Physicochemical properties and cytocompatibility of biomimetically precipitated nanocrystalline calcium phosphate granules

doi:10.12307/2024.487

Abstract

Abstract: BACKGROUND: Artificially synthesized hydroxyapatite ceramic granules are widely used in clinical practice to repair large-volume bone defects. However, the osteogenic effect of hydroxyapatite ceramic granules prepared by high-temperature sintering is limited by their low degradability and bioactivity.
OBJECTIVE: To prepare biomimetically precipitated nanocrystalline calcium phosphate granules by a novel low-temperature deposition technique, and to characterize their physicochemical properties and cytocompatibility.
METHODS: Biomimetically precipitated nanocrystalline calcium phosphate granules were prepared using a modified supersaturated calcium phosphate mineralization solution and a repeated settling and decantation washing method. Hydroxyapatite bioceramic granules were used as the control. The morphology and phase composition of the granules were characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The specific surface area, porosity distribution, hardness and hydrophilicity of the granules were characterized by BET-N2 method, hardness test, and contact angle test. The adsorption properties of the granules for bovine serum albumin and fetal bovine serum protein were determined by bicinchoninic acid assay. The two kinds of granules or granule extracts were co-cultured with human umbilical cord mesenchymal stem cells, and the cell proliferation was detected by MTT assay.
RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed that the surface of the two kinds of particles was slightly rough and accompanied by tiny particles, the surface of the hydroxyapatite bioceramic particles was dense and smooth, and the biomimetically precipitated nanocrystalline calcium phosphate granules were mainly composed of needle/plate crystals with non-uniform nanometer size, and formed a nanopore structure between the crystals. X-ray diffraction and Fourier transform infrared spectroscopy exhibited that compared with hydroxyapatite bioceramic granules, biomimetically precipitated nanocrystalline calcium phosphate granules had smaller crystalline particles, lower crystallinity, and more binding water and carbonic acid groups. Compared with hydroxyapatite bioceramic granules, biomimetically precipitated nanocrystalline calcium phosphate granules had higher specific surface area, better hydrophilicity, lower hardness, and higher protein adsorption capacity. (2) The results of MTT assay showed that the two kinds of granule extracts had no cytotoxicity, human umbilical cord mesenchymal stem cells survived well on the surface of the two kinds of granules, and the biomimetically precipitated nanocrystalline calcium phosphate granules had stronger cell proliferation activity. (3) These findings indicate that compared with hydroxyapatite bioceramic granules, biomimetically precipitated nanocrystalline calcium phosphate granules have better physicochemical properties and cytocompatibility.

Key words: biomimetically precipitated nanocrystalline calcium phosphate granule, hydroxyapatite, bone regeneration and repair, biocompatibility, protein adsorption, human umbilical cord mesenchymal stem cell

CLC Number:

Chen Mingxue, Niu Jianhua, Lin Haiyan, Wu Gang, Wan Ben. Physicochemical properties and cytocompatibility of biomimetically precipitated nanocrystalline calcium phosphate granules[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(22): 3502-3508.

