In vivo biocompatibility and fracture healing of hydroxyapatite-hexagonal boron nitridechitosan- collagen biocomposite coating in rats

The biocompatibility of orthopaedic implants and their effects on fracture healing have key roles for success. In this study, it was aimed to investigate the effects of a novel biocomposite consisting of hydroxyapatite (HA), hexagonal boron nitride (h-BN), chitosan (Cs), and type 1 collagen (Ct1) on biocompatibility and fracture healing in rats. A total of 60 adult male Wistar rats weighing 300?500 g were used in the study. The rats were randomly divided into 2 groups named A (uncoated/control) and B (biocomposite coated). Biocomposite (HA/h-BN/Cs/Ct1) coated and uncoated stainless-steel implants were used as intramedullary pins. Groups A and B were divided into subgroups of A1 and B1 (15th day), A2 and B2 (30th day), A3 and B3 (45th day) according to the date of euthanasia. Clinical, radiographic, haematological, biochemical, and histopathological findings were evaluated by pairwise comparisons. The findings were consistent and similar. No statistically significant difference was found for a finding disturbing the biocompatibility. Histopathological examinations showed that coating biomaterials did not resorb over the course of 15, 30, and 45 days. It is thus revealed that the content is biocompatible. However, it has been concluded that it is necessary to increase the physical strength of the coating surface against sterilization and surgical procedures. As a result, based on the interpretations of the clinical, radiographic, haematological, biochemical, and histopathological findings, the biocompatibility of HA/h-BN/Cs/Ct1 biocomposite materials has been revealed.

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  • 1. Lemons JE. Biomaterials, biomechanics, tissue healing, and immediate-function dental implants. Journal of Oral Implantology 2004; 30 (5): 318-324. doi: 10.1563/0712.1
  • 2. Junker R, Dimakis A, Thoneick M, Jansen JA. Effects of implant surface coatings and composition on bone integration: a systematic review. Clinical Oral Implants Research 2009; 20 (Suppl. 4): 185-206. doi: 10.1111/j.1600-0501.2009.01777.x
  • 3. Darouiche RO, Green G, Mansouri MD. Antimicrobial activity of antiseptic-coated orthopaedic devices. International Journal of Antimicrobial Agents. 1998; 10 (1): 83-86. doi: 10.1016/ S0924-8579(98)00017-X
  • 4. Rupp F, Scheideler L, Olshanska N, De Wild M, Wieland M et al. Enhancing surface free energy and hydrophilicity through chemical modification of microstructured titanium implant surfaces. Journal of Biomedical Materials Research Part A 2006; 76 (2): 323-334. doi: 10.1002/jbm.a.30518
  • 5. Le Guehennec L, Goyenvalle E, Lopez-Heredia MA, Weiss P, Amouriq Y et al. Histomorphometric analysis of the osseointegration of four different implant surfaces in the femoral epiphyses of rabbits. Clinical Oral Implants Research. 2008; 19 (11): 1103-1110. doi: 10.1111/j.1600-0501.2008.01547.x
  • 6. Tozar A, Karahan İH. A comprehensive study on electrophoretic deposition of a novel type of collagen and hexagonal boron nitride reinforced hydroxyapatite/chitosan biocomposite coating. Applied Surface Science. 2018; 452: 322-336. doi: j.apsusc.2018.04.241
