Basic requirements for the toxicity testing of antimicrobial agents

The regulations in different countries on the toxicological testing of antimicrobial agents are similar and harmonized on an international basis. Existing requirements cover both the standard investigations performed with any other new class of therapeutic drug and investigations necessary due to the specific features of anti-infective agents. Such features are the therapeutic target (the microorganisms), the need to provide adequate treatment of patients even during clinical trials, and the potential of the drug to induce certain adverse reactions. The combination of toxicity tests used will be determined by the drug, its class and current knowledge.     

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。     

组织工程软骨缝合技术及缝合材料的研究与进展

背景：软骨组织的生理特征及其功能结构特点表面,其受损后自我康复较难,根据其受伤特点及受损区域可以进行相应缝合,但缝线材料的选择尤为重要,随着可吸收缝线材料的研制与运用,为软骨组织缝合技术的运用提供了更大保障。 目的：文章综述了软骨组织生理特征,医用缝合线材料研究进展及其运用情况,着重介绍了可吸收缝线的材料学特征及其临床运用效果,指导临床合理选择材料和对新材料进行开发或研制。 方法：应用计算机检索CNKI和PubMed数据库中1993-01/2011-01关于软骨损伤及医用缝线材料的文章,在标题和摘要中以“软骨;缝合线;材料”或“Cartilage; Seam; materials”为检索词进行检索。选择文章内容与缝线材料相关,同一领域文献则选择近期发表或发表在权威杂志文章。初检得到193篇文献,根据纳入标准选择30篇文章进行综述。 结果与结论：软骨的生理特征制约着自我修复的效果,一般认为较难恢复,所以大多采用切除手术,但切除手术后期效果不理想,应尽量保留软骨组织,同时研究发现软骨组织周边区域具有一定的自我修复功能,所以对于规则性裂伤进行缝合康复,后期效果较好。但又由于软骨组织特殊的功能结构及需体内手术,所以不可吸收缝合线很难避免二次伤害及手术效果不理想,可吸收缝合线的出现为软骨组织的缝合康复及其缝合技术的发展提供了更大可能和保障,随着缝合技术的进步及新型材料的研制成功,软骨缝合作为一种组织工程修复手段将会得到逐步运用和改良。     

Carcinogenicity testing of antitumor agents

Carcinogenicity testing of antitumor agents in animal bioassays has been proposed because of the potential for carcinogenicity of this class of agents and the expectation that such testing may indicate prospectively the target organs of any related human oncogenesis. The literature reveals the anticipated confirmations in animals of the carcinogenicity of many antitumor agents. Furthermore, these agents have been associated with human tumors in numerous case reports. Review of the literature also indicates the inability of animal studies to predict the sites of carcinogen-induced tumors in man. The carcinogenic risk assessment of antitumor agents should begin with the determination of the ability of the agent to interact with DNA. Those agents which are capable of alkylating or binding DNA should be tested for mutagenic and teratogenic potential. The presumption of carcinogenicity should be made for DNA-reactive, mutagenic/teratogenic antitumor agents without requiring confirmation in long-term carcinogenicity bioassays in large numbers of animals. The inability of carcinogenicity studies in animals to accurately predict potential human tumor sites must also be recognized.     

A novel 3D histotypic cartilage construct engineered by supercritical carbon dioxide decellularized porcine nasal cartilage graft and chondrocytes exhibited chondrogenic capability in vitro

