Abstract
BACKGROUND: Recently, the development of tissue-engineered technique has broadened the study of artificial esophagus. Some investigators have inoculated esophageal epithelial cells cultured in vitro onto compound polymer material and successfully constructed tissue-engineered esophagus.
OBJECTIVE: To investigate the feasibility of tissue-engineered artificial esophagus by combining dog esophageal epithelail cells and an acellularized porcine thoracic aorta allogenic matrix.
DESIGN: An experimental observation.
SETTING: Central Laboratory, Taishan Medical College.
MATERIALS: This study was carried out in the Central Laboratory, Taishan Medical College from June to December in 2004. Three hybrid dogs, 24-hour-old, were provided by the Laboratory Animal Center, Taishan Medical College. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The experimental instruments and reagents were as follows: CO2 incubator (MCO-15AC, SANYO), hypothermal high-speed centrifuge (RC-26, Dupont), trypsin, transferrin, type II collagenase (Gibco), dulbecco's modified eagle's medium (DMEM), DispaseII isolated enzyme, and rat monoclonal anti-keratin antibody (Sigma).
METHODS: Acellularization of porcine aortas was performed by a method of enzyme-detergent. Esophageal epithelial cells of hybrid dogs were in vitro isolated, cultured and proliferated. Next, they were inoculated onto an acellularized porcine thoracic aortas allogenic matrix scaffold. Three and seven days later, the growth of esophageal epithelial cells on the acellularized matrix was observed under an electron microscope.
MAIN OUTCOME MEASURES: Morphology of esophageal epithelial cells cultured in vitro; Biocompatibility of acellular matrix and dog esophageal epithelial cells.
RESULTS: The acellularized procedure resulted in an almost complete removal of the cells and the loose three-dimensional matrix .The acellular matrix could be reseeded with expended esophageal epithelial cells in vitro, and esophageal epithelial cells had the potential of spread and proliferation.
CONCLUSION: Acellular matrix possesses satisfactory biocompatibility for allogenic esophageal epithelial cells. Tissue-engineered artificial esophagus can be generated in vitro by a combination of esophageal epithelial cells and allogenic acellularized matrix.


INTRODUCTION

At present, there have been two kinds of artificial esophagus: acellular matrix and tissue-engineered esophagus. For acellular matrix, time for epithelial cell creeping and substitution is very long (about 3-4months) and regeneration of submucous structure and muscular hyperplasy are poor, so incidence of anastomotic leakage reaches 60%. Moreover, anastomotic stenosis easily forms, and successful rate of surgery is very low [1-5]. Recently, the development of tissue-engineered technique has broadened the study of artificial esophagus [6-12].
In the present study, we designed a new type of tissue-engineered esophagus that combined with dog's esophageal epithelial cells and porcine thoracic aorta acellular matrix.

MATERIALS AND METHODS

Materials
This study was carried out in the Central Laboratory, Taishan Medical College from June to December in 2004. Three hybrid dogs, 24-hour-old, were provided by the Laboratory Animal Center, Taishan Medical College. The protocol was performed in accordance with ethical guidelines for the use and care of animals.
The experimental instruments and reagents were as follows: CO2 incubator (MCO-15AC, SANYO, Japan), hypothermal high-speed centrifuge (RC-26, Dupont), trypsin, transferrin, type II collagenase (Gibco Co., Ltd), dulbecco's modified eagle's medium (DMEM), DispaseII isolated enzyme, and rat monoclonal anti-keratin antibody (Sigma Co.,Ltd., USA).

