Evaluation of apatite-coated chitosan microspheres for bone regeneration

Abstract

The apatite-coated chitosan microspheres were fabricated by incubating chitosan in five times simulated body fluid. The apatite deposited on the microspheres was similar to natural bone mineral, as demonstrated by scanning electron microscopy, X-ray diffraction spectra and Fourier transformed-infrared spectroscopy analyses. Rat bone marrow-derived mesenchymal stem cells (BMSCs) were seeded on apatite-coated microspheres to investigate the effect of this scaffold on cell proliferation and differentiation. BMSCs seeded on uncoated microspheres were served as a control. In vivo experiment was evaluated by transplanting the microspheres loaded with or without BMSCs in 5-mm cranial defects of Wistar rats. Bone regeneration was investigated via micro-CT and histological analysis. It was found that apatite-coated chitosan microspheres could significantly promote the proliferation and alkaline phosphatase activity of BMSCs compared with uncoated microspheres. Histological slices and Micro-CT images at 8 weeks revealed much better regeneration of bone in the apatite-coated microspheres loaded with BMSCs than the control. In addition, the defect filled with pure microspheres induced little new bone formation. Our findings suggest that the apatite-coated chitosan microspheres scaffold is a promising carrier of stem cells for cranial bone tissue engineering.

Keywords

chitosan microspheres / apatite / bone regeneration

References

[1] Yoshimi R, Yamada Y, Ito K, . Self-assembling Peptide Nanofiber Scaffolds, Platelet-rich Plasma, and Mesenchymal Stem Cells for Injectable Bone Regeneration with Tissue Engineering[J]. J. Craniofac. Surg., 2009, 20(5): 1 523-1 530.

[2] Shen H, Hu X, Yang F, . An Injectable Scaffold: rhBMP-2-loaded poly (lactide-co-glycolide)/Hydroxyapatite Composite Microspheres[J]. Acta Biomater., 2010, 6(2): 455-465.

[3] Sukarto A, Yu C, Flynn L E, . Co-delivery of Adipose-derived Stem Cells and Growth Factor-loaded Microspheres in RGD-grafted N-methacrylate Glycol Chitosan Gels for Focal Chondral Repair[J]. Biomacromolecules, 2012, 13(8): 2 490-2 502.

[4] Lee JY, Kim KH, Shin SY, . Enhanced Bone Formation by Transforming Growth Factor-beta1-releasing Collagen/Chitosan Microgranules[J]. J. Biomed. Mater. Res. A, 2006, 76(3): 530-539.

[5] Gunatillake P A, Adhikari R Biodegradable Synthetic Polymers for Tissue Engineering[J]. Eur. Cell. Mater., 2003, 5(1): 1-16.

[6] Yuan Y, Chesnutt B, Utturkar G, . The Effect of Cross-linking of Chitosan Microspheres with Genipin on Protein Release[J]. Carbohydr Polym, 2007, 68(3): 561-567.

[7] Bi L, Cao Z, Hu Y, . Effects of Different Cross-linking Conditions on the Properties of Genipin-cross-linked Chitosan/Collagen Scaffolds for Cartilage Tissue Engineering[J]. J. Mater. Sci. Mater. Med., 2011, 22(1): 51-62.

[8] Frohbergh M E, Katsman A, Botta G P, . Electrospun Hydroxyapatite-containing Chitosan Naofibers Crosslinked with Genipin for Bone Tissue Engineering[J]. Biomaterials, 2012, 33(36): 9 167-9 178.

[9] Du C, Klasens P, Haan R E, . Biomimetic Calcium Phosphate Coatings on Polyactive 1000/70/30[J]. J. Biomed. Mater. Res., 2002, 59(3): 535-546.

[10] Cai Q, Xu Q, Feng Q, . Biomineralization of Electrospun poly (l-lactic acid)/Gelatin Composite Fibrous Scaffold by using a Supersaturated Simulated Body Fluid with Continuous CO2 bubbling[J]. Appl. Surf. Sci., 2011, 257(23): 10 109-10 118.

[11] Kong L, Gao Y, Lu G, . A Study on the Bioactivity of Chitosan/Nano-hydroxyapatite Composite Scaffolds for Bone Tissue Engineering[J]. Eur. Polym. J., 2006, 42(12): 3 171-3 179.

[12] Kang SW, Yang HS, Seo SW, . Apatite-coated poly(Lactic-co-Glycolic Acid) Microspheres as an Injectable Scaffold for Bone Tissue Engineering[J]. J. Biomed. Mater. Res. A, 2008, 85(3): 747-756.

[13] Kim SS, Sun Park M, Jeon O, . Poly(Lactide-co-glycolide)/Hydroxyapatite Composite Scaffolds for Bone Tissue Engineering[J]. Biomaterials, 2006, 27(8): 1 399-1 409.

[14] Ehrlich H, Krajewska B, Hanke T, . Chitosan Membrane as a Template for Hydroxyapatite Crystal Growth in a Model Dual Membrane Diffusion System[J]. J. Memb. Sci., 2006, 273(1): 124-128.

