PRELIMINARY STUDY ON TRANSGENIC CELL SHEET WITH CARTILAGE-DERIVED MORPHOGENETIC PROTEIN 1_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YAOMei ¹ ,  CUIYing ² , WANGYuetian ¹ , ZHANGBen ² , FUWenju ¹

1. Graduate School of Liaoning Medical College, Jinzhou Liaoning, 121000, P. R. China;
2. Department of Otolaryngology, the First Affiliated Hospital of Liaoning Medical College, Jinzhou Liaoning, 121000, P. R. China;

Corresponding author：

CUIYing, Email: yingwu2002@163.com

Keywords：

Cartilage tissue engineering; Cell sheet; Cartilage-derived morphogenetic protein 1; Bone marrow mesenchymal stem cells; Adenovirus transfection

DOI：

10.7507/1002-1892.20140033

Video：

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Objective To construct a transgenic cell sheet of cartilage-derived morphogenetic protein 1 (CDMP-1) by adenovirus vector in vitro and to identify its biological activity. Methods The bone mesenchymal stem cells (BMSCs) were isolated from bone marrow of 1-month-old rabbit, and cultured in vitro. The 3rd-6th generation of BMSCs were used for experiment. The experiment was divided into 3 groups:BMSCs transfected by adenovirus (Ad)-cytomegalovirus (CMV)-human CDMP1 (hCDMP1)-internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) in group A, BMSCs transfected by Ad-CMV-EGFP in group B, and untransfected BMSCs in group C. The expression of green fluorescence was observed in 3 groups under fluorescent inverted microscope. MTT assay was used to detect the proliferation of the cells. The cell sheet was obtained by means of temperature-responsive culture dish for 14 days. The morphological and HE staining observations of the cell sheet were carried out. RT-PCR and Western blot were used to detect the expressions of hCDMP1 and collagen type II at gene and protein levels, while alcian blue staining was used to detect the expression of glycosaminoglycans (GAG). Results Bright green fluorescence was observed in transfected cells at 72 hours under fluorescent inverted microscope, and the transfection efficiency was up to 90%. MTT assay showed approximate S-shaped growth curves in 3 groups, showing no significant difference in the absorbance (A) value among 3 groups within 9 days (P>0.05). The three-dimensional cell sheets were successfully harvested in vitro. The RT-PCR and Western blot showed that there were positive expressions of hCDMP1 and collagen type II in group A and negative expression in other 2 groups. HE staining and alcian blue staining showed that there were rich fibrous tissues, mass extracellular matrix, and dark blue metachromatic granules in group A, but there was less fibrous tissues and no specific blue metachromatic granules in other 2 groups; and the positive expression area was significantly lower and gray scale of GAG was significantly higher in group A than that in groups B and C (P<0.05). Conclusion A transgenic cell sheet of exogenous recombinant hCDMP1 by adenovirus vector can express collagen type II and GAG, so it has chondrogenic capacity. This technology that overcomes limitations in traditional tissue engineering, such as low cell-attachment efficiency and inflammatory reaction, may be a new tissue engineering approach for hard tissue reconstruction and is hopeful to build a large density of tissue engineered cartilage.

Citation： YAOMei, CUIYing, WANGYuetian, ZHANGBen, FUWenju. PRELIMINARY STUDY ON TRANSGENIC CELL SHEET WITH CARTILAGE-DERIVED MORPHOGENETIC PROTEIN 1. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(2): 142-148. doi: 10.7507/1002-1892.20140033 Copy

