EXPERIMENTAL STUDY ON THREE DIMENSINONAL CULTURE OF RABBIT ANNULUS FIBROSUS CELLS ON KLD-12 POLYPEPTIDE NANOFIBER GEL IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIANGXiangchen ¹ ,  SUNJianhua ² , BIANZhengjun ¹ , SHAOHui ¹ , HUANGZhaoqing ¹ , LIXiangchen ¹ , YUYang ¹

1. Shihezi University School of Medicine, Shihezi Xinjiang, 832008, P.R.China;
2. No.3 Department of Orthopedics, the First Affiliated Hospital of the Medical College, Shihezi University;

Corresponding author：

SUNJianhua, Email: 758719985@qq.com

Keywords：

Intervertebral disc degeneration; Tissue engineering; Annulus fibrosus cells; KLD-12 polypeptide; Rabbit

DOI：

10.7507/1002-1892.20160060

Video：

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Objective To investigate the feasibility to culture rabbit annulus fibrosus cells on the KLD-12 polypeptide nanofiber gel so as to search for the seed cells and the scaffolds for tissue engineering. Methods The rabbit annulus fibrosus cells were isolated with pancreatin and cultured; the cells at passage 3 were seeded on the KLD-12 polypeptide nanofiber gel to prepare the KLD-12 polypeptide/annulus fibrosus cells gel. The cell morphology change was observed by inverted microscope. The cell counting kit 8 (CCK-8) was used to detect the cell proliferation, and Calcein-AM/propidium iodide (PI) fluorescent staining to observe the cell vitality. The alcian blue method was used to measure the glycosaminoglycan (GAG) content, immunofluorescence technique to observe the collagen type II level, and real-time fluorescence quantitative PCR (RT-qPCR) to measure the mRNA expressions of Aggrecan and collagen type II. Results The cells on the scaffolds grew well, showing round shape on the scaffolds and spindle or fusiform shape at the edge of the scaffold. The cell proliferation exhibited increasing trend with time, and it was significantly higher at 14 days than the other time points (P < 0.05), and on KLD-12 polypeptide nanofiber gel than on blank gel (P < 0.05). The ratios of living cells were 89.32%±8.58% at 5 days and 97.81%±1.09% at 14 days, showing no significant difference (t=-1.962, P=0.097). The GAG content gradually increased with culture time, reached the peak at 8 days, and then gradually decreased; the GAG content at 5, 8, and 11 days was significantly higher than that at 2 and 14 days (P < 0.05). The level of collagen type II was normal. The mRNA expressions of collagen type II and Aggrecan could be measured at 5 and 14 days; the relative expression levels of collagen type II and Aggrecan mRNA were significantly higher at 14 days than 5 days (P < 0.05). Conclusion The rabbit annulus fibrosus cells on KLD-12 polypeptide nanofiber gel are able to grow well and to produce extracellular matrix, so KLD-12 polypeptide nanofiber gel has the potential to serve as a scaffold for the treatment of intervertebral disc degeneration.

Citation： LIANGXiangchen, SUNJianhua, BIANZhengjun, SHAOHui, HUANGZhaoqing, LIXiangchen, YUYang. EXPERIMENTAL STUDY ON THREE DIMENSINONAL CULTURE OF RABBIT ANNULUS FIBROSUS CELLS ON KLD-12 POLYPEPTIDE NANOFIBER GEL IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(3): 303-308. doi: 10.7507/1002-1892.20160060 Copy