Augmentative and reconstructive rhinoplasty surgical procedures use autologous tissue grafts or synthetic grafts to repair the nasal defect and aesthetic reconstruction. Donor site trauma and morbidity are common in autologous grafts. The desperate need for the production of grafted 3D cartilage tissues as rhinoplasty grafts without the adverse effect is the need of the hour. In the present study, we developed a bioactive 3D histotypic construct engineered with the various ratio of adipose-derived stem cells (ADSC) and chondrocytes together with decellularized porcine nasal cartilage graft (dPNCG). We decellularized porcine nasal cartilage using supercritical carbon dioxide (SCCO2) extraction technology. dPNCG was characterized by H&E, DAPI, alcian blue staining, scanning electron microscopy and residual DNA content, which demonstrated complete decellularization. 3D histotypic constructs were engineered using dPNCG, rat ADSC and chondrocytes with different percentage of cells and cultured for 21 days. dPNCG together with 100% chondrocytes produced a solid mass of 3D histotypic cartilage with significant production of glycosaminoglycans. H&E and alcian blue staining showed an intact mass, with cartilage granules bound to one another by extracellular matrix and proteoglycan, to form a 3D structure. Besides, the expression of chondrogenic markers, type II collagen, aggrecan and SOX-9 were elevated indicating chondrocytes cultured on dPNCG substrate facilitates the synthesis of type II collagen along with extracellular matrix to produce 3D histotypic cartilage. To conclude, dPNCG is an excellent substrate scaffold that might offer a suitable environment for chondrocytes to produce 3D histotypic cartilage. This engineered 3D construct might serve as a promising future candidate for cartilage tissue engineering in rhinoplasty.     

组织工程技术修复损伤关节软骨的研究与应用

背景：软骨是一种无血管的组织,软骨损伤后自身修复能力有限。当前用于治疗关节软骨损伤的方法从保守治疗到手术治疗多种多样。随着组织工程技术的发展,关节软骨的修复又进入了新的高度。 目的：综述组织工程方法修复软骨损伤的新进展。 方法：由第一作者在2013年5月应用计算机检索2000至2013年PubMed 数据库及CNKI 数据库,英文以“cartilage tissue engineering,cartilage defect;stem cell,scaffold;growth factor”为关键词,中文以“软骨组织工程,软骨缺损,干细胞,支架,生长因子”为关键词,选择内容与软骨组织工程、软骨损伤修复相关的文章,同一领域文献则选择近期发表或发表在权威杂志文章,共纳入64篇文献。 结果与结论：软骨组织工程三大要素——种子细胞、支架和细胞因子,三者必须协调发展和互利。现阶段组织工程方法修复关节软骨损伤的研究虽已取得很大进展,但大多停留于实验探索阶段,尚未应用于临床。随着新材料的不断研发,新的组织工程软骨修复材料将兼顾材料学和生物科学的需要,使其更接近机体自身组织生物学特性。在新的技术支持下,动物实验研究也将向临床试验转变,使关节软骨损伤的治疗取得突破性进展。     

Long-term maintenance of human articular cartilage in culture for biomaterial testing

Cartilage is a tissue that derives its unique mechanical and biological properties from the combination of relatively few cells and a large amount of a complex extracellular matrix. Furthermore, cartilage tissue is comparatively slow to respond to changes or harmful influences. To date, the optimal generation and long-term maintenance of cultured human articular cartilage for in vitro testing of biomaterials, poses an experimental difficulty. Experiments using cultured isolated chondrocytes in combination with scaffolds often fail to yield results comparable to the in-vivo situation. Consequently, our aim was to develop a culture method that allows in vitro maintenance of human hyaline cartilage explants in an optimal quality over an extended period of time. Such a culture could, for example, be used to determine the long-term effect of a new scaffold on intact cartilage, as an in vitro model for repair processes and to investigate biomaterial integration. In this study we compared conventional static cultures with and without serum supplementation to a serum-free perfusion culture for the ability to maintain human articular cartilage explants in a morphologically intact and differentiated state over an extended period of time of up to 56 days. Results were evaluated and compared by morphological, histochemical and immunohistochemical methods. The experiments showed that short-term maintenance of cartilage in a differentiated state for up to 14 days is possible under all culture conditions tested. However, best long-term culture results for up to 56 days were obtained with perfusion culture under serum-free conditions. Such a perfusion culture system can be used to perform biocompatabilty tests in vitro by long-term coculture of biomaterial and intact human articular cartilage.     

Diffusion coefficients of articular cartilage for different CT and MRI contrast agents

In contrast enhanced magnetic resonance imaging (MRI) and computed tomography (CT), the equilibrium distribution of anionic contrast agent is expected to reflect the fixed charged density (FCD) of articular cartilage. Diffusion is mainly responsible for the transport of contrast agents into cartilage. In osteoarthritis, cartilage composition changes at early stages of disease, and solute diffusion is most likely affected. Thus, investigation of contrast agent diffusion could enable new methods for imaging of cartilage composition. The aim of this study was to determine the diffusion coefficient of four contrast agents (ioxaglate, gadopentetate, iodide, gadodiamide) in bovine articular cartilage. The contrast agents were different in molecular size and charge. In peripheral quantitative CT experiments, penetration of contrast agent into the tissue was allowed either through the articular surface or through deep cartilage. To determine diffusion coefficients, a finite element model based on Fick's law was fitted to experimental data. Diffusion through articular surface was faster than through deep cartilage with every contrast agent. Iodide, being of atomic size, diffused into the cartilage significantly faster (q < 0.05) than the other three contrast agents, for either transport direction. The diffusion coefficients of all clinical contrast agents (ioxaglate, gadopentetate and gadodiamide) were relatively low (142.8–253.7 μm²/s). In clinical diagnostics, such slow diffusion may not reach equilibrium and this jeopardizes the determination of FCD by standard methods. However, differences between diffusion through articular surface and deep cartilage, that are characterized by different tissue composition, suggest that diffusion coefficients may correlate with cartilage composition. Present method could therefore enable image-based assessment of cartilage composition by determination of diffusion coefficients within cartilage tissue.     

Evaluation of column technology for direct antiglobulin testing

Preliminary reports have suggested that microcolumn technology might be too sensitive for direct antiglobulin testing (DAT). We studied 228 samples from patients with autoimmune diseases and 30 samples from healthy controls to determine the sensitivity of column techniques. Both Sephadex gel and protein A/G columns were compared with manual methods using rabbit or murine polyspecific reagents. Of the 187 samples that were negative by both manual methods, an additional 29 (15%) and 42 (22%) samples gave weakly positive reactions with the Sephadex and protein A/G bead columns, respectively. Subsequently, there was poor correlation between manual and column techniques (r = 0.40-0.61). Acid eluates from these samples were negative. We concluded that the column technology may detect too many weakly positive DATs that are clinically insignificant.     

A novel approach to the development of benchmarking parameters for characterizing cartilage health

This article outlines the motivation and preliminary investigations into a novel method of characterizing cartilage health for potential in vivo application. Current in vivo indentation techniques, which primarily rely on stiffness measurements based on axial data, are unable to adequately distinguish between healthy and degraded tissue. The present in vitro study investigates the effects of controlled artificial degradation on the effective surface stretch, comparing the results with those obtained from the peripheral cartilage surrounding focal osteoarthritis. Results suggest that this technique is highly sensitive, showing a maximum range of 14% effective surface stretch in a normal joint compared with 42% for axial strain measurements. We further demonstrated that the technique can discriminate between degenerative changes and the intrinsic variations in cartilage properties across the normal joint. From these investigations we propose that the relationship between indentation and the in-plane strain field under the indenter can better distinguish degraded tissue than the currently used stiffness techniques.     