1.	Hayashida Y,Nishida K,Yamato M,et al.Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze.Invest Ophthalmol Vis Sci,2006,47(2):552-557.
2.	荆恒,陈欣,李宁毅.细胞片层技术的研究进展.国际口腔医学杂志,2010,37(1):71-73.
3.	荆恒,李宁毅,谭帅,等.骨髓基质干细胞的体外培养及其细胞片层制备.国际口腔医学杂志,2010,37(3):272-274.
4.	王月田,崔颖,潘欣宇,等.重组hCDMP1腺病毒转染骨髓间充质干细胞对其向软骨分化的影响.重庆医学,2012,41(16):1570-1571,1574.
5.	刘永亮,叶钢,易善红,等.BMP7和EGFP重组腺病毒体外转染兔骨髓间充质干细胞的研究.四川医学,2008,29(2):129-131.
6.	Asakawa N,Shimizu T,Tsuda Y,et al.Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering.Biomaterials,2010,31(14):3903-3909.
7.	Nozaki T,Yamato M,Inuma T,et al.Transportation of transplantable cell sheets fabricated with temperature-responsive culture surfaces for regenerative medicine.J Tissue Eng Regen Med,2008,2(4):190-195.
8.	Kaneshiro N,Sato M,Ishihara M,et al.Cultured articular chondrocytes sheets for partial thickness cartilage defects utilizing temperature-responsive culture dishes.Eur Cell Mater,2007,13:87-92.
9.	Okano T.Cell sheet engineering.Rinsho Shinkeigaku,2006,46(11):795-798.
10.	马艳琴,孙子龙,王彬,等.Western blot检测家兔动脉组织蛋白质表达条件的优化.农学学报,2011,1(4):52-56.
11.	张柏青,张仲文,叶启彬.基质诱导自体软骨细胞移植与软骨缺损的修复.中国矫形外科杂志,2008,16(23):1796-1798.
12.	Cui Y,Wang H,Yu MJ,et al.Differentiation plasticity of human fetal articular chondrocytes.Otolaryngol Head Neck Surg,2006,135(1):61-67.
13.	Bai C,Hou LL,Ma Yh,et al.Isolation and characterization of mesenchymal stem cells from chicken bone marrow.Cell Tissue Bank,2013,14(3):437-451.
14.	Swieszkowski W,Tuan BH,Kurzydlowski KJ,et al.Repair and regeneration of osteochondral defects in the articular joints.Biomol Eng,2007,24(5):489-495.
15.	Wang L,Lin F,Wu J,et al.High eiciency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes.Acta Biochim Biophys Sin (Shanghai),2009,41(4):325-334.
16.	Coleman CM,Tuan RS.Growth differentiation factor 5 enhances chondrocyte maturation.Dev Dyn,2003,228(2):208-216.
17.	Coleman CM,Tuan RS.Functional role of growth differentiation factor 5 in chondrogenesis of limb mesenchymal cells.Mech Dev,2003,120(7):823-836.
18.	Katayama R,Wakitani S,Tsumaki N,et al.Repair of articular cartilage defects in rabbits using CDMP1 gene-transfected autologous mesenchymal cells derived from bone marrow.Rheumatology (Oxford),2004,43(8):980-985.
19.	赵贵庆,崔磊,曹谊林.软骨形态发生蛋白1诱导犬真皮成纤维细胞向软骨细胞表型分化的实验研究.组织工程与重建外科杂志,2011,7(3):139-142,150.
20.	刘瑾春,刘霞,曹谊林.体外诱导BMSCs向软骨分化的方法研究进展.中国修复重建外科杂志,2011,25(5):618-623.
21.	Okano T,Yamada N,Sakai H,et al.A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide).J Biomed Mater Res,1993,27(10):1243-1251.
22.	Hirose M,Kwon OH,Yamato M,et al.Creation of desigene shape cell sheets that are noninvasively harvested and moved onto another surface.Biomacromolecules,2000,1(3):377-381.
23.	Yamato M,Hirose M,Harimoto M,et al.Cell and cell sheet manipulation by utilizing biomaterials-development of cell sheet engineering.Tanpakushitsu Kakusan Koso,2000,45(13 Suppl):2156-2161.
24.	Kwon OH,Kikuchi A,Yamato M,et al.Rapid cell sheet detachment from poly (N-isopropylacrylamide)-grafted porous cell culture membranes.J Biomed Mater Res,2000,50(1):82-89.
25.	Kobayashi H,Shimizu T,Yamato M,et al.Fibroblast sheets co-cultured with endothelial progenitor cells improve cardiac function of infarcted hearts.J Artif Organs,2008,11(3):141-147.
26.	Alshammary S,Fukushima S,Miyagawa S,et al.Impact of cardiac stem cell sheet transplantation on myocardial infarction.Surg Today,2013,43(9):970-976.
27.	Du CH,Li NY,Gao N,et al.A preliminary study on the application of bone marrow stromal cell sheet on the formation of functional tissue-engineered bone in dogs.J Oral Maxillofac Surg,2013,71(9):1531.e1-1531.e10.