  • 7. Lee H, Jeong Y, Park S, Jeong S, Kim H et al. Surface properties and cell response of fluoridated hydroxyapatite/TiO 2 coated on Ti substrate. Current Applied Physics 2009; 9 (2): 528-533. doi: 10.1016/j.cap.2008.03.020
  • 8. Raddaha NS, Cordero-Arias L, Cabanas-Polo S, Virtanen S, Roether JA et al. Electrophoretic deposition of chitosan/h-BN and chitosan/h-BN/TiO(2) composite coatings on stainless steel (316L) substrates. Materials (Basel) 2014; 7 (3): 1814- 1829. doi: 10.3390/ma7031814
  • 9. Lahiri D, Singh V, Benaduce AP, Seal S, Kos L et al. Boron nitride nanotube reinforced hydroxyapatite composite: mechanical and tribological performance and in-vitro biocompatibility to osteoblasts. Journal of the Mechanical Behavior of Biomedical Materials 2011; 4 (1): 44-56. doi: 10.1016/j.jmbbm.2010.09.005
  • 10. Durmuş AS, Çeribaşı AO, Can HN. Evaluation of the accelerator effect of coral and platelet rich fibrin on bone healing. Kafkas University Journal of the Faculty of Veterinary Medicine 2019; 25 (2): 193-199. doi: 10.9775/kvfd.2018.20655
  • 11. Mucalo M (editor). Hydroxyapatite (HAp) for Biomedical Applications. Amsterdam, the Netherlands: Elsevier Science; 2015. doi: 10.1016/B978-1-78242-033-0.00014-6
  • 12. Wang D, Chen C, He T, Lei T. Hydroxyapatite coating on Ti6Al4V alloy by a sol-gel method. Journal of Materials Science: Materials in Medicine 2008; 19 (6): 2281-2286. doi: 10.1007/s10856-007-3338-5
  • 13. Tozar A. Bilgisayar destekli optimizasyon kullanilarak biyomimetik yaklaşimla elektroforetik depolanan hidroksiapatit/kitosan/kollajen/h-bn biyokompozit kaplamalarin mekanik, tribolojik ve korozyon özelliklerinin incelenmesi. PhD, Mustafa Kemal University, Hatay, Turkey, 2017 (in Turkish).
  • 14. Croisier F, Jérôme C. Chitosan-based biomaterials for tissue engineering. European Polymer Journal 2013; 49 (4): 780-792. doi: 10.1016/j.eurpolymj.2012.12.009
  • 15. Dimitriou R, Jones E, McGonagle D, Giannoudis PV. Bone regeneration: current concepts and future directions. BMC Medicine 2011; 9 (1): 66. doi: 10.1186/1741-7015-9-66
  • 16. Mina A, Caicedo HH, Uquillas JA, Aperador W, Gutiérrez O et al. Biocompatibility behavior of β-tricalcium phosphatechitosan coatings obtained on 316L stainless steel. Materials Chemistry and Physics 2016; 175: 68-80. doi: 10.1016/j. matchemphys.2016.02.070
  • 17. Gallyamov MO, Chaschin IS, Khokhlova MA, Grigorev TE, Bakuleva NP et al. Collagen tissue treated with chitosan solutions in carbonic acid for improved biological prosthetic heart valves. Materials Science and Engineering: C 2014; 37 (Supplement C): 127-140. doi: 10.1016/j.msec.2014.01.017
  • 18. Campelo CS, Chevallier P, Vaz JM, Vieira RS, Mantovani D. Sulfonated chitosan and dopamine based coatings for metallic implants in contact with blood. Materials Science and Engineering C: Materials for Biological Applications 2017; 72: 682-691. doi: 10.1016/j.msec.2016.11.133
  • 19. Bain JL, Culpepper BK, Reddy MS, Bellis SL. Comparing variable-length polyglutamate domains to anchor an osteoinductive collagen-mimetic peptide to diverse bone grafting materials. International Journal of Oral & Maxillofacial Implants 2014; 29 (6): 1437. doi: 10.11607/jomi.3759
  • 20. Kang S (editor). Development of a porcine collagenhydroxyapatite scaffold as bone graft substitutes. Journal of tissue engineering and regenerative medicine 2014: WileyBlackwell 111 River St, Hoboken 07030-5774, NJ USA.