A novel model of fibroblast-mediated cartilage destruction

In rheumatoid arthritis (RA), fibroblasts have been shown to be crucial for disease progression as well as joint destruction. In the model of human/murine SCID arthritis, synovial explants as well as fibroblasts from human rheumatoid synovial membrane induce destructive arthritis in immunodeficient mice. Hereby, the underlying cartilage destruction is accomplished by murine fibroblasts. Therefore, murine destructive fibroblasts represent a promising tool to investigate destruction of articular cartilage and bone. In this context, a novel destructive murine fibroblast line (LS48) was examined for morphological, ultrastructural, immunological and functional cellular parameters. These cells were injected into knees of SCID mice. Subsequently, the animals were monitored for joint swelling and serological parameters of arthritis by radiological methods. Finally, cartilage destruction was assessed morphologically. Cultured LS48 cells exhibit characteristic features that resemble those of activated synovial fibroblasts in human RA. Expression levels of inducible nitric oxide synthase, interleukin-6, tumour necrosis factor-alpha and matrix metalloproteinases were comparable to those detected in invasive human fibroblasts. The instillation of 5 x 10(5) LS48 cells into the knee joints of SCID mice initiated a rapid progressive process, that caused cartilage destruction within 10 days, and morphological examinations revealed that articular cartilage was infiltrated by the fibroblasts injected previously. In summary, the intra-articular application of LS48 cells represents a rapid and highly reproducible model to investigate the initiation and progression of cartilage destruction in connection with RA therapy and represents an easy-to-handle animal model.     

Methods and agents for testing instruments for measuring pulmonary ventilation function

All-Union Research Institute of Medical Instrumentation, Moscow. Translated from Meditsinskaya Tekhnika, No. 2, pp. 24–29, March–April, 1991.     

Tube and column agglutination technology for autocontrol testing

The incidence of positive autocontrol test results with column agglutination technology is a concern. This study investigates the incidence and significance of positive autocontrols in the ID Micro Typing System (gel) and the Gamma ReACT (ReACT). The study encompassed a total of 1021 randomly selected samples from patients and 95 samples from donors collected during 1 month. The autocontrol testing was carried out according to the manufacturer's instructions for the column agglutination tests. The tube method was carried out using low-ionic-strength solution (LISS). The direct antiglobulin test (DAT) was performed using the tube method, and further investigated with elution studies if warranted. Seventy-nine patient's samples (7.74%) had a positive autocontrol: the gel test, 72 (91.13%); ReACT, 21 (26.58%); and the tube method, 27 (34.18%). Of the 79 positive autocontrols, 44 samples had a negative DAT. Of the samples with positive DAT results, only one possessed a clinically significant antibody, anti-D. Moreover, the same sample also tested positive in all three methods. Column agglutination techniques have increased sensitivity for a positive autocontrol beyond the conventional tube method. However, ReACT and gel tests differ significantly in their frequency of positives. Investigation of the significance of a positive autocontrol in column agglutination technology when the conventional tube method is also positive is suggested.     

免疫抑制剂用于角膜移植排斥反应的临床应用评价

背景：角膜移植免疫排斥反应是一个复杂的反应过程,一般包括宿主对异体组织抗原的致敏和宿主对异体组织抗原的反应两方面。角膜移植失败的主要原因是免疫排斥反应,有效地防治角膜移植术后免疫排斥反应的发生是眼科治疗中亟待解决的实际问题. 目的：文章就目前有关免疫抑制剂在角膜移植免疫排斥反应中的应用作一数据分析。 方法：通过计算机检索Scopus数据库中2002/2011有关免疫抑制剂在角膜移植免疫排斥反应中的应用的文献,检索词为“角膜移植(keratoplasty/corneal transplantation);免疫排斥反应(immunological rejection);免疫抑制剂(immunosuppressant);环孢素A(cyclosporine A,CsA);他克莫司(tacrolimus)或 FK506;雷帕霉素(rapamycin,RAPA)”,共检索文献192篇。 结果与结论：免疫排斥反应的发生是多因素参与的复杂过程,尤其高度血管化的角膜、感染性角膜溃疡、重复移植等高危角膜病变,术后出现排斥反应的概率明显增加,排斥反应发生的时间也相对较早,而且时间跨度较长。角膜移植排斥反应是一个复杂的过程,其预防及治疗已取得了多方面的进展,但排斥反应仍是角膜移植失败的首要原因,所以,专家们在不断尝试应用新的药物和治疗措施改善角膜移植的存活率,其最终目标是使受体在保持正常免疫应答能力的情况下,诱导产生/建立供体特异性免疫耐受。免疫抑制剂在角膜移植排斥反应中的应用不可或缺。     

