In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing

Authors

  • Nicole Fidalgo Paretsis Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Clínica Cirúrgica, São Paulo – SP, Brazil https://orcid.org/0000-0002-9766-4836
  • Vagner Gonçalves Junior Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Patologia, São Paulo – SP, Brazil https://orcid.org/0000-0002-5672-0133
  • Nicolle Gilda Teixeira de Queiroz Hazarbassanov Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Patologia, São Paulo – SP, Brazil https://orcid.org/0000-0002-5692-0565
  • Geissiane Moraes Marcondes Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Clínica Cirúrgica, São Paulo – SP, Brazil https://orcid.org/0000-0003-1919-4143
  • Ana Maria de Guzzi Plepis Universidade de São Paulo, Instituto de Química de São Carlos, São Carlos – SP, Brazil https://orcid.org/0000-0002-4360-8433
  • Virgínia da Conceição Amaro Martins Universidade de São Paulo, Instituto de Química de São Carlos, São Carlos – SP, Brazil
  • Victor Elias Arana-Chavez Universidade de São Paulo, Faculdade de Odontologia, Laboratório de Biologia Oral, São Paulo – SP, Brazil https://orcid.org/0000-0002-6767-7812
  • Joice Fülber Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Clínica Cirúrgica, São Paulo – SP, Brazil https://orcid.org/0000-0001-8989-6779
  • André Luís do Valle De Zoppa Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Clínica Cirúrgica, São Paulo – SP, Brazil https://orcid.org/0000-0002-9852-4843

DOI:

https://doi.org/10.11606/issn.1678-4456.bjvras.2021.179885

Keywords:

Bone regeneration, Propidium iodide assay, Methylthiazol tetrazolium assay, Mesenchymal stem cells, Cytotoxicity

Abstract

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.

Downloads

Download data is not yet available.

References

Abd-Khorsand S, Saber-Samandari S, Saber-Samandari S. Development of nanocomposite scaffolds based on TiO 2 doped in grafted chitosan/hydroxyapatite by freeze drying method and evaluation of biocompatibility. Int J Biol Macromol. 2017;101:51-8. https://doi.org/10.1016/j.ijbiomac.2017.03.067. PMid:28315764.

Ahmad N, Bharatham HB, Hamid ZA, Zulkipli NZ. Cytotoxicity and oxidative stress evaluation of alginate/cockle shell powder Nanobiocomposite bone scaffold on osteoblast. J Sains Kesihat Malaysia. 2017;15(1):97-103. https://doi.org/10.17576/JSKM-2017-1501-12.

Amaral MB, Viana RB, Viana KB, Diagone CA, Denis AB, De Guzzi Plepis AM. In vitro and in vivo response of composites based on chitosan, hydroxyapatite and collagen. Acta Sci Technol. 2020;42(1):1-13.

Azevedo AS, de Sá MJC, Fook MVL, Neto PIN, de Souza OB, Azevedo SS. Hidroxiapatita e quitosana isoladas e associadas à medula óssea no reparo do tecido ósseo em coelhos. Estudo histológico e morfométrico. Ciência Rural St Maria. 2013;43(7):1265-70. https://doi.org/10.1590/S0103-84782013000700019.

Barrientos-Durán A, Nieden NI, Malinin TI. Rodríguez- JC. Carboxyl-modified single-wall carbon nanotubes improve bone tissue formation in vitro and repair in an in vivo rat model. Int J Nanomedicine. 2014;4:4277-91. https://doi.org/10.2147/IJN.S62538. PMid:25246785.

Burk J, Glauche SM, Brehm W, Crovace A, Francioso E, Hillmann A, Schubert S, Lacitignola L. Characterisation and intracellular labelling of mesenchymal stromal cells derived from synovial fluid of horses and sheep. Vet J. 2017;222:1-8. https://doi.org/10.1016/j.tvjl.2017.02.006. PMid:28410670.

Chen L, Hu J, Shen X, Tong H. Synthesis and characterization of chitosan-multiwalled carbon nanotubes/hydroxyapatite nanocomposites for bone tissue engineering. J Mater Sci Mater Med. 2013;24(8):1843-51. https://doi.org/10.1007/s10856-013-4954-x. PMid:23712535.

Dantas TS, Lelis ÉR, Naves LZ, Fernandes-neto AJ, de Magalhães D. Materiais de Enxerto Ósseo e suas Aplicações na Odontologia Bone Graft Materials and their Application in Dentistry. UNOPAR Cient Ciênc Biol Saúde. 2011;13(2):131-6.

Duarte CRA. Avaliação da citotoxicidade in vitro de composições de fosfato de cálcio para uso em reparação óssea [tese]. Lages: Universidade do Estado de Santa Catarina; 2015.

Emara SA, Gadallah SM, Sharshar A. Evaluation of coral wedge and composite as bone graft substitute to induce new bone dormation in a dog tibial defect. J Am Sci. 2013;9(7):526-37.