1. Hayashida Y,Nishida K,Yamato M,et al.Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze.Invest Ophthalmol Vis Sci,2006,47(2):552-557.
2. 荆恒,陈欣,李宁毅.细胞片层技术的研究进展.国际口腔医学杂志,2010,37(1):71-73.
3. 荆恒,李宁毅,谭帅,等.骨髓基质干细胞的体外培养及其细胞片层制备.国际口腔医学杂志,2010,37(3):272-274.
4. 王月田,崔颖,潘欣宇,等.重组hCDMP1腺病毒转染骨髓间充质干细胞对其向软骨分化的影响.重庆医学,2012,41(16):1570-1571,1574.
5. 刘永亮,叶钢,易善红,等.BMP7和EGFP重组腺病毒体外转染兔骨髓间充质干细胞的研究.四川医学,2008,29(2):129-131.
6. Asakawa N,Shimizu T,Tsuda Y,et al.Pre-vascularization of in vitro three-dimensional tissues created by cell sheet engineering.Biomaterials,2010,31(14):3903-3909.
7. Nozaki T,Yamato M,Inuma T,et al.Transportation of transplantable cell sheets fabricated with temperature-responsive culture surfaces for regenerative medicine.J Tissue Eng Regen Med,2008,2(4):190-195.
8. Kaneshiro N,Sato M,Ishihara M,et al.Cultured articular chondrocytes sheets for partial thickness cartilage defects utilizing temperature-responsive culture dishes.Eur Cell Mater,2007,13:87-92.
9. Okano T.Cell sheet engineering.Rinsho Shinkeigaku,2006,46(11):795-798.
10. 马艳琴,孙子龙,王彬,等.Western blot检测家兔动脉组织蛋白质表达条件的优化.农学学报,2011,1(4):52-56.
11. 张柏青,张仲文,叶启彬.基质诱导自体软骨细胞移植与软骨缺损的修复.中国矫形外科杂志,2008,16(23):1796-1798.
12. Cui Y,Wang H,Yu MJ,et al.Differentiation plasticity of human fetal articular chondrocytes.Otolaryngol Head Neck Surg,2006,135(1):61-67.
13. Bai C,Hou LL,Ma Yh,et al.Isolation and characterization of mesenchymal stem cells from chicken bone marrow.Cell Tissue Bank,2013,14(3):437-451.
14. Swieszkowski W,Tuan BH,Kurzydlowski KJ,et al.Repair and regeneration of osteochondral defects in the articular joints.Biomol Eng,2007,24(5):489-495.
15. Wang L,Lin F,Wu J,et al.High eiciency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes.Acta Biochim Biophys Sin (Shanghai),2009,41(4):325-334.
16. Coleman CM,Tuan RS.Growth differentiation factor 5 enhances chondrocyte maturation.Dev Dyn,2003,228(2):208-216.
17. Coleman CM,Tuan RS.Functional role of growth differentiation factor 5 in chondrogenesis of limb mesenchymal cells.Mech Dev,2003,120(7):823-836.
18. Katayama R,Wakitani S,Tsumaki N,et al.Repair of articular cartilage defects in rabbits using CDMP1 gene-transfected autologous mesenchymal cells derived from bone marrow.Rheumatology (Oxford),2004,43(8):980-985.
19. 赵贵庆,崔磊,曹谊林.软骨形态发生蛋白1诱导犬真皮成纤维细胞向软骨细胞表型分化的实验研究.组织工程与重建外科杂志,2011,7(3):139-142,150.
20. 刘瑾春,刘霞,曹谊林.体外诱导BMSCs向软骨分化的方法研究进展.中国修复重建外科杂志,2011,25(5):618-623.
21. Okano T,Yamada N,Sakai H,et al.A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-isopropylacrylamide).J Biomed Mater Res,1993,27(10):1243-1251.
22. Hirose M,Kwon OH,Yamato M,et al.Creation of desigene shape cell sheets that are noninvasively harvested and moved onto another surface.Biomacromolecules,2000,1(3):377-381.
23. Yamato M,Hirose M,Harimoto M,et al.Cell and cell sheet manipulation by utilizing biomaterials-development of cell sheet engineering.Tanpakushitsu Kakusan Koso,2000,45(13 Suppl):2156-2161.
24. Kwon OH,Kikuchi A,Yamato M,et al.Rapid cell sheet detachment from poly (N-isopropylacrylamide)-grafted porous cell culture membranes.J Biomed Mater Res,2000,50(1):82-89.
25. Kobayashi H,Shimizu T,Yamato M,et al.Fibroblast sheets co-cultured with endothelial progenitor cells improve cardiac function of infarcted hearts.J Artif Organs,2008,11(3):141-147.
26. Alshammary S,Fukushima S,Miyagawa S,et al.Impact of cardiac stem cell sheet transplantation on myocardial infarction.Surg Today,2013,43(9):970-976.
27. Du CH,Li NY,Gao N,et al.A preliminary study on the application of bone marrow stromal cell sheet on the formation of functional tissue-engineered bone in dogs.J Oral Maxillofac Surg,2013,71(9):1531.e1-1531.e10.

Chinese Journal of Reparative and Reconstructive Surgery

PRELIMINARY STUDY ON TRANSGENIC CELL SHEET WITH CARTILAGE-DERIVED MORPHOGENETIC PROTEIN 1

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