Figures/Tables 9

References

[1] POH PS, LINGNER T, KALKHOF S, et al. Enabling technologies towards personalization of scaffolds for large bone defect regeneration. Curr Opin Biotechnol. 2022;74:263-270.
[2] SHI H, ZHOU Z, LI W, et al. Hydroxyapatite based materials for bone tissue engineering: A brief and comprehensive introduction. Crystals. 2021;11(2):149.
[3] BALDWIN P, LI D J, AUSTON DA, et al. Autograft, allograft, and bone graft substitutes: clinical evidence and indications for use in the setting of orthopaedic trauma surgery. J Orthop Trauma. 2019;33(4):203-213.
[4] WANG W, YEUNG KW. Bone grafts and biomaterials substitutes for bone defect repair: A review. Bioact Mater. 2017;2(4):224-247.
[5] BALDINI N, DE SANCTIS M, FERRARI M. Deproteinized bovine bone in periodontal and implant surgery. Dent Mater. 2011;27(1):61-70.
[6] PERIĆ KAČAREVIĆ Z, KAVEHEI F, HOUSHMAND A, et al. Purification processes of xenogeneic bone substitutes and their impact on tissue reactions and regeneration. Int J Artif Organs. 2018;41(11):789-800.
[7] RODRÍGUEZ-LUGO V, KARTHIK T, MENDOZA-ANAYA D, et al. Wet chemical synthesis of nanocrystalline hydroxyapatite flakes: effect of pH and sintering temperature on structural and morphological properties. R Soc Open Sci. 2018;5(8):180962.
[8] SARTORI S, SILVESTRI M, FORNI F, et al. Ten‐year follow‐up in a maxillary sinus augmentation using anorganic bovine bone (Bio‐Oss). A case report with histomorphometric evaluation. Clin Oral Implants Res. 2003;14(3):369-372.
[9] MATHINA M, SHINYJOY E, KAVITHA L, et al. A comparative study of naturally and synthetically derived bioceramics for biomedical applications. Mater Today Proc. 2020;26:3600-3603.
[10] 李忠杰,李绍波.磷酸钙人工骨修复骨缺损的研究进展[J].生物骨科材料与临床研究,2021,18(4): 87-91.
[11] GEORGE SM, NAYAK C, SINGH I, et al. Multifunctional hydroxyapatite composites for orthopedic applications: a review. ACS Biomater Sci Eng. 2022;8(8):3162-3186.
[12] YUAN H, VAN BLITTERSWIJK C, DE GROOT K, et al. A comparison of bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) implanted in muscle and bone of dogs at different time periods. J Biomed Mater Res A. 2006;78(1):139-147.
[13] SCALERA F, GERVASO F, SANOSH K, et al. Influence of the calcination temperature on morphological and mechanical properties of highly porous hydroxyapatite scaffolds. Ceram Int. 2013;39(5):4839-4846.
[14] ZHENG Y, WU G, LIU T, et al. A novel BMP2‐coprecipitated, layer‐by‐layer assembled biomimetic calcium phosphate particle: A biodegradable and highly efficient osteoinducer. Clin Implant Dent Relat Res. 2014;16(5):643-654.
[15] LIU T, ZHENG Y, WU G, et al. BMP2-coprecipitated calcium phosphate granules enhance osteoinductivity of deproteinized bovine bone, and bone formation during critical-sized bone defect healing. Sci Rep. 2017;7(1):1-12.
[16] XU G, SHEN C, LIN H, et al. Development, in-vitro characterization and in-vivo osteoinductive efficacy of a novel biomimetically-precipitated nanocrystalline calcium phosphate with internally-incorporated bone morphogenetic protein-2. Front Bioeng Biotechnol. 2022;10: 920696.
[17] WAN B, RUAN Y, SHEN C, et al. Biomimetically precipitated nanocrystalline hydroxyapatite. Nano TransMed. 2022;1(2-4):e9130008.
[18] DUAN R, BARBIERI D, LUO X, et al. Variation of the bone forming ability with the physicochemical properties of calcium phosphate bone substitutes. Biomater Sci. 2018; 6(1):136-145.
[19] ZHU X, FAN H, XIAO Y, et al. Effect of surface structure on protein adsorption to biphasic calcium-phosphate ceramics in vitro and in vivo. Acta Biomater. 2009;5(4):1311-1318.
[20] LI X, DENG Y, CHEN X, et al. Gelatinizing technology combined with gas foaming to fabricate porous spherical hydroxyapatite bioceramic granules. Mater Lett. 2016;185: 428-431.
[21] XU G, WANG T, SHEN C, et al. In-vitro physicochemical characterization of a novel type of bone-defect-filling granules-BpNcCaP in comparison to deproteinized bovine bone (Bio-Oss®). Nano TransMed. 2023;2(1):e9130016.
[22] ŚLÓSARCZYK A, PASZKIEWICZ Z, PALUSZKIEWICZ C. FTIR and XRD evaluation of carbonated hydroxyapatite powders synthesized by wet methods. J Mol Struct. 2005;744:657-661.
[23] 陈鹏.结晶度对羟基磷灰石成骨和骨诱导性影响的研究[D].广州:华南理工大学, 2019.
[24] DALL’ARA E, ÖHMAN C, BALEANI M, et al. The effect of tissue condition and applied load on Vickers hardness of human trabecular bone. J Biomech. 2007;40(14):3267-3270.
[25] ZHU W, IMAMURA H, MARIN E, et al. Effects of annealing in air on microstructure and hardness of hydroxyapatite ceramics. J Phys D Appl Phys. 2021;54(31):315301.
[26] XU Z, LIU C, WEI J, et al. Effects of four types of hydroxyapatite nanoparticles with different nanocrystal morphologies and sizes on apoptosis in rat osteoblasts. J Appl Toxicol. 2012;32(6):429-435.
[27] PATEL N, GIBSON IR, KE S, et al. Calcining influence on the powder properties of hydroxyapatite. J Mater Sci Mater Med. 2001;12:181-188.
[28] ONI OP, HU Y, TANG S, et al. Syntheses and applications of mesoporous hydroxyapatite: a review. Mater Chem Front. 2023;7:9-43.
[29] CHAKRABORTY PK, ADHIKARI J, SAHA P. Variation of the properties of sol–gel synthesized bioactive glass 45S5 in organic and inorganic acid catalysts. Mater Adv. 2021;2(1):413-425.
[30] NAGASAKI T, NAGATA F, SAKURAI M, et al. Effects of pore distribution of hydroxyapatite particles on their protein adsorption behavior. J Asian Ceram Soc. 2017;5(2):88-93.
[31] WANG K, ZHOU C, HONG Y, et al. A review of protein adsorption on bioceramics. Interface Focus. 2012;2(3):259-277.
[32] KLIMEK K, BELCARZ A, PAZIK R, et al. “False” cytotoxicity of ions-adsorbing hydroxyapatite—Corrected method of cytotoxicity evaluation for ceramics of high specific surface area. Mater Sci Eng C. 2016;65:70-79.