Methods
Preparation of porcine thoracic aorta acellular matrix
About 80-100 kg porcine thoracic aorta was harvested from butchery and preserved in D-Hank's balanced salt solution at 4 ℃ for less then 10 minutes. Next, after removal of adventitia, porcine thoracic aorta was soaked in 1% benzalkonium bromide for 3 minutes followed by a thorough lavage by phosphate buffer solution. According to the method from Han et al [6], porcine aorta was cultured for 24 hours with 0.1% trypsin and 0.02% EDTA in a CO2 (0.05 volume fraction)-air incubator at 37℃. Subsequently, it was water-shaken for 176 hours in 1% TritonX-100 at 26 ℃-28℃. The cultured aorta was repeatedly rinsed with phosphate buffer solution, and preserved at 4 ℃in the D-Hank's balanced salt solution supplemented with Ca2+ and Mg2+. Sampling was harvested followed by observation under a scanning electron microscop and haematoxylin-eosin staining.
Harvesting esophageal epithelial cells
A segment of 3-4 cm esophagus was aseptically taken from a euthanized dog born newly. It was longitudinally cut open, and submucous tissue was rejected. Thereafter, it was soaked for 3-5 minutes in 0.05% chlorhexidine and for 2 hours in phosphate buffer solution supplemented with 100 000 U/L penicillin and 100 mg/L streptomycin at 4 ℃. The specimen was treated 4 hours in the digestive supplemented with 0.5% DispaseⅡisolated enzyme and 0.2% type Ⅱcollagenase at 37 ℃. Epithelial lamina was mechanically broken and digested for 15 minutes in the 0.02% trypsin solution at 37 ℃. Single cell suspension was isolated, treated with fetal bovine serum, and filtered with a 300-mesh stainless steel screener. The solution was centrifuged at 1 500 r/min for 5 minutes, and supernatant solution was discarded. Following two washings of phosphate buffer solution, DMEM supplemented with 20% fetal bovine serum, 100 000 U/L penicillin, 100 mg/L streptomycin, 10 mg/L trypsin, 100μg/L hydrocortisone, and 5 mg/L transferrin was added to make into cell suspension. Cells were inoculated into a 50 mL culture flask at 1×108/L and incubated in a CO2 (0.05 volume fraction)-air incubator at 37 ℃. Medium was renewed 6-8 hours later. Subsequently, the cells were digested with 0.05% trypsin and 0.02% EDTA, and passaged at 1∶2 in separate flasks. Passages 2-5 of cells were taken and identified by immunohistochemial keratin.

Cell seeding
Acellular thoracic aorta matrix was dissected into pieces about 1.5 cm× 1.5 cm square. The pieces were placed on the 24-well culture plate, and exposed under an ultraviolet lamp for 24 hours. One or two drops of fetal bovine serum was added at the internal surface of acellular thoracic matrix pieces. Then the tissue culture plate and pieces were placed in the CO2-incubators at 37 ℃ for 2 hours. Esophageal epithelial cells were digested with 0.05% trypsin for 2 to 3 minutes and then centrifuged 5 minutes (1000 r/m). The cells were washed with DMEM 3 times. The proper seeding density was adjusted to 5×109 /L by DMEM in primary culture. The cells were seeded on the surface of the acellular thoracic aorta matrix pieces in the 24-well culture plate. Cells were cultured in DMEM with 10% fetal bovine serum. Cells were expended ex vivo in CO2 (0.05 volume fraction)-air incubators at 37℃. Twelve hours later, medium was renewed, and then it was renewed once every 2-3 days. Sampling was separately performed 3 and 7 days after seeding for haematoxylin-eosin staining and electron microscope observation.

RESULTS

Cell morphology observation
Cell morphological characteristics were studied under an inverted microscope. After 6-8 hours of culture, epithelial esophageal cells at primary culture presented with irregular shape, being ellipse or spherical. Most of cells attached to the wall and formed cell colony. Five or six days later, cells formed cell layer, presenting with a typical "road metal-like" arrangement (Figure 1). Immunohistochemical keratin positive was found.

Gross observation of acellular throacic aorta matrix
After being acellularized, acellular thoracic aorta matrix appeared ivory-white and translucent. Aorta wall became thin and softening, but its elasticity was still well. Its internal surface was smooth (Figure 2).

 

Eight samples were studied by haematoxylin-eosin staining. No cell fragments and blue-stained nucleoli were found under the microscope. Elastic fibers arrayed in order by masson's stained. Among elastic fibers, collagen fiber could be seen (Figure 3). Collagen, elastic fibers and less metamorphic mitochondria could be seen under the scanning election microscope. The fiber of acellular matrix complected to reticulation, and the aperture of reticulation was about 10 μm (Figure 4).

Histological observation after cell seeding
Discontinous cell layers could be seen at the surface of acellular thoracic aorta matrix 3 days after cell seeding. Seven days later, a single continuous cell layer could be seen. Microfilaments in cytoplasm, tight junction as well as desmosomes with keratin filaments among cells and microvillus could be seen under a transmission election microscope.

