【摘要】 目的：探讨脱细胞猪角膜基质体外能否支持兔角膜细胞的生长。
方法：体外培养兔角膜上皮细胞和基质细胞，并接种到制备的脱细胞猪角膜基质上，倒置相差显微镜和组织学观察细胞生长情况。
结果：上皮细胞能在脱细胞猪角膜基质上贴附生长，10d时可形成2～3层的复层结构。基质细胞在脱细胞猪角膜基质上贴附生长后可向材料深层迁徙。
结论：制备的脱细胞猪角膜基质体外可支持兔角膜上皮细胞和基质细胞的生长。
【关键词】 脱细胞猪角膜基质；角膜上皮；角膜基质细胞；组织工程
Support of acellular porcine corneal stroma for growth of corneal epithelium and stromal cell in vitro
XuChu Lin, YanNian Hui, Hao Meng, YongJie Zhang, Yan Jin
Foundation item: This study was supported by the Development of High and New Science and Technology (863 Project) of China (No. 2005AA205241)
Department of Ophthalmology, No. 303 Hospital of Chinese PLA, Nanning 530021, Guangxi Zhuang Autonomous Region, China Research and Development Center for Tissue Engineering, School of Stomatology, the Fourth Military Medical University, Xian 710032, Shaanxi Province, China Department of Ophthalmology, Xijing Hospital, the Fourth Military Medical University, Xian 710032, Shaanxi Province, China
Abstract
AIM: To determine whether acellular porcine cornea stroma (APCS) could support the growth of the rabbit corneal cells in vitro.
METHODS: APCS was prepared. The rabbits corneal epithelium and stromal cells were cultured and seeded on APCS in vitro. The observation of phase contrast photograph and histological examination were performed.
RESULTS: Histological examination showed the epithelium grew on the scaffold of APCS in 23 layers at 10th day. The stromal cells adhered to the surface of the scaffold after 24 hours and invaded into the interlaminar of the material at 5th day.
CONCLUSION: These results indicate that APCS can support the growth and proliferation of the corneal epithelium and stromal cells in vitro.
KEYWORDS: acellular porcine cornea stroma; corneal epithelium; corneal stromal cells; tissue engineering
INTRODUCTION
At present, although the tissue engineering cornea has gained increasing progress, a fully effective prosthetic cornea has not yet been developed [1]. A successful tissueengineered corneal prosthesis must contain a scaffold that fully supports the growth of corneal cells. In the early application of synthetic scaffolds, the surfaces of scaffolds could not be covered by an intact epithelium in vivo, and their applications in tissue engineering were limited [25]. Recently, the application of the natural biologic materials in tissue engineering is widening gradually. The natural materials are beneficial to the growth of cells and can remodel more easily in vivo because of retaining the natural extracellular matrix (ECM) and original structure. In tissue engineering cornea, it has been reported that the natural corneal stroma containing cells is taken as the substrate for the growth of corneal cells in vitro [6,7]. We had prepared acellular porcine corneal stroma (APCS) previously to reduce the antigen and pathogenicity [8]. The aim of the present study was to determine whether APCS could support the growth of the rabbit corneal cells in vitro.
MATERIALS AND METHODS
Experiment Animals The pigs in a local abattoir and New Zealand white rabbits were selected. Rabbits (n=6) with body weight ranged from 2 to 2.5kg were provided by the Experiment Animal Center of the Fourth Military Medical University (Xian, China). The eyes of the animals were healthy, and the gender was not limited. National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH Publication #8523 Rev. 1985) have been observed. The porcine corneas were harvested to prepare APCS. The corneas of the rabbits were selected to collect the rabbit corneal cells.
Preparation of APCS The preparation of APCS was in accordance with a previous report [8].
Culture of the Rabbit Corneal Epithelium Cells The procedure of cell culture was as follows: the lamellar limbal tissue (100μm in deep, 3mm in width) was obtained from the rabbits eyes under an operating microscope. The epithelia were separated from the stroma after a 45minute incubation in Dispase Ⅱ(2.5 mg/mL) at 37℃. The cells were cultured with DMEM/F12 (2∶1) (GIBCO, Grand Island, NY, USA) containing 100mL/L fetal bovine serum (GIBCO), 20μg/L recombinant human EGF (Sigma, St. Louis, MO, USA), 5mg/L insulin (Sigma), 0.18mmol/L adenine (Sigma), 30μg/L cholera toxin (Sigma), 0.5mg/L hydrocortisone (Sigma) and antibiotics (100U/mL penicillin and 100mg/L phytomycin) in a 37℃, 50mL/L CO2 incubator. The culture medium was replaced every 2 days. The result of immunohistochemical detection of AE5 antibody on the epithelial cells cultured was positive (data not shown).
Stromal cells were isolated from rabbits corneas with the following methods: The corneas of the rabbit were harvested, and the epithelium and endothelial cells were removed. The stromal layer left was cut into pieces. After digested with collagenase (GIBCO) for 45 minutes at 37℃, the stromal cells were harvested and cultured with DMEM (GIBCO) containing 100mL/L fetal bovine serum (GIBCO). The culture medium was replaced twice per week.
Seeding of the Corneal Cells of Rabbits on APCS Before the cells being seeded, APCS were soaked in the culture medium for 1 hour and then the medium was blotted. The cell suspension of the third passage epithelium cells was seeded onto the surface adjacent to the epithelium of APCS at a density of 5×104 cells/cm2. Culture medium was not added until the cellscaffold constructs were kept in a 37℃,50mL/L CO2 incubator for 3 hours to allow adhesion of the cells to the scaffolds. The cellscaffold constructs were cultured under media for 1 day and upgraded to airliquid interface at the second day to continue to be cultured. The culture medium was replaced every 2 days. After being cultured for 10 days, the cellscaffold constructs were fixed in 40g/L paraformaldehyde, paraffin embedded, then sectioned and followed by staining with H&E.
The third passage stromal cells of the cornea were seeded onto the stroma side of the APCS (the surface adjacent to the epithelium side was down) at a density of 5×104 cells/cm2 with the same protocol mentioned above. The culture medium was DMEM. After culture for 5 days, the cellscaffold constructs were fixed in 40g/L paraformaldehyde, paraffin embedded, then sectioned and followed by staining with H&E.
RESULTS
Growth of the Corneal Epithelium Cells on APCS in vitroThe cells adhered to the surface of APCS after 24 hours, elongated and presented polygon mostly at 3 days (Figure 1A) and confluenced like the “slabstone” at 10 days. The section of H&E showed the cells grew on the scaffold of APCS in 23 layers at 10 days (Figure 1B).
Growth of the Corneal Stromal Cells on APCS in vitro The stromal cells adhered to the surface of the scaffold after 24 hours and elongated at 3 days (Figure 1C). The cells invaded into the interlaminar of material at 5 days (Figure 1D).
[1] [2] 下一页 |
