骨髓间充质干细胞于软骨组织工程的应用

关节软骨缺损在骨科临床十分常见,目前临床修复软骨缺损的方法很多,但是由于各自固有的缺陷难以达到满意的临床效果,因此探索软骨缺损的修复方法一直是人们不断深入研究的课题。软骨组织工程的发展为软骨缺损的修复提供了新的途径。种子细胞和支架材料是软骨组织的两个基本要素,根据近年来软骨组织工程的研究进展和方向,从骨髓间充质干细胞的诱导方式、诱导机制及研究进展方面进行探讨,证明骨髓间充质干细胞作为种子细胞构建组织工程软骨的优越性。     

软骨组织工程

软骨缺损是临床很难修复的病症之一,本综述了软骨组织工程涉及的软骨细胞分离,细胞培养方式和培养条件。基质材料,聚合物骨架以及软骨组织工程在临床上的应用等方面内容,并针对研究的现状,提出了软骨组织工程存在的问题和发展方向。     

软骨组织工程研究的新进展

软骨组织的再生能力有限 ,组织工程软骨的构建对修复软骨缺损意义重大。本文从四方面介绍了软骨组织工程的研究的新进展 ,包括软骨种子细胞的研究、软骨细胞与支架的体外培养、细胞支架复合物植入体内的研究及软骨细胞移植的临床应用。     

软骨组织工程种子细胞的基础和应用研究进展

软骨种子细胞的老化和缺乏是限制软骨组织工程在临床应用的瓶颈问题。本概述了目前对软骨种子细胞研究的两个方面，扩大种子细胞的来源及预防和延缓种子细胞功能老化，并展望软骨组织工程在临床的应用前景。     

骨软骨组织工程研究进展

骨软骨损伤是临床上常见的关节疾病。由于骨软骨界面的复杂性和软骨自我修复能力匮乏等因素,针对骨软骨损伤的临床治疗局限性较大。组织工程的提出,为骨软骨损伤同步修复提供了新的治疗方式。本文就骨软骨组织工程的三个方面进行综述介绍,它们分别是:种子细胞来源以及培养方式、生长因子的调控以及协同作用、细胞支架的组成类型,文中着重探讨骨软骨修复支架的现状和研究进展,最终期待通过本文综述,可为骨软骨组织工程的进一步发展提供参考。     

软骨组织工程种子细胞及预防其老化的研究进展

软骨组织的修复和再生能力较弱,加之软骨种子细胞易于老化,这些因素限制着软骨组织工程在临床的应用.本文就近年来有关软骨组织工程种子细胞的建立及延缓和预防软骨种子细胞老化的研究进展和前景作一综述.     

软骨组织工程种子细胞的基础和应用研究进展

软骨种子细胞的老化和缺乏是限制软骨组织工程在临床应用的瓶颈问题。本文概述了目前对软骨种子细胞研究的两个方面 :扩大种子细胞的来源及预防和延缓种子细胞功能老化 ,并展望软骨组织工程在监床的应用前景。     

软骨组织工程研究的新进展

软骨组织的再生能力有限，组织工程软骨的构建对修复软骨缺损意义理大。本从四方面介绍了软骨组织工程的研究的新进展，包括软骨种子细胞的研究，软骨细胞与支架的体外培养，细胞支架复合物植入体内的研究及软骨细胞移植的临床应用。     

软骨组织工程种子细胞及预防其老化的研究进展

软骨组织的修复和再生能力较弱，加之软骨种子细胞易于老化，这些因素限制着软骨组织工程在临床的应用。本就近年来有关软骨组织工程种子细胞的建立及延缓和预防软骨种子细胞老化的研究进展和前景作一综述。     

壳聚糖在软骨组织工程的应用

软骨组织工程的出现 ,为解决软骨修复这个临床难题提供了新的方案 ,然而目前软骨组织工程的热点在于寻找一种合适的生物载体材料。壳聚糖 (chitosan) ,一种人工合成的多聚糖材料 ,具有良好的生物相容性、可降解性和生物活性 ,可作为细胞三维生长支架 ,维持种子细胞新陈代谢和生长表型。本文将就壳聚糖的理化特性、生物活性和在软骨组织工程方面应用的状况及前景作一综述。     

Influence of volume of sample processed on detection of Chlamydia trachomatis in urogenital samples by PCR.

