Hydroxyapatite-alginate biocomposite promotes bone mineralization in different length scales in vivo

Abstract

Tissue engineering is a multidisciplinary research area that aims to develop new techniques and/or biomaterials for medical applications. The objective of the present study was to evaluate the osteogenic potential of a composite of hydroxyapatite and alginate in bone defects with critical sizes, surgically made in the calvaria region of rats. The rats (48 adult males), Rattus norvegicus Wistar, were divided into two groups: control (without composite implantation) and experimental (with composite implantation) and analyzed by optical microscopy at the biological time points 15, 45, 90 and 120 d, and transmission electron microscopy 120 d after the implantation of the biomaterial. It was observed that the biomaterial presented a high degree of fragmentation since the first experimental points studied, and that the fragments were surrounded by new bone after the duration of the project. These areas were studied by analytical transmission electron microscopy using an energy dispersive X-ray spectrometer. Three regions could be distinguished: (1) the biomaterial rich in hydroxyapatite; (2) a thin contiguous region containing phosphorus but without calcium; (3) a region of initial ossification containing mineralizing collagen fibrils with a calcium/phosphorus ratio smaller than the particles of the composite. The intermediate region (without calcium or containing very low amounts of calcium), which just surrounded the composite had not been described in the literature yet, and is probably associated specifically to the biocomposite used. The high performance of the biomaterial observed may be related to the fact that alginate molecules form highly anionic complexes and are capable of adsorbing important factors recognized by integrins from osteoblasts. Regions of fibrotic tissue were also observed mainly in the initial experimental points analyzed. However, it did not significantly influence the final result. In conclusion, the biomaterial presents a great potential for application as bone grafts in the clinical area.

Keywords

bone engineering / bone healing / hydroxyapatite / alginate / biocomposite / analytical microscopy

References

[1] Weiner S, Wagner H D. The material bone: structure mechanical function relations. Annual Review of Materials Science, 1998, 28: 271-298

[2] Allen M R, Hock J M, Burr D B. Periosteum: biology, regulation, and response to osteoporosis therapies. Bone, 2004, 35(5): 1003-1012

[3] Sodek J, McKee M D. Molecular and cellular biology of alveolar bone. Periodontology, 2000, 24: 99-126

[4] Boskey A L. The organic and inorganic matrices. In: Höllinger J O, Einhorn T A, Doll B A, . Bone Tissue Engineering. 1st ed. Boca Raton, USA: CRC Press, 2005

[5] Young M F. Bone matrix proteins: their function, regulation, and relationship to osteoporosis. Osteoporosis International, 2003, 14: S35-S42

[6] Landis J L, Silver H S. The structure and function of normally mineralizing avian tendons. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2002, 133: 1135-1157

[7] Rho J Y, Kuhn-Spearing L, Zioupos P. Mechanical properties and the hierarchical structure of bone. Medical Engineering & Physics, 1998, 20(2): 92-102

[8] de Long W G, Einhorn T A, Koval K, . Bone, grafts and bone graft substitutes in orthopedic trauma surgery — a critical analysis. Journal of Bone and Joint Surgery-American Volume, 2007, 89A(3): 649-658

[9] Bernard G W. Healing and repair of osseous defects. Dental Clinics of North America, 1991, 35(3): 469-478

[10] Munting E, Mirtchi A A, Lemaitre J. Bone repair of defects filled with a phosphocalcic hydraulic cement—an in vivo Study. Journal of Materials Science-Materials in Medicine, 1993, 4(3): 337-344

[11] Ono I, Tateshita T, Satou M, . Treatment of large complex cranial bone defects by using hydroxyapatite ceramic implants. Plastic and Reconstructive Surgery, 1999, 104(2): 339-349

[12] Cardoso A K M V, Barbosa Jr. A A, Miguel F B, . Histomorphometric analysis of tissue responses to bioactive glass implants in critical defects in rat calvaria. Cells Tissues Organs, 2006, 184: 128-137

[13] Suh H. Tissue restoration, tissue engineering and regenerative medicine. Yonsei Medical Journal, 2000, 41: 681-684

[14] Lavik E, Langer R. Tissue engineering: current state and perspectives. Applied Microbiology and Biotechnology, 2004, 65(1): 1-8

[15] Neumann M, Epple M. Composites of calcium phosphate and polymers as bone substitution materials. European Journal of Trauma, 2006, 32: 125-131