DISCUSSION

The structure of macromolecule polymer is simple and the component is singleness. The macromolecule polymer cannot provide the appropriate extracellular matrix and ultrastructure for different kinds of cell proliferation. Adherence between cells and extracellular matrix is the base of cell proliferation [3, 13-15]. It is said that collagen I is prominent in the submucosa and in the muscular septa, collagen III is in the lamina propria and the submocusa [16]. After being acellularized, cells and soluble protein of dog's carotid were washed. Its histological changes and ultrastructure were observed [17]. Collagen I, collagen III and elastic protein are the main components. The components of the carotid are similar to natural extracelullar matrix. Its immunity is low. Acellular thoracic aorta matrix and small intestinal submucosa had been used for bladder and esophagus defect repair in animal models [18-20]. The experimental results showed that the acellular thoracic aorta matrix had good compatibility and could accelerate cell proliferation. In the present study, porcine thoracic aortas acellular matrix was used as the seeding patch of esophageal epithelial cells. The results showed acellular thoracic aorta matrix preserved the main structure and components of extracellular matrix and was propitious to the growth and proliferation of esophageal epithelial cells. The preparation process of acellular thoracic aorta matrix was comparatively simple, but the material was abundant. Acellular thoracic aorta matrix can be used as the matrix of tissue-engineered esophagus.
Adherence between cells is the base of cell growth and proliferation. Because Ca2+ and Mg2+ act an important rule in cell adherence, so preserving acellular thoracic aorta matrix in D-Hank's balanced salt solution supplemented with Ca2+ and Mg2+ is a good choice. Fetal bovine serum added at acellular thoracic aorta matrix pieces can increase cell adherence. Fetal bovine serum not only provides protein, which can combine metal ion, but also neutralizes the toxicity of trypsin and triton X-100. In the culture of epithelial esophageal cells, isolated enzyme was initially used to separate squamous epithelium from basal interstitial substance, and then digestive passage culture was performed. Results demonstrated that many epithelial esophageal cells could be used by such a method [21-22].
In order to culture high proliferation cell clone, the seeding density cannot be less than 1×108 /L. In the present study, the seeding density was adjusted to 1×109 /L. It was found that cell growth and proliferation were well at this seeding density.

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体外构建犬组织工程化食管的
初步试验☆

张 哲1，张 璐1，牛晓光2，尹志伊1，何宝亮1，王伦青1
1青岛市市立医院胸外科，山东省青岛市 266071; 2泰安市中心医院， 山东省泰安市 271000
张 哲☆，男，1969年生，山东省临沭县人，汉族，1991年泰山医学院毕业，博士，副主任医师，主要从事胸外科研究。
摘要
背景：近年来组织工程技术的发展为人工食管的研究开辟了新的思路，有研究者应用体外培养的食管黏膜上皮细胞接种于复合高分子材料上，构建组织工程食管获得成功。
目的：探讨应用体外培养的犬食管黏膜上皮细胞种植于猪的主动脉脱细胞基质支架上，构建组织工程化人工食管的可行性。
设计：观察实验。
单位：泰山医学院中心实验室。
材料：实验于2004-06/2004-12在泰山医学院中心实验室完成，选用3只出生24 h 内杂种犬，由泰山医学院动物园提供。 实验过程中对动物的处置过程符合动物伦理学标准。二氧化碳培养箱MCO-15AC（ SANYO），RC-26低温高速离心机（杜邦）。胰蛋白酶、转铁蛋白、II型胶原酶（GIBCO）；DMEM、DispaseII分离酶、鼠抗人角蛋白单克隆抗体 （Sigma）。
方法：用酶-去污剂法对猪主动脉进行脱细胞处理，体外分离、培养、扩增新生杂种犬的食管黏膜上皮细胞，接种于去细胞基质支架体外培养，种植后3天、1周通过组织学及电镜观察食管黏膜上皮细胞在脱细胞基质支架上的生长情况。
主要观察指标：体外培养的食管黏膜上皮细胞的形态；脱细胞基质与犬食管上皮细胞的生物相容性。
结果： 酶化学除垢剂法能使猪主动脉细胞脱落，基质三维结构变疏松。 体外扩增的犬食管黏膜上皮细胞可以种植在脱细胞基质上并能生长，增殖。
结论：脱细胞的基质与犬食管上皮细胞具有良好的生物相容性，能结合在一起并形成人工食管移植体。
关键词：组织工程；人工食管；细胞外基质；食管黏膜上皮细胞
中图分类号: R318 文献标识码: A 文章编号: 1673-8225(2008)12-02181-04
张哲，张璐，牛晓光，尹志伊，何宝亮，王伦青. 体外构建犬组织工
程化食管的初步试验[J].中国组织工程研究与临床康复,2008,12(11):
2181-2184
[www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2181(ps).pdf]
(Edited by Cao ZA/Song LP/Wang L)