PROLIFERATION AND CHONDROGENIC DIFFERENTIATION OF PRECARTILAGINOUS STEM CELLS IN SELFASSEMBLING PEPTIDE NANOFIBER SCAFFOLDS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LUO Wen ¹ , FAN Jiannan ² , YE Chuan ²

1Graduate School of Guiyang Medical University, Guiyang Guizhou, 550004, P.R.China;;
2Department of Orthopedics, Affiliated Hospital of Guiyang Medical University. Corresponding author: LUO Wen, E-mail: rovine1984@yahoo.com.cn;

Corresponding author：

Keywords：

Tissue engineered scaffold; Self-assembl ing peptide nanofiber scaffold; Precartilaginous stem cells; Articular cartilage defect repair

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Objective To construct a new type of self-assembling peptide nanofiber scaffolds—RGDmx, and to study the cell compatibility of the new scaffolds and the proliferation and chondrogenic differentiation of precartilaginous stem cells
(PSCs) in scaffolds. Methods PSCs were separated and purified from newborn Sprague Dawley rats by magnetic activated cell sorting and indentified by immunohistochemistry and immunofluorescent staining. The RGDmx were constructed by mixing KLD-12 and KLD-12-PRG at volume ratio of 1 ∶ 1. PSCs at passage 3 were seeded into the KLD-12 scaffold (control group) and RGDmx scaffold (experimental group). The proliferation of PSCs in 2 groups were observed with the method of cell counting kit (CCK) -8 after 1, 3, 7, and 14 days after culture. The RGDmx were constructed by mixing KLD-12-PRG and KLD-12 at different volume ratios of 0, 20%, 40%, 60%, 80%, and 100% and the prol iferation of PSCs was also observed. The complete chondrogenic medium (CCM) was used to induce chondrogenic differentiation of PSCs in different scaffolds. The differentiation of PSCs was observed by toluidine blue staining and RT-PCR assay. Results PSCs were separated and purified successfully, which were identified by immunohistochemistry and immunofluorescent staining methods. The results of CCK-8 showed that the absorbance (A) value in the experimental group increased gradually and reached the highest at 7 days; the A value in the experimental group was significantly higher than that in the control group at 7 days and 14 days (P lt; 0.05). Meanwhile, the A value in the RGDmx scaffold with a volume ratio of 40% was significantly higher than those in others (P lt; 0.05). After 14 days of induction culture with CCM, the toluidine blue staining results were positive in 2 groups; the results of RT-PCR showed
that the expression levels of collagen type II and the aggrecan in the experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion The self-assembling peptide nanofiber scaffold—RGDmx is an ideal scaffold for tissue engineer because it has good cell compatibility and more effective properties of promoting the differentiation of PSCs to chondrocytes.

Citation： LUO Wen,FAN Jiannan,YE Chuan. PROLIFERATION AND CHONDROGENIC DIFFERENTIATION OF PRECARTILAGINOUS STEM CELLS IN SELFASSEMBLING PEPTIDE NANOFIBER SCAFFOLDS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(12): 1505-1511. doi: Copy