Development and testing of a human collagen graft material

Human Type I collagen was extracted from placenta using pepsin and salt fractionation. The collagen was characterized by SDS-PAG electrophoresis dispersed in acidic medium, freeze-dried, and cross-linked in an 0.25% glutaraldehyde solution pH 4.5 for 2 days. After washing for 7 days and freeze drying the resultant collagen sponge was tested with regard to mechanical, physical, enzymatic degradation properties and biological responses. The modulus of elasticity was found to be 289 +/- 10 g/mm2 and the sponge was insoluble in water, buffered saline, or tissue culture medium over a period of 6 weeks with swelling occurring at less than 5% of volume. The sponge had a high fluid binding capacity, amounting to 56 +/- 5 mL tissue culture medium per gram of dry weight. Bacterial collagenase produced slow degradation of the sponge with complete disappearance by 24 h only when high concentrations (200 units enzyme per mg of the collagen sponge) were used. Cytotoxicity studies using human gingival and periodontal ligament fibroblasts revealed less than 5% apparent cytotoxicity or proliferation. Subcutaneous implantation was followed by resorption and vascularization over a period of 6-8 weeks. It was concluded that the collagen sponge prepared from human Type I collagen has potential as a graft material in oral surgical procedures.     

A novel simulation model for engineered cartilage growth in static systems

A novel mathematical model to simulate the growth of engineered cartilage in static systems is proposed. This model is based on material balances for the involved species (glycosaminoglycan and collagen, both pertaining to extracellular matrix), as well as mass-structured population balance for simulating cell growth and its proliferation within the scaffold. This model may simulate tissue growth on static culture taking place in Petri dishes, static flasks, and well plates for different types of scaffolds (i.e., poly(glycolic acid) [PGA], PGA/poly(l-lactic acid), and collagen sponge). This work aimed to demonstrate that the model approach proposed in previous works, regarding engineered cartilage growth on PGA scaffolds performed in rotating bioreactors, may also be applied to different scaffolds and system configurations. In particular, the balance equation for simulating collagen production is introduced, as well as the use of spatial averaging over the spatial region to compare experimental data with the model. Experimental data from the literature in terms of cells, glycosaminoglycans, and collagen content have been successfully compared with model results, thus demonstrating the validity of the proposed model, as well as its predictive capability.     

ProtEx technology for the generation of novel therapeutic cancer vaccines

Therapeutic vaccines present an attractive alternative to conventional treatments for cancer. However, tumors have evolved various immune evasion mechanisms to modulate innate, adaptive, and regulatory immunity for survival. Therefore, successful vaccine formulations may require a non-toxic immunomodulator or adjuvant that not only induces/stimulates innate and adaptive tumor-specific immune responses, but also overcomes immune evasion mechanisms. Given the paramount role costimulation plays in modulating innate, adaptive, and regulatory immune responses, costimulatory ligands may serve as effective immunomodulating components of therapeutic cancer vaccines. Our laboratory has developed a novel technology designated as ProtEx^™ that allows for the generation of recombinant costimulatory ligands with potent immunomodulatory activities and the display of these molecules on the cell surface in a rapid and efficient manner as a practical and safe alternative to gene therapy for immunomodulation. Importantly, the costimulatory ligands not only function when displayed on tumor cells, but also as soluble proteins that can be used as immunomodulatory components of conventional vaccine formulations containing tumor-associated antigens (TAAs). We herein discuss the application of the ProtEx^™ technology to the development of effective cell-based as well as cell-free conventional therapeutic cancer vaccines.