Ferreira WS, Costa CMR, Ferreira SRB, Nunes SF. Propriedades estruturais e eletrônicas da hidroxiapatita a partir de cálculos de primeiros princípios. Engevista. 2017;19(1):194-201. https://doi.org/10.22409/engevista.v19i1.820.

Fidalgo TK S, Barcelos R, Petrópolis DB, Azevedo BR, Primo LG. Silva Filho FC e. Citotoxidade de diferentes concentrações de hipoclorito de sódio sobre osteoblastos humanos Citotoxicity of different amounts of sodium hypochlorite on human cultured osteoblasts. Biologia (Bratisl). 2009;57(3):317-21.

Freitas GP, Lopes HB, Almeida ALG, Abuna RPF, Gimenes R, Souza LEB, Covas DT, Beloti MM, Rosa AL. Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue. Calcif Tissue Int. 2017;101(3):312-20. https://doi.org/10.1007/s00223-017-0282-3. PMid:28451713.

Fülber J, Maria DA, Silva LCLC, Massoco CO, Agreste F, Baccarin RYA. Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment. Stem Cell Res Ther. 2016;7(1):35. https://doi.org/10.1186/s13287-016-0294-3. PMid:26944403.

Gupta SK, Kumar R, Mishra NC. Influence of quercetin and nanohydroxyapatite modifications of decellularized goat-lung scaffold for bone regeneration. Mater Sci Eng C. 2017;71:919-28. https://doi.org/10.1016/j.msec.2016.10.085. PMid:27987789.

Horn MM, Martins VCA, Plepis AMG. Interaction of anionic collagen with chitosan: effect on thermal and morphological characteristics. Carbohydr Polym. 2009;77(2):239-43. https://doi.org/10.1016/j.carbpol.2008.12.039.

Jarcho M, Bolen H, Thomas MB, Bobick J, Kay JF, Doremus RH. Hydroxylapatite Synthesis and Characterization in Sense Polycristalline Forms. J Mater Sci. 1976;11(11):2027-35. https://doi.org/10.1007/PL00020328.

Jorge JH, Giampaoli ET, Pavarina AC. Citotoxicidade dos Materiais Dentários. Rev Odontol UNESP. 2004;33(2):65-8.

Lee CC, Hirasawa N, Garcia KG, Ramanathan D, Kim KD. Stem and progenitor cell microenvironment for bone regeneration and repair. Regen Med. 2019;14(7):693-702. https://doi.org/10.2217/rme-2018-0044. PMid:31393221.

Marcondes GM, Nobrega FS, Corrêa L, Arana-chavez VE, Plepis AMG, Martins VCA, Zoppa ALV. Avaliação da interação biológica entre compósito de quitosana, colágeno e hidroxiapatita e tecido ósseo ovino. Arq Bras Med Vet Zootec. 2016;68(6):1531-8. https://doi.org/10.1590/1678-4162-8824.

Masson AO, Nascimento MHM, Lombello CB. Análise comparativa de diferentes métodos de citotoxicidade in vitro. In: Proceedings of the 24º Congresso Brasileiro de Engenharia Biomédica – CBEB; 2014 Out 13-17; Uberlândia; 2014. p. 2484–7.

Milori FP, Quitzan J, de Souza RS, Cirio SM, Dornbusch PT, do Prado AMRB. Placas ósseas confeccionadas a partir de diáfise cortical equina na osteossíntese femoral em coelhos. Pesq Vet Bras. 2013;33(10):1201-7. https://doi.org/10.1590/S0100-736X2013001000005.

Nóbrega FS. Avaliação da interação biológica entre polímero de poliuretana de mamona acrescido de carbonato de cálcio e tecido ósseo de equinos [tese]. São Paulo: Faculdade de Medicina Veterinária e Zootecnica, Universidade de São Paulo; 2014. https://doi.org/10.11606/T.10.2014.tde-12112014-093337.

Notodihardjo FZ, Kakudo N, Kushida S, Suzuki K, Kusumoto K. Bone regeneration with BMP-2 and hydroxyapatite in critical size calvarial defects in rats. J Craniomaxillofac Surg. 2012;40(3):287-91. https://doi.org/10.1016/j.jcms.2011.04.008. PMid:21737289.

Pan L, Pei X, He R, Wan Q, Wang J. Multiwall carbon nanotubes/polycaprolactone composites for bone tissue engineering application. Colloids Surf B Biointerfaces. 2012;93:226-34. https://doi.org/10.1016/j.colsurfb.2012.01.011. PMid:22305638.

Paretsis NF, Arana-Chavez VE, Correa L, Peplis AMG, Martins VCA, Cortopassi SRG, Zoppa ALV. Avaliação histológica e histomorfométrica da regeneração óssea a partir da utilização de biomateriais em tíbias de ovinos. Pesq Vet Bras. 2017;37(12):1537-44. https://doi.org/10.1590/s0100-736x2017001200029.

Patel S, Gheewala N, Suthar A, Shah A. In-vitro cytotoxicity activity of solanum nigrum extract against Hela cell line and Vero cell line. Int J Pharm Pharm Sci. 2009;1(Suppl. 1):38-46.

Portinho CP, Collares MVM, Silva FLH, Nardi NB, Pinto RA, Siqueira E, Morellato G, Sumino K. Reconstrução de calota craniana com células - tronco mesenquimais indiferenciadas: estudo experimental. Rev Soc Bras Cir Plást. 2006;21(3):161-5.

Ramesh S, Tan CY, Hamdi M, Sopyan I, Teng WD. The influence of Ca/P ratio on the properties of hydroxyapatite bioceramics. In Proceedings SPIE 6423, International Conference on Smart Materials and Nanotechnology in Engineering; 2007 Nov 1. Harbin, China. p. 64233A.

Reis ECC, Borges APB, Fonseca CC, Martinez MMM, Eleotério RB, Morato GO, Oliveira PM. Biodegradation of a Hydroxyapatite-polyhydroxybutyrate Composite. Braz Arch Biol Technol. 2010;53(4):817-26. https://doi.org/10.1590/S1516-89132010000400010.

Schönberger T, Hahn J, Kasten P, Südkamp NP, Fechner K, Pearce S, Niemeyer P. A novel software-based evaluation method for objective quantification of bone regeneration in experimental bone defects. Eur Cell Mater. 2007;14:92.

Shin US, Yoon I-K, Lee G-S, Jang W-C, Knowles JC, Kim H-W. Carbon nanotubes in nanocomposites and hybrids with hydroxyapatite for bone replacements. J Tissue Eng. 2011;2011:1-10. https://doi.org/10.4061/2011/674287. PMid:21776341.

Spin-Neto R, Coletti FL, de Freitas RM, Pavone C, Campana-filho SP, Marcantonio RAC. Chitosan-based biomaterials used in critical-size bone defects : radiographic study in rat ’ s calvaria. Rev Odontol UNESP. 2012;41(5):312-7. https://doi.org/10.1590/S1807-25772012000500003.

Sun T, Wang M, Shao Y, Wang L, Zhu Y. The effect and osteoblast signaling response of trace silicon Doping Hydroxyapatite. Biol Trace Elem Res. 2018;181(1):82-94. https://doi.org/10.1007/s12011-017-1031-1. PMid:28456913.

Tavakol S, Nikpour MR, Amani A, Soltani M, Rabiee SM, Rezayat SM, Chen P, Jahanshahi M. Bone regeneration based on nano-hydroxyapatite and hydroxyapatite/chitosan nanocomposites: an in vitro and in vivo comparative study. J Nanopart Res. 2013;15(1):1-16. https://doi.org/10.1007/s11051-012-1373-8.

Tavaria FK, Costa EM, Pina-Vaz I, Carvalho MF, Pintado MM. A quitosana como biomaterial odontológico: estado da arte. Rev Bras Eng Bioméd. 2013;29(1):110-20. https://doi.org/10.4322/rbeb.2013.002.

Tsuchiya N, Sato S, Kigami R, Kawano E, Takane M, Arai Y, Ito K, Ogiso B. Effect of a chitosan sponge impregnated with platelet-derived growth factor on bone augmentation beyond the skeletal envelope in rat calvaria. J Oral Sci. 2014;56(1):23-8. https://doi.org/10.2334/josnusd.56.23. PMid:24739704.

Türk S, Altınsoy I, Çelebi Efe G, Ipek M, Özacar M, Bindal C. 3D porous collagen/functionalized multiwalled carbon nanotube/chitosan/hydroxyapatite composite scaffolds for bone tissue engineering. Mater Sci Eng C Mater Biol Appl. 2018;92:757-68. https://doi.org/10.1016/j.msec.2018.07.020. PMid:30184804.

Venkatesan J, Qian Z, Ryu B, Ashok Kumar N, Kim S-K. Kumarc, Nanjundan Ashok Kima S-K. Preparation and characterization of carbon nanotube-grafted-chitosan – Natural hydroxyapatite composite for bone tissue engineering. Carbohydr Polym. 2011;83(2):569-77. https://doi.org/10.1016/j.carbpol.2010.08.019.

Downloads

Published

2021-06-07

Issue

Vol. 58 (2021)

Section

FULL ARTICLE

License

Copyright (c) 2021 Brazilian Journal of Veterinary Research and Animal Science

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

The journal content is authorized under the Creative Commons  BY-NC-SA  license (summary of the license: https://creativecommons.org/licenses/by-nc-sa/4.0 | full text of the license: https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode) 

Funding data

How to Cite

1.
Paretsis NF, Gonçalves Junior V, Hazarbassanov NGT de Q, Marcondes GM, Plepis AM de G, Martins V da CA, et al. In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing. Braz. J. Vet. Res. Anim. Sci. [Internet]. 2021 Jun. 7 [cited 2024 Apr. 27];58:e179885. Available from: https://www.revistas.usp.br/bjvras/article/view/179885