Evaluation of the proliferation and viability rates of nucleus pulposus cells of human intervertebral disk in fabricated chitosan-gelatin scaffolds by freeze drying and freeze gelation methods

Karimi, Zeinab; Ghorbani, Masoud; Hashemibeni, Batool; Bahramian, Hamid

Evaluation of the proliferation and viability rates of nucleus pulposus cells of human intervertebral disk in fabricated chitosan-gelatin scaffolds by freeze drying and freeze gelation methods

Authors

¹ Student of Medicine, School of Medicine and Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran

² Applied Biotechnology Researches Center, Pajooheshgah, Baqiatallah University of Medical Sciences; Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

³ Department of Anatomy and Molecular Biology, Medicine School, Isfahan University of Medical Sciences, Tehran, Iran

Abstract

Background: Low back pain is one of the most significant musculoskeletal diseases of our time. Intervertebral disk herniation and central degeneration of the disk are two major reasons for low back pain, which occur because of structural impairment of the disk. The reduction of cell count and extracellular matrix, especially in the nucleus pulposus, causes disk degeneration. Different scaffolds have been used for tissue repairing and regeneration of the intervertebral disk in tissue engineering. Various methods are used for fabrication of the porosity scaffolds in tissue engineering. The freeze drying method has disadvantages such as: It is time consuming, needs high energy, and so on.
The freeze-gelation method can save a great deal of time and energy, and large-sized porous scaffolds can be fabricated by this method. In this study, proliferation of the nucleus pulposus (NP) cells of the human intervertebral disk are compromised in the fabricated Chitosan-gelatin scaffolds by freeze drying and freeze gelation methods.
Materials and Methods: The cells were obtained from the nucleus pulposus by collagenase enzymatic hydrolysis. They were obtained from patients who were undergoing open surgery for discectomy in the Isfahan Alzahra Hospital. Chitosan was blended with gelatin. Chitosan polymer, solution after freezing at -80°C, was immersed in sodium hydroxide (NaOH) solution. The cellular suspension was transferred to each scaffold and cultured in plate for 14 days. Cell viability and proliferation were investigated by Trypan blue and MTT assays.
Results: The MTT and Trypan blue assays demonstrated that cell viability and the mean of the cell number showed a significant difference between three and fourteen days, in both scaffolds. Accordingly, there was a significantly decrease in the fabricated chitosan-gelatin scaffold by the freeze-drying method.
Conclusion: The fabricated chitosan-gelatin scaffold by the freeze-gelation method prepared a better condition for proliferation of NP cells when compared with the fabricated chitosan–gelatin scaffold by the freeze drying method.