1.	Samartzis D, Karppinen J, Mok F, et al. A population-based study of juvenile disc degeneration and its association with overweight and obesity, low back pain, and diminished functional status. J Bone Joint Surg (Am), 2011, 93(7): 662-670.
2.	Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
3.	Gruber HE, Hanley EN Jr. Analysis of aging and degeneration of the human intervertebral disc: comparison of Surgical specimens with normal controls. Spine (Phila Pa 1976), 1998, 23(7): 751-757.
4.	Rannou F, Lee TS, Zhou RH, et al. Intervertebral disc degeneration: the role of the mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload. J Am J Pathol, 2004, 164(3): 915-924.
5.	Li J, Liu C, Guo Q, et al. Regional variations in the cellular, biochemical, and biomechanical characteristics of rabbit annulus fiborous. PLoS One, 2014, 9(3): e91799.
6.	谢健, 童培建, 肖鲁伟, 等.兔髓核与纤维环细胞生物学特性差异的研究.中国骨伤, 2013, 26(6): 481-485.
7.	Sun J, Zheng Q, Wu Y, et al. Biocompatibility of KLD-12 peptide hydrogel as a scaffold in tissue engineering of intervertebral discs in rabbits. J Huazhong Univ Sci Technol Med Sci, 2010, 30(2): 173-177.
8.	Sun JH, Zheng QX, Wu YC, et al. Culture of nucleus pulposus cells from intervertebral disc on self-assembling KLD-12 peptide hydrogel scaffold. Materials Science and Engineering C, 2010, 30(7): 975-980.
9.	Zhang Lan-lan, Song Hong, Zhao Xiao-jun. 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.
10.	Xie M, Yang S, Win HL, et al. Rabbit annulus fibrosus cell apoptosis induced by mechanical overload via a mitochondrial apoptotic pathway. J Huazhong Univ Sci Technolog Med Sci, 2010, 30(3): 379-384.
11.	Bian Z, Sun J. Development of a KLD-12 polypeptide/TGF-β1-tissue scaffold promoting the differentiation of mesenchymal stem cell into nucleus pulposus-like cells for treatment of intervertebral disc degeneration. J Int J Clin Exp Pathol, 2015, 8(2): 1093-1103.
12.	Schleich C, Müller-Lutz A, Matuschke F, et al. Glycosaminoglycan chemical exchange saturation transfer of lumbar intervertebral discs in patients with spondyloarthritis. J Magn Reson Imaging, 2015, 42(4): 1057-1063.
13.	Lee P, Tran K, Chang W, et al. Influence of chondroitin sulfate and hyaluronic acid presence in nanofibers and its alignment on the bone marrow stromal cells: cartilage regeneration. J Biomed Nanotechnol, 2014, 10(8): 1469-1479.
14.	Haschtmann D, Ferguson SJ, Stoyanov JV. BMP-2 and TGF-β3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossification of the annulus fibrosus. J Eur Spine J, 2012, 21(9): 1724-1733.
15.	Turner KG, Ahmed N, Santerre JP, et al. Modulation of annulus fibrosus cell alignment and function on oriented nanofibrous polyurethane scaffolds under tension. Spine J, 2014, 14(3): 424-434.
16.	Colombini A, Lopa S, Ceriani C, et al. In vitro characterization and in vivo behavior of human nucleus pulposus and annulus fibrosus cells in clinical-grade fibrin and collagen-enriched fibrin gels. Tissue Eng Part A, 2015, 21(3-4): 793-802.
17.	Saad L, Spector M. Effects of collagen type on the behavior of adult canine annulus fibrosus cells in collagen-glycosaminoglycan scaffolds. J Biomed Mater Res A, 2004, 71(2): 233-241.
18.	Reza AT, Nicoll SB. Hydrostatic pressure differentially regulates outer and inner annulus fibrosus cell matrix production in 3D scaffolds. Ann Biomed Eng, 2008, 36(2): 204-213.
19.	Iu J, Santerre JP, Kandel RA. Inner and outer annulus fibrosus cells exhibit differentiated phenotypes and yield changes in extracellular matrix protein composition in vitro on a polycarbonate urethane scaffold. Tissue Eng Part A, 2014, 20(23-24): 3261-3269.
20.	Xu B, Xu H, Wu Y, et al. Intervertebral disc tissue engineering with natural extracellular matrix-derived biphasic composite scaffolds. PLoS One, 2015, 10(4): e0124774.
21.	Jiang X, Wang K, Ding M, et al. Quantitative grafting of peptide onto the nontoxic biodegradable waterborne polyurethanes to fabricate peptide modified scaffold for soft tissue engineering. J Mater Sci Mater Med, 2011, 22(4): 819-827.
22.	Raghunath J, Georgiou G, Armitage D, et al. Degradation studies on biodegradable nanocomposite based on poly caprolactone/ polycarbonate (80:20%) polyhedral oligomeric silsesquioxane. J Biomed Mater Res A, 2009, 91(3): 834-844.
23.	Jin KM, Kurz B, Huang H, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix pruduction and cell division: implications for catilage tissue repair. J Proc Natl Acad Sci USA, 2002, 99(15): 9996-10001.
24.	Shi DH, Cai DZ, Zhou CR, et al. Development and potential of a biomimetic chitosan/type II collagen scaffold for cartilage tissue engineering. J Chin Med J (Engl), 2005, 118(17): 1436-1443.

