenous overproduction of hyaluronan resulted in the opposite effect. We suggest that the addition of exogenous HA to collagen gels represents a model for the therapeutic administration of HA, whereas the addition of excess HA to a tissue via the endogenous overexpression of has represents a model for the pathological accumulation of HA.     

Nanoparticles and microparticles as vaccine-delivery systems

Vaccine-delivery systems are generally particulate (e.g., emulsions, microparticles and liposomes) and have comparable dimensions to the pathogens, which the immune system evolved to combat. Increasingly more sophisticated delivery systems are being developed in which immunostimulatory adjuvants may be incorporated with the antigen. The rationale for this approach is to ensure that both the antigen and adjuvant are delivered into the same population of antigen-presenting cells. Enhancement of adjuvant activity through the use of micro- and nanoparticulate delivery systems is particularly exciting, as synergistic effects are often seen resulting in immune responses stronger than those elicited by the adjuvant or delivery system alone. Micro- and nanoparticles also offer the possibility of enhancement of their uptake by appropriate cells through manipulation of their surface properties. The next important step in the development of many of the experimental microparticle- and nanoparticle-based technologies will be evaluation of effectiveness in human trials.     

Evaluation of a thin and mechanically stable collagen cell carrier

The biological function of adherent cell populations strongly depends on the physical and biochemical properties of extracellular matrix molecules. Therefore, numerous biocompatible cell carriers have been developed to specifically influence cell attachment, proliferation, cellular differentiation, and tissue formation for diverse cell culture applications and cell-based therapies. In the present study, we evaluated the mechanical and the cell biological properties of a novel, thin, and planar collagen scaffold. The cell carrier is based on fibrillar bovine collagen type I and exhibits a low material thickness coupled with a high mechanical stability as measured by tensile tests. The influence of this new biomaterial on cell viability, proliferation, and cell differentiation was analyzed using 5-bromo-2-deoxyuridine (BrdU) proliferation assay, immunocytochemistry, water-soluble tetrazolium salt-1 assay (WST-1), live cell imaging, and electron microscopy. Cell culture experiments with the human osteosarcoma cell line Saos-2, human mesenchymal stem cells, and rodent cardiomyocytes demonstrated the in vitro biocompatibility of this chemically noncrosslinked scaffold. Both the mechanical characteristics and the in vitro biocompatibility of this collagen type I carrier facilitate the engineering of thin transferable tissue constructs and offer new possibilities in the fields of cell culture techniques, tissue engineering, and regenerative medicine.     

Nanocrystalline hydroxyapatite and calcium sulphate as biodegradable composite carrier material for local delivery of antibiotics in bone infections

The use of polymethylmetacrylate beads for local delivery of antibiotics requires a second surgical procedure for their removal and resorbable calcium sulphate exhibits cytotoxic effects. In this work, a bioresorbable composite of calcium sulphate and nanoparticulate hydroxyapatite (PerOssal was studied regarding its antibiotic release properties and biocompatibility. Material characteristics of plain PerOssal and pure calcium sulphate pellets were studied using scanning and electron microscopy and X-ray methods. Pellets were soaked with gentamicin and vancomycin, respectively. Release properties of both antibiotics from both materials were investigated over 10 days. Quantitative and qualitative cytotoxic assays were performed for biocompatibility testing. Specific surface was 106 m(2)/g for PerOssal and 2.2 m(2)/g for pure calcium sulphate. Almost complete elution of gentamicin was found for both carrier materials (94.7% for PerOssal vs. 95.8% for calcium sulphate) within 10 days, whereas vancomycin release was higher for PerOssal (96.3% vs. 74.8%). PerOssal showed higher initial and lower release after approximately 5 days compared to calcium sulphate. No significant in vitro cytotoxic differences were found between PerOssal and nontoxic cell culture medium. Calcium sulphate showed cytotoxic effects in two out of four tests. PerOssal exhibits excellent properties regarding resorption, biocompatibility, and antibiotic release.     

Synthesis and characterization of collagen/hyaluronan/chitosan composite sponges for potential biomedical applications