  • 21. Włodarczyk-Biegun MK, Werten MW, de Wolf FA, van den Beucken JJ, Leeuwenburgh SC et al. Genetically engineered silk-collagen-like copolymer for biomedical applications: Production, characterization and evaluation of cellular response. Acta Biomaterialia 2014; 10 (8): 3620-3629. doi: 10.1016/j.actbio.2014.05.006
  • 22. Cen L, Liu W, Cui L, Zhang W, Cao Y. Collagen tissue engineering: development of novel biomaterials and applications. Pediatric Research 2008; 63 (5): 492-496. doi: 10.1203/PDR.0b013e31816c5bc3
  • 23. Mushahary D, Wen C, Kumar JM, Lin J, Harishankar N et al. Collagen type-I leads to in vivo matrix mineralization and secondary stabilization of Mg-Zr-Ca alloy implants. Colloids and Surfaces B: Biointerfaces 2014; 122: 719-728. doi: 10.1016/j. colsurfb.2014.08.005
  • 24. Del Turco S, Ciofani G, Cappello V, Gemmi M, Cervelli T et al. Cytocompatibility evaluation of glycol-chitosan coated boron nitride nanotubes in human endothelial cells. Colloids and Surfaces B: Biointerfaces 2013; 111: 142-149. doi: 10.1016/j. colsurfb.2013.05.031
  • 25. Ciofani G, Ricotti L, Danti S, Moscato S, Nesti C et al. Investigation of interactions between poly-L-lysinecoated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation. International Journal of Nanomedicine 2010; 5: 285. doi: 10.2147/IJN.S9879
  • 26. Engler M, Lesniak C, Damasch R, Ruisinger B, Eichler J. Hexagonal boron nitride (hBN): applications from metallurgy to cosmetics. In: CFI Ceramic Forum International; Baden, Germany; 2007.
  • 27. Tozar A, Karahan İH. A comparative study on the effect of collagen and h-BN reinforcement of hydroxyapatite/chitosan biocomposite coatings electrophoretically deposited on Ti6Al-4V biomedical implants. Surface and Coatings Technology 2018; 340: 167-176. doi: 10.1016/j.surfcoat.2018.02.034
  • 28. Costa CM, Bernardes G, Ely JB, Porto LM. Proposal for access to the femur in rats. International Journal of Biotechnology and Molecular Biology Research 2011; 2 (4): 73-79.
  • 29. Whelan DB, Bhandari M, Stephen D, Kreder H, McKee MD et al. Development of the radiographic union score for tibial fractures for the assessment of tibial fracture healing after intramedullary fixation. Journal of Trauma and Acute Care Surgery 2010; 68 (3): 629-632. doi: 10.1097/TA.0b013e3181a7c16d
  • 30. Luna LG. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York, NY, USA: Blakiston Division, McGraw-Hill; 1968.
  • 31. Allen HL, Wase A, Bear W. Indomethacin and aspirin: effect of nonsteroidal anti-inflammatory agents on the rate of fracture repair in the rat. Acta Orthopaedica Scandinavica 1980; 51 (1- 6): 595-600. doi: 10.3109/17453678008990848
  • 32. Estai MA, Soelaiman IN, Shuid AN, Das S, Ali AM et al. Histological changes in the fracture callus following the administration of water extract of Piper sarmentosum (Daun Kadok) in estrogen-deficient rats. Iranian Journal of Medical Sciences 2011; 36 (4): 281.
  • 33. Naddaf H, Baniadam A, Esmaeilzadeh S, Ghadiri A, Pourmehdi M et al. Histopathologic and radiographic evaluation of the electroacupuncture effects on ulna fracture healing in dogs. Open Veterinary Journal 2014; 4 (1): 44-50.
