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.	荊恒,陳欣,李寧毅.細胞片層技術的研究進展.國際口腔醫學雜志,2010,37(1):71-73.
3.	荊恒,李寧毅,譚帥,等.骨髓基質干細胞的體外培養及其細胞片層制備.國際口腔醫學雜志,2010,37(3):272-274.
4.	王月田,崔穎,潘欣宇,等.重組hCDMP1腺病毒轉染骨髓間充質干細胞對其向軟骨分化的影響.重慶醫學,2012,41(16):1570-1571,1574.
5.	劉永亮,葉鋼,易善紅,等.BMP7和EGFP重組腺病毒體外轉染兔骨髓間充質干細胞的研究.四川醫學,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.	馬艷琴,孫子龍,王彬,等.Western blot檢測家兔動脈組織蛋白質表達條件的優化.農學學報,2011,1(4):52-56.
11.	張柏青,張仲文,葉啟彬.基質誘導自體軟骨細胞移植與軟骨缺損的修復.中國矯形外科雜志,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.	趙貴慶,崔磊,曹誼林.軟骨形態發生蛋白1誘導犬真皮成纖維細胞向軟骨細胞表型分化的實驗研究.組織工程與重建外科雜志,2011,7(3):139-142,150.
20.	劉瑾春,劉霞,曹誼林.體外誘導BMSCs向軟骨分化的方法研究進展.中國修復重建外科雜志,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. 荊恒,陳欣,李寧毅.細胞片層技術的研究進展.國際口腔醫學雜志,2010,37(1):71-73.
3. 荊恒,李寧毅,譚帥,等.骨髓基質干細胞的體外培養及其細胞片層制備.國際口腔醫學雜志,2010,37(3):272-274.
4. 王月田,崔穎,潘欣宇,等.重組hCDMP1腺病毒轉染骨髓間充質干細胞對其向軟骨分化的影響.重慶醫學,2012,41(16):1570-1571,1574.
5. 劉永亮,葉鋼,易善紅,等.BMP7和EGFP重組腺病毒體外轉染兔骨髓間充質干細胞的研究.四川醫學,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. 馬艷琴,孫子龍,王彬,等.Western blot檢測家兔動脈組織蛋白質表達條件的優化.農學學報,2011,1(4):52-56.
11. 張柏青,張仲文,葉啟彬.基質誘導自體軟骨細胞移植與軟骨缺損的修復.中國矯形外科雜志,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. 趙貴慶,崔磊,曹誼林.軟骨形態發生蛋白1誘導犬真皮成纖維細胞向軟骨細胞表型分化的實驗研究.組織工程與重建外科雜志,2011,7(3):139-142,150.
20. 劉瑾春,劉霞,曹誼林.體外誘導BMSCs向軟骨分化的方法研究進展.中國修復重建外科雜志,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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