Cells, scaffolds and growth factors are three main components of a tissue-engineered construct. Collagen type I, a major protein of the extracellular matrix (ECM) in mammals, is a suitable scaffold material for regeneration. Another important constituent of the ECM, hyaluronic acid (hyaluronan, HA), has been used for medical purposes due to its hydrogel properties and biodegradability. Chitosan is a linear polysaccharide comprised of β1- to β4-linked d-glucosamine residues, and its potential as a biomaterial is based on its cationic nature and high charge density in solution. This study was conducted to evaluate the characteristics of scaffolds composed of different ratios of type I comb collagen and chitosan with added HA in order to obtain the optimum conditions for the manufacture of collagen–hyaluronan–chitosan (Col–HA–Ch; comprising collagen, HA and chitosan mixed in different ratios: 10:1:0, Col10HACh0; 9:1:1, Col9HACh1; 8:1:2, Col8HACh2; 7:1:3, Col7HACh3; 6:1:4, Col6HACh4; and 5:1:5, Col5HACh5) composite porous scaffolds. Microstructural observation of the composite scaffolds was performed using scanning electron microscopy. The mean pore diameters ranged from 120 to 182 μm and decreased as the chitosan composition increased. All scaffolds showed high pore interconnectivity. Swelling ratio measurements showed that all specimens could bind 35- to 40-fold of physiological fluid and still maintain their form and stability. For tensile strength, the optimal ratio of collagen and chitosan was 9:1. Thermal stability was investigated using a differential scanning calorimeter and showed that Col5HACh5 and Col6HACh4 were significantly more stable than the other groups. In enzymatic sensitivity, a steady increase in the biostability of the scaffolds was achieved as the chitosan concentration was increased. In biocompatibility testing, the proliferation of the fibroblasts cultured in Co-HA-Ch tri-copolymer scaffolds was high. Overall, we observed the 9:1:1 mixing ratio of collagen, hyaluronan and chitosan to be optimal for the manufacture of complex scaffolds. Furthermore, Col–HA–Ch tri-polymer scaffolds, especially Col9HACh1, could be developed as a suitable scaffold material for tissue engineering applications.     

A role for hyaluronan in macrophage accumulation and collagen deposition after bleomycin-induced lung injury

Elevated concentrations of hyaluronan (HA) are associated with the accumulation of macrophages in the lung after injury. We have investigated the role of HA in the inflammatory and fibrotic responses to lung injury using the intratracheal instillation of bleomycin in rats as a model. After bleomycin-induced lung injury, both HA content in bronchoalveolar lavage (BAL) and staining for HA in macrophages accumulating in injured areas of the lung were maximal at 4 d. Increased HA in BAL correlated with increased locomotion of isolated alveolar macrophages. HA-binding peptide was able to specifically block macrophage motility in vitro. Importantly, systemic administration of HA-binding peptide to rats before injury not only decreased alveolar macrophage motility and accumulation in the lung, but also reduced lung collagen alpha (I) messenger RNA and hydroxyproline contents. We propose a model in which HA plays a critical role in the inflammatory response and fibrotic consequences of acute lung injury.     

Genetically modified Tomato aspermy virus 2b protein as a tumor-targeting siRNA delivery carrier

In nature, there exist a wide range of dsRNA-binding proteins that have different binding modes for small interfering RNA (siRNA) as well as structural differences, and some of these proteins have potential as effective siRNA delivery carriers. In order to deliver siRNA into cancer cells, a dsRNA-binding 2b protein derived from Tomato aspermy virus was genetically modified by fusing the integrin-targeting RGD peptide to its C-terminus, and biosynthesized. The resulting 2b-RGD protein possesses distinct characteristics favorable for biomedical applications of siRNA: (i) high affinity for siRNA, (ii) siRNA protection against RNases in serum, (iii) low cytotoxicity compared to the polycationic polymers often employed in conventional siRNA carriers, (iv) specific binding to integrins on cancer cells, and the ability to pass through the cell membrane via endocytosis, and (v) the ability to facilitate cytosolic release of siRNA. Here, we demonstrate that the 2b-RGD/siRNA complexes have great potential as a tumor-targeting siRNA delivery carrier and suggest their possible therapeutic applications for cancer treatment.     

Preparation of poly(ethylene glycol)-introduced cationized gelatin as a non-viral gene carrier

The objective of this study was to prepare cationized gelatins grafted with poly(ethylene glycol) (PEG) (PEG-cationized gelatin) and evaluate the in vivo efficiency as a non-viral gene carrier. Cationized gelatin was prepared by chemical introduction of ethylenediamine to the carboxyl groups of gelatin. PEG with one terminal of active ester group was coupled to the amino groups of cationized gelatin to prepare PEG-cationized gelatins. Electrophoretic experiments revealed that the PEG-cationized gelatin with low PEGylation degrees was complexed with a plasmid DNA of luciferase, in remarked contrast to that with high PEGylation degrees. When the plasmid DNA complexed with the cationized gelatin or PEG-cationized gelatin was mixed with deoxyribonuclease I (DNase I) in solution to evaluate the resistance to enzymatic degradation, stronger protection effect of the PEG-cationized gelatin was observed than that of the cationized gelatin. The complex of plasmid DNA and PEG-cationized gelatin had an apparent molecular size of about 300 nm and almost zero surface charge. These findings indicate that the PEG-cationized gelatin–plasmid DNA complex has a nano-order structure where the plasmid DNA is covered with PEG molecules. When the PEG-cationized gelatin–plasmid DNA complex was intramuscularly injected, the level of gene expression was significantly increased compared with the injection of plasmid DNA solution. It is concluded that the PEG-cationized gelatin was a promising non-viral gene carrier to enhance gene expression in vivo.