1.	Zhang S, Holmes TC, DiPersio CM, et al. Self-complementary oligopeptide matrices support mammalian cell attachment. Biomaterials, 1995, 16(18): 1385-1393.
2.	Giorgio C, Patricia S, Ehud G. Peptide self-assembly at the nanoscale: a challenging target for computational and experimental biotechnology. Trends Biochem, 2007, 25(5): 211-218.
3.	Davis ME, Motion JP, Narmoneva DA, et al. Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells. Circulation, 2005, 111(4): 442-450.
4.	Ma Z, Kotaki M, Inai R, et al. Potential of nanofiber matrix as tissue-engineering scaffolds. Tissue Eng, 2005, 11(1-2): 101-109.
5.	Jung Y, Kim SH, Kim YH, et al. The effects of dynamic and three-dimensional environments on chondrogenic differentiation of bone marrow stromal cells. Biomed Mater, 2009, 4(5): 055009.
6.	Rosenberg MD. Cell guidance by alterations in monomolecular films. Science, 1963, 139(3553): 411-412.
7.	Shin H, Zygourakis K, Farach-Carson MC, et al. Attachment, proliferation, and migration of marrow stromal osteoblasts cultured on biomimetic hydrogels modified with an osteopontin-derived peptide. Biomaterials, 2004, 25(5): 895-906.
8.	Hayashibara T, Hiraga T, Yi B, et al. A synthetic peptide fragment of human MEPE stimulates new bone formation in vitro and in vivo. J Bone Miner Res, 2004, 19(3): 455-462.
9.	Shin H, Jo S, Mikos AG. Biomimetic materials for tissue engineering. Biomaterials, 2003, 24(24): 4353-4364.
10.	Shin H, Jo S, Mikos AG. Modulation of marrow stromal osteoblast adhesion on biomimetic oligo[poly (ethylene glycol) fumarate] hydrogels modified with Arg-Gly-Asp peptides and a poly (ethyleneglycol) spacer. J Biomed Mater Res, 2002, 61(2): 169-179.
11.	Alsberg E, Anderson KW, Albeiruti A, et al. Cell-interactive alginate hydrogels for bone tissue engineering. J Dent Res, 2001, 80(11): 2025-2029.
12.	Roberts C, Chen CS, Mrksich M, et al. Using mixed self-assembled monolayers presenting RGD and (EG)3OH groups to characterize long-term attachment of bovine capillary endothelial cells to surfaces. J Am Chem Soc, 1998, 120(26): 6548-6555.
13.	Robinson D, Hasharoni A, Cohen N, et al. Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells. Clin Orthop Relat Res, 1999, (367 Suppl): S163-175.
14.	郭风劲, 陈安民, 黄仕龙. 免疫分选软骨前体细胞并诱导永生化的研究. 中华实验外科杂志, 2007, 24(2): 226-228.
15.	Horii A, Wang X, Gelain F, et al. Biological designer self-Assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration. PLoS ONE, 2007, 2(2): e190.
16.	Yokoi H, Kinoshita T, Zhang S. Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc Natl Acad Sci U S A, 2005, 102(24): 8414-8419.
17.	张兰兰, 宋鸿, 赵晓军. Self-assembling short-peptide hydrogel for three-dimensional culture of rabbit articular chondrocytes in vitro. 中国组织工程研究与临床康复, 2008, 12(49): 9779-9782.
18.	Barry F, Boynton RE, Liu B, et al. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res, 2001, 268(2): 189-200.
19.	游洪波, 陈安民. 增强型绿色荧光蛋白基因转染及TGFβ3诱导前软骨干细胞向成软骨方向分化. 中华小儿外科杂志, 2004, 25(6): 545-549.
20.	孙凯. TGF-β3诱导大鼠前软骨干细胞向软骨细胞特性方向分化及其在KLD-12自组装肽纳米凝胶中的培养. 武汉: 华中科技大学, 2010.
21.	游洪波, 陈安民, 程浩. 免疫磁性细胞分选技术分离纯化新生大鼠骨骺前软骨干细胞. 中华创伤杂志, 2004, 20(10): 606-608.
22.	Sun J, Zheng Q. Experimental study on self-assembly of KLD-12 peptide hydrogel and 3-D culture of MSC encapsulated within hydrogel in vitro. J Huazhong Univ Sci Technol Med Sci, 2009, 29(4): 512-516.
23.	Wei X, Gao J, Messner K. Maturation-dependent repair of untreated osteochondrol defects in the rabbit knee. J Biomed Mat Res, 1997, 34(1): 63-72.
24.	Zhang S, Holmes T, Lockshin C, et al. Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc Natl Acad Sci U S A, 2003, 90(8): 3334-3338.
25.	Tatebe M, Nakamura R, Kagami H, et al. Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit. Cytotherapy, 2005, 7(6): 520-530.
26.	Jin XB, Luo ZJ, Wang J. Treatment of rabbit growth plate injuries with an autologous tissue-engineered composite. An experimental study. Cells Tissues Organs, 2006, 183(2): 62-67.
27.	黄志刚, 李峰, 游洪波, 等. 转化生长因子-β3 诱导大鼠前软骨干细胞成软骨分化的量效作用. 华中医学杂志, 2008, 32(3): 185-187.
28.	de Wynter EA, Coutinbo LH, Pei X, et al. Comparison of purity and enrichment of CD34+ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems. Stem Cells, 1995, 13(5): 524-532.
29.	Zhang S. Emerging biological materials through molecular self-assembly. Biotechnol Adv, 2002, 20(5-6): 321-339.
30.	Holmes T, de Lacalle S, Su X, et al. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Pro Natl Acad Sci U S A, 2000, 97(12): 6728-6733.
31.	Aggeli A, Bell M, Boden N, et al. Responsive gels formed by the spontaneous self-assembly of peptides into polymeric beta-sheet tapes. Nature, 1997, 386(6622): 259-262.
32.	Gelain F, Bottai D, Vescovi A, et al. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PLoS One, 2006, 1: e119.
33.	Bokhari MA, Akay G, Zhang S, et al. The enhancement of osteoblast growth and differentiation in vitro on a peptide hydrogel-polyHIPE polymer hybrid material. Biomaterials, 2005, 26(25): 5198-5208.
34.	Kisiday J, Jin M, Kurz B, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair. Proc Natl Acad Sci U S A, 2002, 99(15): 9996-10001.
35.	张衣北, 陈安民, 郭风劲, 等. 骨骺干细胞的体外培养以及生长特性观察. 中国康复, 2006, 2(1): 79-82.

