Experimental study of tissue-engineered urethra with collagen / chitosan composite as scaffolds

In this article we investigated the preparation of tissue-engineered urethra by using the urethral epithelial subculture cells of male New Zealand young rabbits. We inoculated the epithelial cells of urinary mucosa of male New Zealand young rabbits on collagen, chitosan and collagen chitosan composite as scaffolds to prepare tissue-engineered urethra. The results of inverted phase contrast microscope, HE staining and scanning electron microscope of three kinds of tissue-engineered urethra were compared. What’s more, we reported a new method for quantitative and rapid detection of epithelial cell activity of urinary mucosa in situ by Interactive Laser Cytometer. The collagen chitosan composite was more similar to the extracellular matrix of mammalian. Its three-dimensional porous structure had a high area volume ratio, which was conducive to cell adhesion, growth and metabolism. In vitro, the urethral epithelial cells had been cultured on collagen chitosan composite, and the tissue-engineered urethra was successfully prepared, which laid a solid foundation for further in vivo experiments.


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Urethral reconstruction continues to be a challenging field for urologists. 45 Urethral stricture is a disease characterized by a pathological narrowing of the urethra. 46 Treatment for this condition often requires surgery using autologous grafts 47 (urethroplasty). It is common practice to use patient's own tissue like genital and 48 extragenital skin, bladder and buccal mucosa as a source of the graft [1]. Whilst for 3 49 some conditions only one or few procedures are standard, over 300 techniques are 50 known for urethral stricture repair [2]. This diversity illustrates the complexity of 51 these conditions and also indicates the lack of one perfect procedure. However, all of 52 these substitutes have limitations compared to the autologous urethral tissue, which 53 can lead to complications (e.g. stricture formation, graft failure). Also, the amount of 54 tissue that can be harvested from a donor site is limited; especially in the case of long 55 defects, this could pose a problem [3]. To overcome these problems, alternative 56 materials for urethral reconstruction have been explored. 57 In the field of regenerative medicine, tissue engineering (TE) is defined as "an 58 interdisciplinary field that applies the principles of engineering and life sciences 59 toward the development of biological substitutes that restore, maintain, or improve 60 tissue function or a whole organ" [4]. As early as the 1980s the first steps were made 61 in culturing urothelial cells. Initially, these cultures were used as an in vitro system to 62 study the effects of exogenous substances on tissue [5]. When TE started to evolve, 63 the aim of culturing tissues changed to the replacement of damaged or absent organs. 64 The rationale behind this latter strategy is that, with a limited amount of material (e.g.

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Tissue engineered urethra with collagen as scaffolds 84 We took one 8-hole sterile culture plate. The collagen substrate was cut into the 85 bottom of the culture plate (0.28cm 2 ) according to the corresponding size, which had 86 been irradiated and sterilized by high-efficiency ultraviolet sterilizer. The urethral 87 epithelial subculture cells of male New Zealand young rabbits were seeded on 88 collagen matrix with a density of 0.7×10 4 / hole. 0.2ml mixed culture medium was 89 added into the culture hole, and cells were cultured in 34℃ , 5% CO 2 and saturated 90 humidity cell incubator. The growth of the epithelial cells was observed under the 5 91 inverted phase contrast microscope every day. The culture medium was changed once 92 every 2 days. Cells had been cultured for 7 days.

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Tissue engineered urethra with chitosan as scaffolds 94 We took one 8-hole sterile culture plate. The chitosan matrix was cut into the 95 bottom of the culture plate (0.28cm2) according to the corresponding size, which had    Quantitative test on computer: the focus plane of each specimen was consistent, 149 magnification was 10 × 2.5, frame was 10, and PMT was 250. Each specimen was  Microscopically, the number of cells on the collagen chitosan composite scaffolds 167 was more than that on the collagen and chitosan scaffolds when the cells were 168 cultured for 7 days. It shows that the collagen chitosan composite scaffolds was more 169 similar to the extracellular matrix of mammalian, and its three-dimensional porous 170 structure had a high area volume ratio, which was conducive to cell adhesion, growth 171 and metabolism.  However, the number of cells on the chitosan as scaffolds was less, which were also 212 round or oval. which is a quantitative method in situ.

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The cultured urethral epithelial cells of male New Zealand young rabbits were 362 capable of extensive expansion in vitro, and can be used for urethral reconstruction.

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The collagen chitosan composite was more similar to the extracellular matrix of 364 mammalian. Its three-dimensional porous structure had a high area volume ratio, 365 which was conducive to cell adhesion, growth and metabolism. In order to form a