Natural diatomite particles: Size-, dose- and shape- dependent cytotoxicity and reinforcing effect on injectable bone cement

doi:10.1016/j.jmst.2017.04.020

Abstract

Abstract:

Natural diatomite (DT) is the ancient deposit of diatom skeleton with many regular pores of 50-200 nm and also an abundant source of biogenic silica. Although silica is considered biologically safe and there is an increasing interest of using natural diatomite for biomedical applications, the toxicity information about natural diatomite is still missing. Here, cytotoxicity of natural diatomite on osteoblasts and fibroblasts were compared to hydroxyapatite and the relationships between cytotoxicity and diatomite sizes, dose, geometry or impurity were systematically investigated. Cell adhesion and interaction with diatomite particles were also fluorescently observed. The results clearly suggested a size-, dose- and shape-dependent cytotoxicity of natural diatomite. Disk-shaped diatomite particles with average size of 30 μm in diameter revealed the least toxicity, while the diatomite particles with irregular shapes and sizes less than 10 μm were remarkably toxic. Diatomite particles with proper sizes were then selected to investigate the reinforcing effect on injectable calcium phosphate bone cement. Results showed that diatomite significantly improved the compressive strength of bone cement but did not alter the injectability of the cement. This work provided important biocompatibility information of natural diatomite and demonstrated the feasibility of using selected diatomite as bone implant material.

Key words: Biocompatibility, Size and shape effect, Bone cement, Particle, Silica, Kyphoplasty

Xiang Zhang, Huilin Yang, Song Li, Gaowu Qin, Lei Yang. Natural diatomite particles: Size-, dose- and shape- dependent cytotoxicity and reinforcing effect on injectable bone cement[J]. J. Mater. Sci. Technol., 2018, 34(6): 1044-1053.

Figures/Tables 10

Fig. 1. SEM images and DLS results showing the morphology and particle size distributions of segregated natural diatomite: (a) unsegregated DT, (b) 30DT-disk, (c) 30DT-ring, (d) 25DT, (e) 10DT, and (f) 3DT.

Table 1 Size distribution and elemental composition of different diatomite and synthetic HA.

		DT	30DT-disk	30DT-ring	25DT	10DT	3DT	HA
Particle size (μm)	D₁₀	12.3	20.7	11.0	15.0	4.0	0.8	2.61
	D₅₀	31.9	32.7	28.1	25.5	9.7	3.6	11.27
	D₉₀	66.3	49.9	48.5	43.1	26.3	10.0	34.87
Impurity Content (ppm)	K	72	4.2	34.2	26.1	35.7	69.2	NT
	Fe	44.5	10.5	21.0	19.7	27.6	4.8	NT
	Al	OL	OL	OL	OL	OL	OL	NT

Fig. 2. Results of osteoblast (a and b) and fibroblast (c and d) proliferation on different diatomite samples and HA measured by CCK8 assay after culturing for 1 day (a and c) and 3 days (b and d). α-MEM + PBS was used as controls (indicated by the dash lines) and its proliferation rate on the first day was set to 100%, while results of diatomite and HA were normalized to it. Data = mean ± S.D. (n = 3), *p < 0.01.

Fig. 3. Toxic effects of diatomite and HA on osteoblasts (a and b) and fibroblasts (c and d) measured by LDH assay after culturing for 1 day (a and c) and 3 days (b and d). α-MEM + PBS was used as controls (indicated by the dash lines). Data = mean ± S.D. (n = 3), *p < 0.01.

Fig. 4. Fluorescence microscopy images of osteoblasts cultured on cover glass (a) and with 30DT-disk (b) and 3DT (c) particles at a concentration of 0.1 mg/ml after 1 day. Cells were pre-stained with Cell-tracker before cell culturing. (d) Average osteoblast spreading areas on glass, 30DT-disk and 3DT after culturing for 6 h, 12 h and 24 h, respectively. Data = mean ± S.D., *p < 0.01.

Fig. 5. Fluorescence microscopy images of fibroblasts cultured on cover glass (a) and with 30DT-disk (b) and 3DT (c) particles at a concentration of 0.1 mg/ml after 1 day. Cells were pre-stained with Cell-tracker before cell culturing. (d) Average fibroblast spreading areas on glass, 30DT-disk and 3DT after culturing for 6 h, 12 h and 24 h, respectively. Data = mean ± S.D., *p < 0.01.

Fig. 6. Interaction between osteoblasts and 30DT-disk particles. (a) and (d) Schematics of the position relationship between osteoblast and diatomite particle. (b) and (e) Optical microscopy images viewed from the bottom, while (c) and (f) fluorescence microscopy images viewed from the top. Cells are pre-stained and then cultured with diatomite particle for 12 h.

Fig. 7. SEM images of (a) DT, (b) SBF-treated DT and (c) HA-treated DT. Insets in (b) and (c) showing results of EDS analyses of the particle surface. XRD spectra of (d) DT, (e) SBF-treated DT and (f) HA-treated DT.

Table 2 Mechanical properties, injectability, and setting properties of diatomite-modified CPC.

	Compressive strength (MPa)	Compressive modulus (MPa)	Injectability (%)	Initial setting time, t_i (min)	Final setting time, t_f (min)
CPC	15.62 ± 0.98	748.04 ± 72.97	15 ± 1	3.61 ± 1.1	18.6 ± 1.2
Starch/CPC	18.73 ± 2.14	950.54 ± 215.54	\	18.4 ± 2.4	36.8 ± 3.1
SBF-treated DT30/CPC	25.17 ± 2.13	1404.96 ± 188.22	21 ± 0.5	6.74 ± 1.4	22.6 ± 2.3
HA-treated DT30/CPC	24.54 ± 1.42	1496.9 ± 231.92	28 ± 2.2	7.06 ± 1.6	24.5 ± 2.1
SBF-treated DT30/starch/CPC	29.47 ± 2.45	1711.24 ± 216.95	82 ± 2.2	20.2 ± 2.6	38.7 ± 3.4
HA-treated DT30/starch/CPC	34.74 ± 4.59	1771.36 ± 486.94	90 ± 2.2	21.2 ± 2.3	39.5 ± 2.4

Fig. 8. SEM images of (a) fracture surface of HA-treated 30DT/CPC and (b) osteoblast adhesion morphology on the HA-treated 30DT/CPC. Cells were cultured on the cement sample for 24 h.

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