Keywords

References

1.	Waddell G. Low back pain: A twentieth century health care enigma. Spine (Phila Pa 1976) 1966;21:2820-5.
2.	Boss N, Rieder R, Schade V, Spartt KF, Semmer N, Aebi M. The diagnostic accuracy of magnetic resonance imaging, work perception, and psychosocial factors in identifying symptomatic descherniations. Spine 1995;20:2613-25.
3.	Oegema TR Jr. The role of disk cell heterogeneity in determining disk biochemistry: A speculation. Biochem Soc Trans 2002;30:839-44.
4.	Hunter CJ, Matyas JR, Duncan NA. The notochordal cell in the nucleus pulposus: A review in the context of tissue engineering. Tissue Eng 2003;9:667-77.
5.	Roberts S, Evans H, Trivedi J. Histology and pathology of the human intervertevral disk. J Bone Joint Surg Am 2006;88:10-4.
6.	Sobajima S, Vadala G, Shimer A, Kim JS, Gilbertson LG, Kang JD. Feasibility of a stem cell therapy for intervertebral disk degeneration. Spine J 2008;8:888-96.
7.	Tabato Y. Recent progress in tissue engineering. Drug Diskov Today 2001;6:483-7.
8.	Tsuchiya K, Chen G, Ushida T, Matsuno T, Tateishi T. The effect of coculture of chondrocytes with mesenchymal stem cells on their cartilaginous phenotype in vitro. Mat Sci Eng 2004;24:391-6.
9.	Bronzino JD. Tissue engineering and artificial organs. 3^rd ed. Vol. 37. The biomedical engineering handbood. Hartford, Connecticut, U.S.A Tylorand Francis; 2007. p. 1-8.
10.	Vunjak-Novakovic G, Freshney RI. Culture of cells for tissue engineering. Vol. 14. New York: Wiley-lis; 2006. p. 131-55.
11.	Haringham T, Tew S, Murdoch A. Tissue engineering: Chondrocyes and cartilage. Arthritis Res 2002;4:63-8.
12.	Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: A review. Biomaterials 2000;21:2589-98.
13.	Khor E, Lim LY. Implantable applications of chitin and chitosan. Biomaterials 2003;24:2339-49.
14.	Laiji A, Sohrabi A, Hungerford DS, Frondoza CG. Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. J Biomed Mater Res 2000;51:586-95.
15.	Elder SH, Nettles DL, Bumgardner JD. Synthesis and characterization of chitosan schffolds for cartilage-tissue engineering. Methods Mol Biol 2004;238:41-8.
16.	Chenite A, Chaput C, Wang D, Combes C, Buschmann MD, Hoemann CD, et al. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials 2000;21:2155-61.
17.	Huang Y, Onyeri S, Siewe M, Moshaveghian A, Sundararajan V. Madihally:In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials 2005;26:7616-27.
18.	Ho MH, Kuo PY, Hsieh HJ, Hsien TY, Hou LT, Lai JY, et al. Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods. Biomaterial 2004;25:129-38.
19.	Hsieh CY, Tsai SP, Ho MH, Wang DM, Liu CE, Hsieh CH, et al. Analysis of freeze-gelation and cross-linking processes for preparing porous chitosan scaffolds. Carbohydr Polym 2007;67:124-32.
20.	Renani HB, Ghorbani M, Beni BH, Karimi Z, Mirhosseini M, Zarkesh H, et al. Determination and comparison of specifics of nucleus pulposus cells of human intervertebral disk in alginate and chitosan-gelatin scaffolds. Adv Biomed Res 2012;1:81. [PUBMED]
21.	Griffon DJ, Sedighi MR, Schaeffer DV, Eurell JA, Johnson AL. Chitosan scaffolds: Interconnective pore size and cartilage engineering. Acta Biomater 2006;2:313-20.
22.	Miranda SC, Silva GA, Hell RC, Martins MD, Alves JB, Goes AM. Three-dimensional culture of rat BMMSCs in a porous chitosan-gelatin caffold: A promising association for bone tissue engineering in oral reconstruction. Arch Oral Biol 2011;56:1-15.
23.	Roughley P, Hoemann C, DesRosiers E, Mwale F, Antoniou J, Alini M. The potential of chitosan-based gels containing intervertebral disk cells for nucleus pulposus supplenetation. Biomaterials 2007;27:388-96.
24.	Mao J, Zhao L, De Yao K, Shang Q, Yang G, Cao Y. Study of novel chitosan-gelatin artificial skin in vitro. J Biomed Mater Res A 2003;64:301-8.
25.	Mao JS, Zhao LG, Yin YJ, Yao KD. Structure and properties of bilayer chitosan-gelatin scaffolds. Biomaterials 2003;24:1067-74.
26.	Chen T, Embree HD, Brown EM, Taylor MM, Payne GF. Enzyme-catalyzed gel formation of gelatin and chitosan: Potential for in situ applications. Biomaterials 2003;24:2831-41.
27.	Ghorbani M, Bahramian H, Beni BH, Karimi Z, Esmaeel N, Salehi M. Comparison of the production of extracellular matrix in nucleus pulposus of intervertebral disk in alginate and chitosan-gelatin scaffolds medical school journal. Advanced biomedical researches 2013;31:1-10.
28.	Nwe N, Furuike T, Tamura H. The mechanical and biological properties of chitosan scaffolds for tissue regeneration templates are significantly enhanced by chitosan from gongronella butleri. Materials 2009;2:374-98.
29.	Bertolo A, Mehr M, Aebli N, Baur M, Ferguson SJ, Stoyanov JV. Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells. Eur Spine J 2012;21(Suppl 6):S826-38.
30.	Griffon DJ, Sedighi MR, Schaeffer DV, Eurell JA, Johnson AL. Chitosan scaffolds: Interconnective pore size and cartilage engineering. Acta Biomater 2006;2:313-20.
31.	Jiankang H, Dichen L, Yaxiong L, Bo Y. Preparation of chitosan-gelatin hybrid scaffolds with well-organized microstructures for hepatic tissue engineering. Acta Biomater 2009;5:453-61.
32.	Dang JM, Sun DD, Shin-Ya Y, Sieber AN, Kostuik JP, Leong KW. Temperature-responsive hydroxybutyl chitosan for the culture of mesenchymal stem cells and intervertebral disk cells. Biomaterials 2006;27:406-18.

Advanced Biomedical Research