Biocompatibility of various hydoxyapatite scaffolds evaluated by proliferation of rat's bone marrow mesenchymal stem cells: an in vitro study
Background: Scaffold (biomaterial) biocompatibility test should be performed in vitro prior to in vivo stem cell application in animal or clinical trial. These test consists of direct and indirect toxicity test (MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]). Those tests were used to identify cell morphological changes, cell-substrate adhesion impairment, and reduction in cell proliferation activity.
Methods: The tested scaffolds were hydroxyapatite-calcium sulphate (HA-CaSO4) (scaffold I), nano-particular HA paste (scaffold II), synthetic HA granule (scaffold III), bovine HA granule (scaffold IV), and morsellized bovine xenograft (scaffold V). Direct contact toxicity test and MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were performed on those groups. In direct contact toxicity test, we put granules of various scaffolds within plates and incubated together with mesenchymal stem cells (MSCs). In MTT assay we included phenol 20 mg/mL and 100 mg/mL group as positive control. Morphology, cell adhesion impairment, and cell growth were monitored daily until day-7. Cells counting in the direct contact toxicity test was conducted on day-7.
Results: There were no changes on 24 hours observation after direct contact. On day-7, an impairment of cell adhesion to plastic substrates, changes in cell morphology, and cell death were observed, especially in scaffold I, scaffold II, and scaffold V. In MTT assay, only scaffold I, phenol 20 mg/mL, and phenol 100 mg/mL showed more than 50% inhibition at 24-hour and 7-day-observation. Extracts from scaffold II, III, IV, and V did not affect the viability and proliferation of bone marrow MSCs (inhibition value < 50%). Scaffold II, III, IV and V were proven non-cytotoxic and have good biocompatibility in vitro, no statistical significant differences were observed among the scaffold groups (p > 0.05).
Conclusion: We understand which scaffold was nontoxic or the least toxic to MSCs in vitro. Scaffold IV (bovine HA granule) showed the least toxic effect to rat's bone marrow MSCs on direct contact test and MTT assay. (Med J Indones. 2013;22:202-8. doi: 10.13181/mji.v22i4.600)
Lehmann G, Palmero P, Cacciotti I, Pecci R, Campagnolo L, Bedini R, et al. Design, production and biocompatibility of nanostructured porous HAp and Si-HAp ceramics as three-dimensional scaffolds for stem cell culture and differentiation. Ceramics-Silikaty. 2010;54(2):90-6.
Hollister SJ, Taboas JM, Schek RM, Lin CY, Chu TM. Design and fabrication of bone tissue engineering scaffolds. In: Hollinger JO, Einhorn TA, Doll BA, Sfeir C, editors. Bone tissue engineering. Bocaraton: CRC Press; 2005.p.167-94.
Radu A, Eleonora C, Lucian A, Georgeta C, Virginia V, Cristiana T. In vitro biocompatibility testing of implantable biomaterials. Roum Biotechnol Lett. 2008;13(4):3863-72.
Nuss KM, von Rechenberg B. Biocompatibility issues with modern implants in bone – a review for clinical orthopedics. Open Orthop J. 2008;2:66-78. http://dx.doi.org/10.2174/1874325000802010066
Kim do K, Lee SJ, Cho TH, Hui P, Kwon MS, Hwang SJ. Comparison of a synthetic bone substitute composed of carbonated apatite with an anorganic bovine xenograft in particulate forms in a canine maxillary augmentation model. Clin Oral Implants Res. 2010;21(12):1334-44. http://dx.doi.org/10.1111/j.1600-0501.2010.01953.x
Bong S, Choon K, Kug S, Hyuk J, Hyun S, Sung S. Osteoduction at porous hydroxyapatite with variuos pore configurations. Biomaterials. 2000;21:1291-8. http://dx.doi.org/10.1016/S0142-9612(00)00030-2
Rhodes N. Biocompatibility testing of tissue engineered products. Vox Sanguinis. 2004;87:161-3. http://dx.doi.org/10.1111/j.1741-6892.2004.00477.x
Idris BS, Danmark S, Wistrand F, Arvidson K, Albertsson AC, Bolstad AI, et al. Biocompatibility of polyester scaffold with fibroblast and osteoblast-like cells for bone tissue engineering. J Bioactive Compatible Polymers. 2010;25(6):567-83. http://dx.doi.org/10.1177/0883911510381368
Silva GA, Marques AP, Gomes ME, Coutinho OP, Reis R, editors. Cytotoxicity screening of biodegradable polymeric systems. Texas: CRC Press; 2004.
Louisia S, Stromboni M, Meunier A, Sedel L, Petite H. Coral grafting supplemented with bone marrow. J Bone Joint Surg Br. 1999;81(4):719-24. http://dx.doi.org/10.1302/0301-620X.81B4.9358
Fotakis G, Timbrell JA. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett. 2006;160(2):171-7. http://dx.doi.org/10.1016/j.toxlet.2005.07.001
Zhu Y, Zhang X, Zhu J, Zhao Q, Li Y, Li W, et al. Cytotoxicity of phenol red in toxicity assays for carbon nanoparticles. Int J Mol Sci. 2012;13(10):12336-48. http://dx.doi.org/10.3390/ijms131012336
Dias RCM, Goes AM, Serakides R, Ayres E, Orefice RL. Porous biodegradable polyurethane nanocomposites: preparation, characterization, and biocompatibility tests. Mat Res. 2010;13(2):211-8. http://dx.doi.org/10.1590/S1516-14392010000200015
Kan I, Melamed E, Offen D. Integral therapeutic potential of bone marrow mesenchymal stem cells. Curr Drug Targets. 2005;6(1):31-41. http://dx.doi.org/10.2174/1389450053344902
Mastrangelo F, Nargi E, Carone L, Dolci M, Caciagli F, Ciccarelli R, et al. Tridimensional response of human follicular dental stem cells onto synthetic hydroxyapatite scaffolds. J Health Sci. 2008;54(2):154-61. http://dx.doi.org/10.1248/jhs.54.154
Rauschmann MA, Wichelhaus TA, Stirnal V, Dingeldein E, Zichner L, Schnettler R, et al. Nanocrystalline hydroxyapatite and calcium sulphate as biodegradable composite carrier material for local delivery of antibiotics in bone infections. Biomaterial. 2005;26(15):2677-84. http://dx.doi.org/10.1016/j.biomaterials.2004.06.045
Zheng W. Preparation and characterization of tri-calcium phosphate scaffolds with tunnel-like macro-pores for bone tissue engineering [thesis]. Mount Pleasant (MI): Queensland Univ of Technology; 2011.
Copyright (c) 2013 Achmad F. Kamal, Diah Iskandriati, Ismail H. Dilogo, Nurjati C. Siregar, Errol U. Hutagalung, R. Susworo, Achmad A. Yusuf, Adang Bachtiar
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