  • 34. He J, Huang T, Gan L, Zhou Z, Jiang B et al. Collageninfiltrated porous hydroxyapatite coating and its osteogenic properties: in vitro and in vivo study. Journal of Biomedical Materials Research Part A 2012; 100 (7): 1706-15. doi: 10.1002/ jbm.a.34121
  • 35. Negroiu G, Piticescu RM, Chitanu GC, Mihailescu IN, Zdrentu L et al. Biocompatibility evaluation of a novel hydroxyapatitepolymer coating for medical implants (in vitro tests). Journal of Materials Science: Materials in Medicine 2008; 19 (4): 1537- 1544. doi: 10.1007/s10856-007-3300-6
  • 36. Chan KW, Wong HM, Yeung KWK, Tjong SC. Polypropylene Biocomposites with Boron Nitride and Nanohydroxyapatite Reinforcements. Materials (Basel) 2015; 8 (3): 992-1008. doi: 10.3390/ma8030992
  • 37. Skott M, Andreassen TT, Ulrich-Vinther M, Chen X, Keyler DE et al. Tobacco extract but not nicotine impairs the mechanical strength of fracture healing in rats. Journal of Orthopaedic Research 2006; 24 (7): 1472-1479. doi: 10.1002/jor.20187
  • 38. Haffner-Luntzer M, Muller-Graf F, Matthys R, Abaei A, Jonas R et al. In vivo evaluation of fracture callus development during bone healing in mice using an mri-compatible osteosynthesis device for the mouse femur. Journal of Visualized Experiments: JoVE 2017; (129). doi: 10.3791/56679
  • 39. Frydman GH, Marini RP, Bakthavatchalu V, Biddle KE, Muthupalani S et al. Local and systemic changes associated with long-term, percutaneous, static implantation of titanium alloys in rhesus macaques (Macaca mulatta). Comparative Medicine 2017; 67 (2): 165-175.
  • 40. DiCarlo EF, Bullough PG. The biologic responses to orthopedic implants and their wear debris. Clinical Materials 1992; 9 (3-4): 235-260. doi: 10.1016/0267-6605(92)90104-2
  • 41. Turgut K. Veteriner Klinik Laboratuvar Teşhis. Konya, Turkey: Bahçıvanlar Basım Sanayi A Ş; 2000 (in Turkish).
  • 42. Carlotti APCP, Bohn D, Matsuno AK, Pasti DM, Gowrishankar M et al. Indicators of lean body mass catabolism: emphasis on the creatinine excretion rate. QJM: An International Journal of Medicine 2008; 101 (3): 197-205. doi: 10.1093/qjmed/hcm127
  • 43. Nathwani RA, Pais S, Reynolds TB, Kaplowitz N. Serum alanine aminotransferase in skeletal muscle diseases. Hepatology 2005; 41 (2): 380-382. doi: 10.1002/hep.20548
  • 44. Golub EE, Boesze-Battaglia K. The role of alkaline phosphatase in mineralization. Current Opinion in Orthopaedics 2007; 18 (5): 444-448. doi: 10.1097/BCO.0b013e3282630851
  • 45. Wang ZL, Yan YH, Wan T, Yang H. Poly (L-lactic acid)/ hydroxyapatite/collagen composite coatings on AZ31 magnesium alloy for biomedical application. Proceedings of the Institution of Mechanical Engineers. Part H, Journal of Engineering in Medicine 2013; 227 (10): 1094-1103. doi: 10.1177/0954411913493845
  • 46. Ou KL, Wu J, Lai WFT, Yang CB, Lo WC et al. Effects of the nanostructure and nanoporosity on bioactive nanohydroxyapatite/reconstituted collagen by electrodeposition. Journal of Biomedical Materials Research Part A 2010; 92 (3): 906-912.