1. Samartzis D, Karppinen J, Mok F, et al. A population-based study of juvenile disc degeneration and its association with overweight and obesity, low back pain, and diminished functional status. J Bone Joint Surg (Am), 2011, 93(7): 662-670.
2. Buckwalter JA. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976), 1995, 20(11): 1307-1314.
3. Gruber HE, Hanley EN Jr. Analysis of aging and degeneration of the human intervertebral disc: comparison of Surgical specimens with normal controls. Spine (Phila Pa 1976), 1998, 23(7): 751-757.
4. Rannou F, Lee TS, Zhou RH, et al. Intervertebral disc degeneration: the role of the mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload. J Am J Pathol, 2004, 164(3): 915-924.
5. Li J, Liu C, Guo Q, et al. Regional variations in the cellular, biochemical, and biomechanical characteristics of rabbit annulus fiborous. PLoS One, 2014, 9(3): e91799.
6. 谢健, 童培建, 肖鲁伟, 等.兔髓核与纤维环细胞生物学特性差异的研究.中国骨伤, 2013, 26(6): 481-485.
7. Sun J, Zheng Q, Wu Y, et al. Biocompatibility of KLD-12 peptide hydrogel as a scaffold in tissue engineering of intervertebral discs in rabbits. J Huazhong Univ Sci Technol Med Sci, 2010, 30(2): 173-177.
8. Sun JH, Zheng QX, Wu YC, et al. Culture of nucleus pulposus cells from intervertebral disc on self-assembling KLD-12 peptide hydrogel scaffold. Materials Science and Engineering C, 2010, 30(7): 975-980.
9. Zhang Lan-lan, Song Hong, Zhao Xiao-jun. 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.
10. Xie M, Yang S, Win HL, et al. Rabbit annulus fibrosus cell apoptosis induced by mechanical overload via a mitochondrial apoptotic pathway. J Huazhong Univ Sci Technolog Med Sci, 2010, 30(3): 379-384.
11. Bian Z, Sun J. Development of a KLD-12 polypeptide/TGF-β1-tissue scaffold promoting the differentiation of mesenchymal stem cell into nucleus pulposus-like cells for treatment of intervertebral disc degeneration. J Int J Clin Exp Pathol, 2015, 8(2): 1093-1103.
12. Schleich C, Müller-Lutz A, Matuschke F, et al. Glycosaminoglycan chemical exchange saturation transfer of lumbar intervertebral discs in patients with spondyloarthritis. J Magn Reson Imaging, 2015, 42(4): 1057-1063.
13. Lee P, Tran K, Chang W, et al. Influence of chondroitin sulfate and hyaluronic acid presence in nanofibers and its alignment on the bone marrow stromal cells: cartilage regeneration. J Biomed Nanotechnol, 2014, 10(8): 1469-1479.
14. Haschtmann D, Ferguson SJ, Stoyanov JV. BMP-2 and TGF-β3 do not prevent spontaneous degeneration in rabbit disc explants but induce ossification of the annulus fibrosus. J Eur Spine J, 2012, 21(9): 1724-1733.
15. Turner KG, Ahmed N, Santerre JP, et al. Modulation of annulus fibrosus cell alignment and function on oriented nanofibrous polyurethane scaffolds under tension. Spine J, 2014, 14(3): 424-434.
16. Colombini A, Lopa S, Ceriani C, et al. In vitro characterization and in vivo behavior of human nucleus pulposus and annulus fibrosus cells in clinical-grade fibrin and collagen-enriched fibrin gels. Tissue Eng Part A, 2015, 21(3-4): 793-802.
17. Saad L, Spector M. Effects of collagen type on the behavior of adult canine annulus fibrosus cells in collagen-glycosaminoglycan scaffolds. J Biomed Mater Res A, 2004, 71(2): 233-241.
18. Reza AT, Nicoll SB. Hydrostatic pressure differentially regulates outer and inner annulus fibrosus cell matrix production in 3D scaffolds. Ann Biomed Eng, 2008, 36(2): 204-213.
19. Iu J, Santerre JP, Kandel RA. Inner and outer annulus fibrosus cells exhibit differentiated phenotypes and yield changes in extracellular matrix protein composition in vitro on a polycarbonate urethane scaffold. Tissue Eng Part A, 2014, 20(23-24): 3261-3269.
20. Xu B, Xu H, Wu Y, et al. Intervertebral disc tissue engineering with natural extracellular matrix-derived biphasic composite scaffolds. PLoS One, 2015, 10(4): e0124774.
21. Jiang X, Wang K, Ding M, et al. Quantitative grafting of peptide onto the nontoxic biodegradable waterborne polyurethanes to fabricate peptide modified scaffold for soft tissue engineering. J Mater Sci Mater Med, 2011, 22(4): 819-827.
22. Raghunath J, Georgiou G, Armitage D, et al. Degradation studies on biodegradable nanocomposite based on poly caprolactone/ polycarbonate (80:20%) polyhedral oligomeric silsesquioxane. J Biomed Mater Res A, 2009, 91(3): 834-844.
23. Jin KM, Kurz B, Huang H, et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix pruduction and cell division: implications for catilage tissue repair. J Proc Natl Acad Sci USA, 2002, 99(15): 9996-10001.
24. Shi DH, Cai DZ, Zhou CR, et al. Development and potential of a biomimetic chitosan/type II collagen scaffold for cartilage tissue engineering. J Chin Med J (Engl), 2005, 118(17): 1436-1443.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON THREE DIMENSINONAL CULTURE OF RABBIT ANNULUS FIBROSUS CELLS ON KLD-12 POLYPEPTIDE NANOFIBER GEL IN VITRO

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