In the present study, it was demonstrated that the sensitivity of the PCR for the detection of Chlamydia trachomatis is influenced by the volume of the clinical sample which is processed in the PCR. An adequate sensitivity for PCR was established by processing at least 4%, i.e., 80 microliters, of the clinical sample volume per PCR. By using this preparation procedure, 1,110 clinical samples were evaluated by PCR and by cell culture, and results were compared. After discordant analysis, cell culture resulted in a sensitivity of 79.1% and PCR resulted in a sensitivity of 92.7%. Furthermore, it was shown that treatment with antibiotics immediately resulted in negative cell culture results but that PCR could give positive results up to 2 weeks posttreatment.     

Solid-phase C1q-directed bacterial capture followed by PCR for detection of Chlamydia trachomatis in clinical specimens.

An antigen capture system based on the binding of bacteria to solid-phase immobilized complement C1q followed by PCR for detection of Chlamydia trachomatis in clinical samples was developed and clinically evaluated. Comparison of C1q-directed antigen capture PCR with cell culture and direct PCR on 71 consecutive clinical specimens revealed an identical sensitivity. In this group, all 11 cell culture-positive samples were positive by direct PCR and C1q-directed antigen capture PCR. In addition, two samples found negative by cell culture were found positive by both direct PCR and C1q-directed antigen capture PCR. To further assess the sensitivity of C1q-directed antigen capture PCR, 20 clinical samples with one to five inclusions in cell culture and 20 clinical samples with 6 to 20 inclusions in cell culture were tested. Results obtained showed sensitivities of 95 and 90% for clinical samples with 6 to 20 and 1 to 5 inclusions in cell culture, respectively. Using C1q-coated solid phases, C1q-binding Chlamydia particles can be concentrated from large volumes with concomitant removal of inhibitors of PCR, allowing the use of large volumes of clinical samples for clinical testing. Since C1q has been shown to bind to a range of gram-negative bacteria, the newly developed technique has utility for a broad range of bacteria.     

Determination of cell line suitability for rapid isolation of herpes simplex virus

Herpes simplex virus (HSV) is one of the most commonly identified viruses in the clinical laboratory. HSV is of clinical consequence because of its ability to produce such life-threatening infections as encephalitis and neonatal disease. A variety of cell lines are currently being used for detection of HSV in cell culture. This study compared several cell lines simultaneously in a 96-well cell culture system to determine which lines demonstrated viral CPE most quickly following infection. In considering speed at which viral CPE is demonstrated, our results showed that primary rabbit kidney cells surpassed all other cell lines at 48 h post-viral infection. Alternative cell lines demonstrated to be suitable by this study were human fetal foreskin, human embryonic lung, and Vero cells.     

On-chip testing device for electrochemotherapeutic effects on human breast cells

A microfabricated cell-based testing device for electrochemotherapy (ECT) has been developed by miniaturizing the widely used clinical electroporator with a two-needle array into two-dimensional planar electrodes while keeping the similarity of the electric field strength distribution. In this device, all the biological processes from cell culture to electroporation and final cell-based assays were carried out on a chip using a conventional 2D cell culture method, and the multiple electrochemotherapeutic assays could be realized by exploiting the six electroporation sites in a single device. With the proposed platform, the electroporation rate was evaluated with propidium iodide and cell proliferation after 48 h of electrochemotherapy with bleomycin was determined with T47D human breast ductal carcinoma cell line in various electric field strengths and drug concentrations. This microsystem has several advantages over conventional cuvette type electroporation assay, such as multiple assays on a chip, on-chip based operation from cell culture to final assay, and having similar electric field distribution as that of the clinical electroporator. As the clinical trials of electrochemotherapy are being carried out, this new platform is expected to have valuable applications in basic in vitro ECT studies, drug discovery, and development of clinical ECT equipment.     

改良人颅咽管瘤原代细胞培养方法

目的　探索一种改良人颅咽管瘤细胞培养方法。 方法　根据文献报道和自己的经验,改良既往文献报道的颅咽管瘤细胞培养方法,对两种病理类型的人颅咽管瘤细胞进行原代培养,同时对其进行纯化与传代。通过观察细胞镜下形态、细胞免疫组织化学染色以及细胞增殖实验对原代细胞进行鉴定。 结果　改良后的培养方法培养出的颅咽管瘤细胞从组织块中爬出,排列紧密,透亮且折光性强。细胞免疫组化显示,两种病理类型的细胞均为颅咽管瘤细胞。增殖实验显示,细胞在体外仍具有较好的增殖和分裂的能力。 结论　本研究改良了人颅咽管瘤原代细胞的培养方法,具有简单、经济、成功率高等特点,为颅咽管瘤基础研究提供了良好的保证。     

Corneal regeneration by transplantation of corneal epithelial cell sheets fabricated with automated cell culture system in rabbit model

We have performed clinical applications of cell sheet-based regenerative medicine with human patients in several fields. In order to achieve the mass production of transplantable cell sheets, we have developed automated cell culture systems. Here, we report an automated robotic system utilizing a cell culture vessel, cell cartridge. The cell cartridge had two rooms for epithelial cells and feeder layer cells separating by porous membrane on which a temperature-responsive polymer was covalently immobilized. After pouring cells into this robotic system, cell seeding, medium change, and microscopic examination during culture were automatically performed according to the computer program. Transplantable corneal epithelial cell sheets were successfully fabricated in cell cartridges with this robotic system. Then, fabricated cell sheets were transplanted onto ocular surfaces of rabbit limbal epithelial stem cell deficiency model after 6-h transportation using a portable homothermal container to keep inner temperature at 36 °C. Within one week after transplantation, normal corneal epithelium was successfully regenerated. This automatic cell culture system would be useful for industrialization of tissue-engineered products for regenerative medicine.     

Comparison of HeLa 229 and McCoy cell cultures for detection of Chlamydia trachomatis in clinical specimens.

Consecutive clinical specimens of Chlamydia trachomatis (1,048) were inoculated in parallel on DEAE-dextran- and cycloheximide-treated HeLa 229 cells and cycloheximide-treated McCoy cells. HeLa 229 cell culture detected 113 positive specimens, and McCoy cell culture detected 103 positive specimens. This difference is not significant. However, HeLa 229 cell culture yielded significantly more inclusions than McCoy cell culture in the 95 specimens positive in both cell types (P = 0.042). For routine diagnostic purposes, a choice for one of the cell types may be determined by local preferences.     

Cell culture compared with broth for detection of Trichomonas vaginalis.

Trichomonas vaginalis can be grown in cell culture. We studied the growth kinetics of T. vaginalis in McCoy cell culture compared with that in a conventional broth medium (Diamond TYI-S-33 medium supplemented with 10% heat-inactivated bovine serum [TYI]). In the presence of McCoy cells and two parts cell culture medium to one part TYI, a peak concentration of 2 X 10(6) to 6 X 10(6) T. vaginalis per ml was consistently achieved with inocula as low as three T. vaginalis cells per ml. Without cells, this medium did not support growth of T. vaginalis. T. vaginalis in TYI in 1-ml vials with or without McCoy cells demonstrated poor growth. In tubes containing 10 ml of TYI, inocula grew to 2 X 10(6) to 6 X 10(6) T. vaginalis per ml, but at least 3 X 10(3) T. vaginalis per tube was required to initiate growth. Thus, in vitro, cell culture was more sensitive than TYI broth in detecting low numbers of T. vaginalis. In a subsequent clinical comparison of broth and cell culture for isolation of T. vaginalis from 188 vaginal specimens and 21 urethral specimens from men, the results were in agreement for 206 specimens (98.6%). There were no situations in which culture was negative and a saline preparation showed motile trichomonads. For women, using a positive culture as the indicator of true positivity, the sensitivity of detection of T. vaginalis was 83% with the Pappenheim stain and 77% with saline preparations. These studies show that cell culture can be used for isolation of T. vaginalis from clinical specimens; it gave results comparable to those of broth culture for the group of mainly symptomatic women. Further studies should be performed to determine its utility in clinical populations such as asymptomatic women and men with and without symptoms, in which T. vaginalis is more likely to be present in low numbers.     

Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model

Conventional periodontal regeneration methods remain insufficient to attain complete and reliable clinical regeneration of periodontal tissues. We have developed a new method of cell transplantation using cell sheet engineering and have applied it to this problem. The purpose of this study was to investigate the characteristics of human periodontal ligament (HPDL) cell sheets retrieved from culture on unique temperature-responsive culture dishes, and to examine whether these cell sheets can regenerate periodontal tissues. The HPDL cell sheets were examined histologically and biochemically, and also were transplanted into a mesial dehiscence model in athymic rats. HPDL cells were harvested from culture dishes as a contiguous cell sheet with abundant extracellular matrix and retained intact integrins that are susceptible to trypsin-EDTA treatment. In the animal study, periodontal ligament-like tissues that include an acellular cementum-like layer and fibrils anchoring into this layer were identified in all the athymic rats transplanted with HPDL cell sheets. This fibril anchoring highly resembles native periodontal ligament fibers; such regeneration was not observed in nontransplanted controls. These results suggest that this technique, based on the concept of cell sheet engineering, can be useful for periodontal tissue regeneration.     

Rapid Shell Vial Culture Technique for Detection of Enteroviruses and Adenoviruses in Fecal Specimens: Comparison with Conventional Virus Isolation Method

Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.