1. Zhang S, Holmes TC, DiPersio CM, et al. Self-complementary oligopeptide matrices support mammalian cell attachment. Biomaterials, 1995, 16(18): 1385-1393.
2. Giorgio C, Patricia S, Ehud G. Peptide self-assembly at the nanoscale: a challenging target for computational and experimental biotechnology. Trends Biochem, 2007, 25(5): 211-218.
3. Davis ME, Motion JP, Narmoneva DA, et al. Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells. Circulation, 2005, 111(4): 442-450.
4. Ma Z, Kotaki M, Inai R, et al. Potential of nanofiber matrix as tissue-engineering scaffolds. Tissue Eng, 2005, 11(1-2): 101-109.
5. Jung Y, Kim SH, Kim YH, et al. The effects of dynamic and three-dimensional environments on chondrogenic differentiation of bone marrow stromal cells. Biomed Mater, 2009, 4(5): 055009.
6. Rosenberg MD. Cell guidance by alterations in monomolecular films. Science, 1963, 139(3553): 411-412.
7. Shin H, Zygourakis K, Farach-Carson MC, et al. Attachment, proliferation, and migration of marrow stromal osteoblasts cultured on biomimetic hydrogels modified with an osteopontin-derived peptide. Biomaterials, 2004, 25(5): 895-906.
8. Hayashibara T, Hiraga T, Yi B, et al. A synthetic peptide fragment of human MEPE stimulates new bone formation in vitro and in vivo. J Bone Miner Res, 2004, 19(3): 455-462.
9. Shin H, Jo S, Mikos AG. Biomimetic materials for tissue engineering. Biomaterials, 2003, 24(24): 4353-4364.
10. Shin H, Jo S, Mikos AG. Modulation of marrow stromal osteoblast adhesion on biomimetic oligo[poly (ethylene glycol) fumarate] hydrogels modified with Arg-Gly-Asp peptides and a poly (ethyleneglycol) spacer. J Biomed Mater Res, 2002, 61(2): 169-179.
11. Alsberg E, Anderson KW, Albeiruti A, et al. Cell-interactive alginate hydrogels for bone tissue engineering. J Dent Res, 2001, 80(11): 2025-2029.
12. Roberts C, Chen CS, Mrksich M, et al. Using mixed self-assembled monolayers presenting RGD and (EG)3OH groups to characterize long-term attachment of bovine capillary endothelial cells to surfaces. J Am Chem Soc, 1998, 120(26): 6548-6555.
13. Robinson D, Hasharoni A, Cohen N, et al. Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells. Clin Orthop Relat Res, 1999, (367 Suppl): S163-175.
14. 郭风劲, 陈安民, 黄仕龙. 免疫分选软骨前体细胞并诱导永生化的研究. 中华实验外科杂志, 2007, 24(2): 226-228.
15. Horii A, Wang X, Gelain F, et al. Biological designer self-Assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration. PLoS ONE, 2007, 2(2): e190.
16. Yokoi H, Kinoshita T, Zhang S. Dynamic reassembly of peptide RADA16 nanofiber scaffold. Proc Natl Acad Sci U S A, 2005, 102(24): 8414-8419.
17. 张兰兰, 宋鸿, 赵晓军. Self-assembling short-peptide hydrogel for three-dimensional culture of rabbit articular chondrocytes in vitro. 中国组织工程研究与临床康复, 2008, 12(49): 9779-9782.
18. Barry F, Boynton RE, Liu B, et al. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res, 2001, 268(2): 189-200.
19. 游洪波, 陈安民. 增强型绿色荧光蛋白基因转染及TGFβ3诱导前软骨干细胞向成软骨方向分化. 中华小儿外科杂志, 2004, 25(6): 545-549.
20. 孙凯. TGF-β3诱导大鼠前软骨干细胞向软骨细胞特性方向分化及其在KLD-12自组装肽纳米凝胶中的培养. 武汉: 华中科技大学, 2010.
21. 游洪波, 陈安民, 程浩. 免疫磁性细胞分选技术分离纯化新生大鼠骨骺前软骨干细胞. 中华创伤杂志, 2004, 20(10): 606-608.
22. Sun J, Zheng Q. Experimental study on self-assembly of KLD-12 peptide hydrogel and 3-D culture of MSC encapsulated within hydrogel in vitro. J Huazhong Univ Sci Technol Med Sci, 2009, 29(4): 512-516.
23. Wei X, Gao J, Messner K. Maturation-dependent repair of untreated osteochondrol defects in the rabbit knee. J Biomed Mat Res, 1997, 34(1): 63-72.
24. Zhang S, Holmes T, Lockshin C, et al. Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc Natl Acad Sci U S A, 2003, 90(8): 3334-3338.
25. Tatebe M, Nakamura R, Kagami H, et al. Differentiation of transplanted mesenchymal stem cells in a large osteochondral defect in rabbit. Cytotherapy, 2005, 7(6): 520-530.
26. Jin XB, Luo ZJ, Wang J. Treatment of rabbit growth plate injuries with an autologous tissue-engineered composite. An experimental study. Cells Tissues Organs, 2006, 183(2): 62-67.
27. 黄志刚, 李峰, 游洪波, 等. 转化生长因子-β3 诱导大鼠前软骨干细胞成软骨分化的量效作用. 华中医学杂志, 2008, 32(3): 185-187.
28. de Wynter EA, Coutinbo LH, Pei X, et al. Comparison of purity and enrichment of CD34+ cells from bone marrow, umbilical cord and peripheral blood (primed for apheresis) using five separation systems. Stem Cells, 1995, 13(5): 524-532.
29. Zhang S. Emerging biological materials through molecular self-assembly. Biotechnol Adv, 2002, 20(5-6): 321-339.
30. Holmes T, de Lacalle S, Su X, et al. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds. Pro Natl Acad Sci U S A, 2000, 97(12): 6728-6733.
31. Aggeli A, Bell M, Boden N, et al. Responsive gels formed by the spontaneous self-assembly of peptides into polymeric beta-sheet tapes. Nature, 1997, 386(6622): 259-262.
32. Gelain F, Bottai D, Vescovi A, et al. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PLoS One, 2006, 1: e119.
33. Bokhari MA, Akay G, Zhang S, et al. The enhancement of osteoblast growth and differentiation in vitro on a peptide hydrogel-polyHIPE polymer hybrid material. Biomaterials, 2005, 26(25): 5198-5208.
34. Kisiday J, Jin M, Kurz B, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair. Proc Natl Acad Sci U S A, 2002, 99(15): 9996-10001.
35. 张衣北, 陈安民, 郭风劲, 等. 骨骺干细胞的体外培养以及生长特性观察. 中国康复, 2006, 2(1): 79-82.

Chinese Journal of Reparative and Reconstructive Surgery

PROLIFERATION AND CHONDROGENIC DIFFERENTIATION OF PRECARTILAGINOUS STEM CELLS IN SELFASSEMBLING PEPTIDE NANOFIBER SCAFFOLDS

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