  • 47. Akkouch A, Zhang Z, Rouabhia M. A novel collagen/ hydroxyapatite/poly(lactide-co-ε-caprolactone) biodegradable and bioactive 3D porous scaffold for bone regeneration. Journal of Biomedical Material Research Part A 2011; 96 (4): 693-704. doi: 10.1002/jbm.a.33033
  • 48. Göncü Y, Gecgin M, Bakan F, Ay N. Electrophoretic deposition of hydroxyapatite-hexagonal boron nitride composite coatings on Ti substrate. Materials Science and Engineering: C 2017; 79: 343-353. doi: 10.1016/j.msec.2017.05.023
  • 49. Emanet M, Kazanc E, Cobandede Z, Culha M. Boron nitride nanotubes enhance properties of chitosan-based scaffolds. Carbohydrate Polymers 2016; 151: 313-320. doi: 10.1016/j. carbpol.2016.05.074
  • 50. Atila A, Halici Z, Cadirci E, Karakus E, Palabiyik SS et al. Study of the boron levels in serum after implantation of different ratios nano-hexagonal boron nitride-hydroxyapatite in rat femurs. Materials Science and Engineering: C 2016; 58: 1082- 1089. doi: 10.1016/j.msec.2015.09.041
  • 51. Przekora A, Ginalska G. Biological properties of novel chitosanbased composites for medical application as bone substitute. Open Life Sciences 2014; 9 (6): 634-641. doi: 10.2478/s11535- 014-0297-y
  • 52. Di Martino A, Sittinger M, Risbud MV. Chitosan: a versatile biopolymer for orthopaedic tissue-engineering. Biomaterials 2005; 26 (30): 5983-5990. doi: 10.1016/j. biomaterials.2005.03.016
  • 53. Gopi D, Nithiya S, Shinyjoy E, Rajeswari D, Kavitha L. Carbon nanotubes/carboxymethyl chitosan/mineralized hydroxyapatite composite coating on Ti-6Al-4V alloy for improved mechanical and biological properties. Industrial & Engineering Chemistry Research 2014; 53 (18): 7660-7669. doi: 10.1021/ie403903q
  • 54. Chang C, Peng N, He M, Teramoto Y, Nishio Y et al. Fabrication and properties of chitin/hydroxyapatite hybrid hydrogels as scaffold nano-materials. Carbohydrate Polymers 2013; 91 (1): 7-13. doi: 10.1016/j.carbpol.2012.07.070
  • 55. Madhumathi K, Binulal N, Nagahama H, Tamura H, Shalumon K et al. Preparation and characterization of novel β-chitin-hydroxyapatite composite membranes for tissue engineering applications. International Journal of Biological Macromolecules 2009; 44 (1): 1-5. doi: 10.1016/j. ijbiomac.2008.09.013
  • 56. Madhumathi K, Shalumon K, Rani VD, Tamura H, Furuike T et al. Wet chemical synthesis of chitosan hydrogel-hydroxyapatite composite membranes for tissue engineering applications. International Journal of Biological Macromolecules 2009; 45 (1): 12-15. doi: 10.1016/j.ijbiomac.2009.03.011
  • 57. Kumar PS, Srinivasan S, Lakshmanan VK, Tamura H, Nair S et al. β-Chitin hydrogel/nano hydroxyapatite composite scaffolds for tissue engineering applications. Carbohydrate Polymers 2011; 85 (3): 584-591. doi: 10.1016/j.carbpol.2011.03.018
  • 58. Ling T, Lin J, Tu J, Liu S, Weng W et al. Mineralized collagen coatings formed by electrochemical deposition. Journal of Materials Science: Materials in Medicine 2013; 24 (12): 2709- 2718. doi: 10.1007/s10856-013-5028-9
  • 59. Al-Saadi S, Banerjee PC, Anisur MR, Raman RKS. Hexagonal boron nitride impregnated silane composite coating for corrosion resistance of magnesium alloys for temporary bioimplant applications. Metals 2017; 7 (12): 518. doi: 10.3390/ met7120518
  • 60. Nagarajan S, Belaid H, Pochat-Bohatier C, Teyssier C, Iatsunskyi I et al. Design of boron nitride/gelatin electrospun nanofibers for bone tissue engineering. ACS Applied Materials & Interfaces 2017; 9 (39): 33695-33706. doi: 10.1021/acsami.7b13199