| BackgroundDue to trauma, disease, genetic defects, and some surgical operations due to urethral defect is a common disease of Urology the urethral tissue defects often lead to urethral scarring, urethral stricture, voiding dysfunction, sometimes the merger of the lower urinary tract recurrent infections. Patients are often very painful, this time only the urethral tissue reconstruction in order to solve the patient suffering the only way out. For urinary tract tissue reconstruction, a variety of methods, including simple materials transplantation, autologous tissue transplants, such as autologous oral mucosal graft composite cell tissue engineering urethral tissue transplantation, tissue engineering the urethral tissue defects in the urinary tract treatment in this area has showing a distinct advantage.Tissue engineering as a rapid rise in recent years, a new technology, new ideas and methods to repair urethral tissue defects. Following in order to more clearly understand the tissue engineering technology, let us look back at the history of the development of tissue engineering;1994, American scholars Vacanti first use of tissue engineering technology successfully prepared the Mavericks have some form of structure-activity bone. Subsequently, the scholars comprehensive development of tissue engineering, including access to seed cells and scaffold for the development, the role of growth factors, cell-scaffold dimensional culture. Tissue engineering and technical advantages include:building a supply graft with cell biological activity, the source of biological material is wide, and can be shaped according to the required prefabricated, if the use of autologous cells can mate to reduce immunogenicity, while avoiding the concurrent autologous tissue defect psychosis. Study of tissue-engineered urethra, scientists have a more in-depth research in the urethral defect tissue engineering technology, has achieved initial results confirmed the feasibility of the urethral tissue engineering technique to repair the urethral defect. The rapid development of tissue engineering indicates that modern medicine by organ transplant era into the era of organ reconstruction.Although the prospects for the development of tissue engineering urethra is another encouraging urethral tissue engineering research are still experiencing a lot of problems, the ideal seed cells as one of the key issues of tissue engineering, which restricts the development of urethral tissue engineering; at the moment, urethral tissue engineering research, epidermal cells, the epithelial cells of the oral mucosa has been occupied as seed cells for the urethra tissue engineering epithelium, but these seed cells ideal the urethra tissue engineering seed cells still far apart; regardless of the epidermal cells or oral epithelial cells, must be caused by the patient’s own tissue defects, epidermal cells and oral epithelial cells belong to adult cells in vitro were cultured amplification capabilities are very limited. In recent years as a popular study of embryonic stem cells or iPS cells, although strong in vitro expansion and differentiation ability, but are not suitable because it may appear tumorigenic tissue build. This study attempts to explore hair follicle stem cells as seed cells feasibility, first in basic and clinical research, a number of scholars reported that hair follicle stem cells into epidermal cells specific sum instead damage the epidermis; Secondly, the hair follicle stem cells As adult stem cells retain stem cell should be strong in vitro expansion capacity, while avoiding the application of embryonic stem cells may appear tumorigenicity. More importantly, the hair follicle stem cells can be in the process of obtaining the maximum to avoid tissue damage to the donor.This study is one of the core content sorting enrichment hair follicle stem cells, therefore, the sorting of hair follicle stem cell enrichment methods, and amplification law of the hair follicle cells, to provide guidance for urethral tissue engineering building became the study must be the primary problem, therefore, the sorting of hair follicle stem cells enriched on the basis of the experimental part of this research paper. Also constitutes the contents of the first part of the present research papers. At the same time, the hair follicle cells in vitro amplification regular has been inextricably linked with stem cell research. We intend to explore the hair follicle stem cell sorting enrichment methods, recorded at the same time the law of the hair follicle stem cells in vitro amplification. We choose the source of rabbit beard hair follicle stem cells in vitro amplification regular count method and cell culture by growth kinetics to study its potential for amplification. Clone formation rate, Keratinl9, Keratinl5, CD200, the expression in case authentication Evaluation stars selected as to obtain the hair follicle stem cell content and purity assessment sorting method; analysis of hair follicle stem cells in the process of in vitro amplification of the content change; preliminary explore hair follicle stem cells in vitro amplification mechanism.According to the existing research reports, the compound epithelial tissue engineering urethral stent superior to the pure scaffolds, the epithelial cells can not be ignored. Epidermal cells, oral epithelial cells have been reported for build tissue engineering urethral mucosa epithelium; fat stem cells, bone marrow stem cells has also been reported for the Construction of tissue engineering urethral submucosa tissue. A variety of cells used in tissue engineering urethra complex built all have different advantages, however, if the use of epidermal cells, oral mucosal cells as seed cells, the source of cells and the number of cells are relatively limited. Therefore, to ensure the urethral mucosa layer of tissue engineering to achieve more desired results, a new source of seed cells is needed to continue to explore important one. Relatively rich source of hair follicle stem cells, hair follicle stem cells collected acquisition process is relatively easy, and more importantly, is the biological characteristics of the hair follicle stem cells and epidermal stem cells are very similar. Therefore, the hair follicle stem cells is to build a good candidate cells for tissue engineering urethra; In other words, the hair follicle stem cells is likely to become a new source of urethral tissue engineering seed cells; Although the biological characteristics of the hair follicle stem cells and epidermal stem cells is very similar. However, the biology of stem cells of the hair follicle stem cells and the urethral mucosa phenotypic differences are not very clear. Therefore, to explore the phenotypic differences and compare hair follicle stem cells and urethral mucosa stem cell biology is one of the important problems awaiting solution. Moreover, in the last part of the study, we have established a method of hair follicle stem cell sorting enrichment. A the quality relatively stable hair follicle stem cells.-Oriented part of the research carried out to lay the foundation. More importantly, the hair follicle stem cells and urethral mucosa stem cell biology phenotype comparative studies have rarely been reported. Therefore, we need to further compare the hair follicle stem cells and the urethral mucosa stem cell biological phenotypes.The seed cells and scaffold materials the necessary indispensable factors in tissue engineering, tissue engineering scaffolds of a variety of methods. According to the source material can be divided into:natural scaffold materials and synthetic scaffold materials; natural materials in tissue engineering represents:acellular dermal calcined coral, acellular bladder acellular small intestinal organized under. Synthetic scaffolds representative of polyglycolic acid, also known as polyglycolic acid (polyglycolic acids, PGA), poly lactic acid (polylactic acid, PLA), and poly (glycolic acid)[poly (D, of L-lactic-co-glycolic) acid] PLAG, tricalcium phosphate (tricalcium phosphate, TCP), and so on. Tissue engineering scaffold material that is used in different types of human tissues, according to the material purpose, functions and organizations to have their specific alternative design. Tissue engineering scaffolds include:skin, cartilage, nerves, blood vessels, bone, and artificial organs such as the liver, spleen, kidney, bladder and other tissue scaffold material. Liver, pancreas, kidneys, urinary system using tissue engineering scaffold materials for biodegradable materials, research in this area and use of materials, mainly natural proteins, polysaccharides, synthetic polymers biodegradable materials. E.g.:a bracket for constructing tissue engineering liver plasma fibrinogen, and polylactic acid, for the urethral tissue engineering polyglycolic acid and so on. BAMG off as bladder cell matrix and urinary system tissue approximation very high degree, applies to the construction of urethral tissue engineering. BAMG as a natural acellular material must also clear the nature of its biological research.Provide a theoretical basis, and parameters to guide when the seed cells and biological materials for tissue engineering urethra build, we can carry out the application of the hair follicle stem cells BAMG of build the tissue engineering urethra repairing rabbit urethral defect animal model experimental research. Of course, the ultimate goal of our research services in clinical research results. In other words, the hair follicle stem cells as well as the research base of the bladder acellular matrix, and should meet the needs of the construction of tissue engineering urethra. In summary, we will in the present study, to explore the use of stem cells of the hair follicle origin build tissue engineering feasibility of the urethra, and artificial tissue engineered urethra, animal models for rabbit urethral defect. To promote the development and application of the urethral tissue engineering parameters and theoretical basis. And hope eventually to patients early clinical application of this research project. Human services, to the benefit of mankind.ObjectiveWe conducted this study’s purpose is to explore the in vitro isolation and enrichment of cultured hair follicle stem cells optimized conditions, comparing hair follicle stem cells and urethral mucosal epithelial source stem cells of the biological characteristics of the difference, and further study the differentiation of hair follicle stem cells and build tissue engineering urethral mucosa layer of feasibility, and eventually used in repairing rabbit urethral defect. For the clinical treatment of urethral defects, repair the urethra to provide a theoretical basis and guidance.MethodThe experimental study of hair follicle stem cells derived from New Zealand white rabbits, the rabbits were anesthetized with intravenous pentobarbital, rabbit bilateral tentacles Ministry layer of skin under sterile conditions. Blunt dissection of a single hair follicle tissue. The "two-step enzyme method to obtain a single cell from a single hair follicle tissue, and is used for a variety of detection; digested single cells inoculated into2×108/L density culture flasks, placed in5%of CO2,37℃humidified incubator in culture medium was changed every3d until the cells reached90%confluence1:3passage. Flow cytometry respectively sub-elect diameter<10um and CD200(+)/CD34(-) table type of hair follicle stem cells and urethral mucosa epithelial stem cells, separation cultured rabbit hair follicle stem cells and urethral mucosa epithelial stem cells, In vitro primary culture amplification record and depict the growth curve of the rabbit hair follicle stem cells and urethral mucosa stem cells, the maximum amplification of the two cell ratio; colony βforming ability; use flow cytometry sorting latter two cell K19, p63,1-integrin of The positive expression rates.Experimental animals rabbits were sacrificed overdose anesthetic (pentobarbital100mg/kg), full cut rabbit bladder tissue, bladder exstrophy, scraping the mucous layer, in50ml phosphate-buffered saline (PBS, pH value of7.0,10mmo1/L) and0.1%sodium azide mixture was stirred for5-6h, the cells were dissolved, to remove the basement membrane and detrusor. Rinsed with PBS, in50ml of1mol/L NAC1solution (containing deoxyribonuclease2000kU) in was stirred for6-8h, the cells were thoroughly dissolved, cellular components fully released. In50ml of4%sodium deoxycholate and0.1%sodium azide-containing mixture was stirred for5-6h, to dissolve the cell membrane lipid bilayer, and intracellular membrane lipids. Repeat step3made BAMG BAMG washed3times with PBS and then stored in a solution of10%neomycin.Sorting enriched hair follicle stem cells were seeded on tissue engineering scaffold, build tissue engineering urethra. Vitro attempt urethral mucosal epithelial stem cells to construct tissue engineered urethra. Urethra application histology, fluorescent staining tissue engineered hair follicle stem cells and urethral mucosa epithelial stem cells in vitro. The various categories of tissue engineering urethra using scanning electron microscopy, the distribution of cells in the bracket, the hair follicle stem cells build tissue engineering urethral repair2.5cm rabbit urethra defect, histological, urethrography observed defects.ResultsThe flow cytometer display:in the analyzed samples, there is obviously the two groups of particles, the group of the parameters of the forward scattered light distribution broader particle group, we will it is judged as a cell sample, another group of particles of the small rectangular area within distribution of forward scattered light parameters than the concentration of the particles, the the group particle diameter size relatively uniform, but at the same time expressing three fluorescent.Hair follicle stem cells after sorting by flow cytometry, was transferred to a petri dish10cm in diameter, and then placed under inverted phase contrast microscope observation. The same magnification, unsorted cell population, visible cell diameter sizes. According to the small diameter of the cell sorting cell groups, smaller and relatively homogeneous. Large and relatively homogeneous groups of cell sorting of large cell diameter volume.Maximum amplification multiples rabbit hair follicle stem cells and urethral mucosa stem cells (6.1±0.8)×104-fold and (7.1±1.1) x103times; Cell markers, cell size, the composite parameter sorting and colony-forming cell population unsorted area were:205.37±33.18(mm2),197.72±25.31(mm2),359.79±59.57(mm2),3.10±1.07(mm2). Composite parameter cell population sorting clone form the largest area, and there is a statistically significant difference (P<0.05), with the remaining three sets of data. By the cell marker (the Marker sort), cell size (Small size), the composite parameter sorting (Marker+small size) ways and unsorted (Unsort) was obtained in a cell population after clone after staining visible:sorting the obtained composite parameter the number of cell clones and the area was significantly greater than the other three groups. The unsorted cell clones obvious.The hair follicle stem cells and urethral mucosa stem cells in vitro tissue engineering to build a urethra HE staining. Hair follicle stem cells and urethral mucosa stem cells in stratified epithelium-like structure can be formed on the bracket similar organization built by the two types of cells in the histological structure. Submucosal βno obvious scar tissue enriched hair follicle stem cells express K19, p631-integrin urethral stent has good biocompatibility, hair follicle cells to form stratified epithelium-like structure in the bracket AE1/AE3positive staining. Scanning electron microscopy showed cells with the bracket firmly attached, moderate stretching and matrix secretion, hair follicle stem cells to construct tissue engineering urethra repairing rabbit urethral defect to good effect, while the control group for the fibrous scar tissue repair. The hair follicle stem cells and urethral mucosa stem cells in vitro tissue engineering of building a urethra immunofluorescence AE1/AE3staining. Clearly visible in the epithelial tissue build green fluorescent protein.Histological staining of tissue engineering scaffold No cell within the structures in the acellular matrix material, the material is collagen fibers rendered natural distribution state available to the cell holding the pore structure between the fibers. Simplex bladder acellular material tensile strength higher elastic modulus with close to the normal tissue of Pa, it is possible to meet the organization building material needs. The results showed that the off cell matrix material cells and hair follicle stem cells in vitro co-culture, cells with good material compatibility, cell full adhesion with the surface of the material, and the normal secretion of extracellular matrix, cells attached to the material on the cells are able to migrate freely.The acellular material implanted in animals not cause immune rejection of animal tissues, and description of the material to various inflammatory substances in the process has been thorough treatment. At the same time, also the body further validate bladder acellular matrix material with the cells in the body there is a good tissue compatibility. Visible cells in the stratified epithelium-like structure, the hair follicle stem cells to build the organization structure similar in histology and normal urethral tissue can form on the bracket. Submucosal no obvious scar tissue.Conclusion By flow cytometry on the cell surface markers as well as the cell diameter dual parameter optimization, the hair follicle stem cells may be sorting enriched. Rabbit hair follicle stem cells and urethral mucosa stem cells with a similar phenotype, can be used as the urethra tissue engineering seed cells is used to construct tissue engineered urethra, seed cells get the hair follicle stem cells better than the urethral mucosa stem cells. Enriched rabbit hair follicle stem cells to construct tissue engineered urethral repair is better than not enriched hair follicle stem cells build tissue engineering urethral repair effect.Application enriched rabbit hair follicle stem cells to construct tissue engineering urethra can be used rabbit urethral defect repair treatment. The repair effect is good, there is further possible for the clinical study. |
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