[15] Kong L, Gao Y, Lu G, . A Study on the Bioactivity of Chitosan/Nano-hydroxyapatite Composite Scaffolds for Bone Tissue Engineering[J]. Eur. Polym. J., 2006, 42(12): 3 171-3 179.

[16] Davis HE, Rao R R, He J, . Biomimetic Scaffolds Fabricated from Apatite-coated Polymer Microspheres[J]. J. Biomed. Mater. Res. A, 2009, 90(4): 1 021-1 031.

[17] Chou YF, Chiou WA, Xu Y, . The Effect of pH on the Structural Evolution of Accelerated Biomimetic Apatite[J]. Biomaterials, 2004, 25(22): 5 323-5 331.

[18] Xu L, Lv K, Zhang W, . The Healing of Critical-size Calvarial Bone Defects in Rat with rhPDGF-BB, BMSCs, and Beta-TCP Scaffolds[J]. J. Mater. Sci. Mater. Med., 2012, 23(4): 1 073-1 084.

[19] Sohn JY, Park JC, Um YJ, . Spontaneous Healing Capacity of Rabbit Cranial Defects of Various Sizes[J]. J. Periodontal Implant Sci., 2010, 40(4): 180-187.

[20] Chang C, Peng N, He M, . Fabrication and Properties of Chitin/Hydroxyapatite Hybrid hydrogels as Scaffold Nano-materials[J]. Carbohydr Polym., 2013, 91(1): 7-13.

[21] Thein-Han W, Misra R Biomimetic Chitosan-nanohydroxyapatite Composite Scaffolds for Bone Tissue Engineering[J]. Acta Biomater., 2009, 5(4): 1 182-1 197.

[22] Manjubala I, Scheler S, Bössert J, . Mineralisation of Chitosan Scaffolds with Nano-apatite Formation by Double Diffusion Technique[J]. Acta Biomater., 2006, 2(1): 75-84.

[23] Bagheri-Khoulenjani S, Mirzadeh H, Etrati-Khosroshahi M, . Particle Size Modeling and Morphology Study of Chitosan/Gelatin/Nanohydroxyapatite Nanocomposite Microspheres for Bone Tissue Engineering[J]. J. Biomed. Mater. Res. A, 2013, 101(6): 1 758-1 767.

[24] Li B, Wang Y, Jia D, . Gradient Structural Bone-like Apatite Induced by Chitosan Hydrogel Via Ion Assembly[J]. J. Biomater. Sci. Polym. Ed., 2011, 22(4–6): 505-517.

[25] Chen Y, Mak AF, Li J, . Formation of Apatite on poly(alpha-Hydroxy acid) in an Accelerated Biomimetic Process[J]. J. Biomed. Mater. Res. B Appl. Biomater., 2005, 73(1): 68-76.

[26] Zong C, Xue D, Yuan W, . Reconstruction of Rat Calvarial Defects with Human Mesenchymal Stem Cells and Osteoblast-like Cells in poly-lactic-co-glycolic Acid Scaffolds[J]. Eur. Cell. Mater., 2010, 20: 109-120.

[27] Kim J, Kim IS, Cho TH, . Bone Regeneration Using Hyaluronic Acid-based Hydrogel with Bone Morphogenic Protein-2 and Human Mesenchymal Stem Cells[J]. Biomaterials, 2007, 28(10): 1 830-1 837.

[28] Salamon A, Toldy E, Nagy L, . [The Role of Adult Bone Marrow Derived Mesenchymal Stem Cells in the Repair of Tissue Injuries][J]. Orv. Hetil., 2012, 153(46): 1 807-1 815.

[29] Tang Y, Zhao Y, Wong CS, . Apatite-coated Three-dimensional Fibrous Scaffolds and Their Osteoblast Response[J]. J. Biomed. Mater. Res. A, 2013, 101(3): 674-683.

[30] Salgado A, Figueiredo J, Coutinho O, . Biological Response to Premineralized Starch Based Scaffolds for Bone Tissue Engineering[J]. J. Mater. Sci. Mater. Med., 2005, 16(3): 267-275.

[31] Ge S, Zhao N, Wang L, . Bone Repair by Periodontal Ligament Stem Cellseeded Nanohydroxyapatite-chitosan Scaffold[J]. Int. J. Nanomedicine, 2012, 7: 5 405-5 414.

[32] Galli C, Piemontese M, Lumetti S, . The Importance of WNT Pathways for Bone Metabolism and Their Regulation by Implant Topography[J]. Eur. Cell. Mater., 2012, 24: 46-59.

[33] Mendonca DB, Miguez PA, Mendonca G, . Titanium Surface Topography Affects Collagen Biosynthesis of Adherent Cells[J]. Bone, 2011, 49(3): 463-472.

[34] Chai YC, Roberts SJ, Schrooten J, . Probing the Osteoinductive Effect of Calcium Phosphate by Using an in vitro Biomimetic Model[J]. Tissue Eng. Part A, 2011, 17(7–8): 1 083-1 097.