[16] Eiselt P, Yeh J, Latvala R K, . Porous carriers for biomedical applications based on alginate hydrogels. Biomaterials, 2000, 21(19): 1921-1927

[17] Anselme K. Osteoblast adhesion on biomaterials. Biomaterials, 2000, 21(7): 667-681

[18] Ribeiro C C, Barrias C C, Barbosa M A. Calcium phosphate-alginate microspheres as enzyme delivery matrices. Biomaterials, 2004, 25: 4363-4373

[19] Thian E S, Loh N H, Khor K A, . Microstructures and mechanical properties of powder injection molded Ti-6Al-4V/HA powder. Biomaterials, 2002, 23(14): 2927-2938

[20] Gombotz W R, Wee S F. Protein release from alginate matrices. Advanced Drug Delivery Reviews, 1998, 31(3): 267-285

[21] Boontheekul T, Kong H J, Mooney D J. Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. Biomaterials, 2005, 26(15): 2455-2465

[22] Klock G, Pfeffermann A, Ryser C, . Biocompatibility of mannuronic acid-rich alginates. Biomaterials, 1997, 18(10): 707-713

[23] Kuo C K, Ma P X. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties. Biomaterials, 2001, 22(6): 511-521

[24] Rees D A. Polyssacharide shapes and their interactions-some recent advances. Pure and Applied Chemistry, 1981, 53: 1-14

[25] Aslani P, Kennedy R A. Studies on diffusion in alginate gels: 1. Effect of cross-linking with calcium or zinc ions on diffusion of acetaminophen. Journal of Controlled Release, 1996, 42(1): 75-82

[26] Miguel F B, Cardoso A K M V, Barbosa A A, . Morphological assessment of the behavior of three-dimensional anionic collagen matrices in bone regeneration in rats. Journal of Biomedical Materials Research Part B - Applied Biomaterials, 2006, 78B(2): 334-339

[27] Yuan H P, van den Doel M, Li S H, . A comparison of the osteoinductive potential of two calcium phosphate ceramics implanted intramuscularly in goats. Journal of Materials Science - Materials in Medicine, 2002, 13(12): 1271-1275

[28] Pelissier P, Villars F, Mathoulin-Pelissier S, . Influences of vascularization and osteogenic cells on heterotopic bone formation within a madreporic ceramic in rats. Plastic and Reconstructive Surgery, 2003, 111(6): 1932-1941

[29] Mastrogiacomo M, Muraglia A, Komlev V, . Tissue engineering of bone: search for a better scaffold. Orthodontics & Craniofacial Research, 2005, 8(4): 277-284

[30] Leventouri T. Synthetic and biological hydroxyapatites: crystal structure questions. Biomaterials, 2006, 27(18): 3339-3342

[31] Benaqqa C, Chevalier J, Saädaoui M, . Slow crack growth behavior of hydroxyapatite ceramics. Biomaterials, 2005, 26: 6106-6112

[32] Rezwan K, Chen Q Z, Blaker J J, . Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials, 2006, 27(18): 3413-3431

[33] Wang M. Developing bioactive composite materials for tissue replacement. Biomaterials, 2003, 24(13): 2133-2151

[34] Schmitz J P, Schwartz Z, Hollinger J O, . Characterization of rat calvarial nonunion defects. Acta Anatomica, 1990, 138(3): 185-192

[35] Ferreira G R, Cestari T M, Granjeiro J M, . Lack of repair of rat skull critical size defect treated with bovine morphometric protein bound to microgranular bioabsorbable hydroxyapatite. Brazilian Dental Journal, 2004, 15(3): 175-180

[36] Intini G, Andreana S, Intini F E, . Calcium sulfate and platelet-rich plasma make a novel osteoinductive biomaterial for bone regeneration. Journal of Translational Medicine, 2007, 5: 1-13

[37] Wu T J, Huang H H, Lan C W, . Studies on the microspheres comprised of reconstituted collagen and hydroxyapatite. Biomaterials, 2004, 25: 651-658

[38] Thomsen P, Esposito M, Gretzer C, . Inflammatory response to implanted materials. In: Davies J E. Bone Engineering.1st Ed. Toronto, Canada. Squared incorporated, 2000

[39] Yang L, Zhang Y, Cui F Z. Two types of mineral-related matrix vesicles in the bone mineralization of zebrafish. Biomedical Materials, 2007, 2(1): 21-25

Acknowledgements

We thank the financial support from CNPq and FAPERJ Brazilian Agencies.

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg