The main objective of this study is to characterize the thermal, mineralization, and osteoblast cells response of pearl nanocrystallites. The results obtained from X-ray diffraction, FTIR spectra demonstrate that the pearl nano-crystallites can induce the formation of an HA layer on their surface in SBF, even after only short soaking periods. The in vitro cell response to nano-grade pearl powders is assessed by evaluating the cytotoxicity against a mouse embryonic fibroblast cell line and by characterizing the attachment ability and alkaline phosphatase activity of mouse bone cells (MC3T3-E1, abbreviated to E1) and bone marrow stromal precursor (D1) cells. The cytotoxicities of pearls were tested by the filtration and culture of NIH-3T3 mouse embryonic fibroblast cells. The viability of the cultured cells in media containing pearl crystallites for 24 and 72 h is greater than 90%. The bone cells seen in these micrographs are elongated and align predominately along the pearl specimen. The cells observed in these images also appeared well attached and cover the surface almost completely after 1 h. The pearl nanocrystallites had a positive effect on the osteogenic ability of ALP activity, and this promoted the osteogenic differentiation of MSCs significantly at explanations.
Pearl, which is composed of nacre, is produced in an active physiological environment by molluscs. Pearl, nacre, and bone are all biomineralization products of organisms. Pearl and nacre are basically composed of calcium carbonate (aragonite, CaCO3), whereas bone mainly consists of calcium phosphate or hydroxyl apatite (HA); although their components are different, parts of the complex machineries that direct their formation may be homologous [
In this study, morphology evaluation using the Brunauer, Emmett, and Teller (BET) method was performed in conjunction with high resolution transmission electron microscope (HRTEM). The mineralization of pearl powders was tested by immersing the pearl samples in simulated body fluid (SBF). The formation of a crystalline phase on the surface of the pearls was identified by scanning electron microscopy and X-ray diffraction. The in vitro cell response to nanograde pearl powders is assessed by evaluating the cytotoxicity against a mouse embryonic fibroblast cell line and by characterizing the attachment ability and alkaline phosphatase activity of mouse bone cells (MC3T3-E1, abbreviated to E1) and bone marrow stromal precursor (D1) cells.
The nanoparticles were prepared using the wet polish method with a ball grinding machine (Just Nanotech Co., JBM-B035, Taiwan). A mixture of dry ingredients composed of 8 g of pearl powder and 1.6 g of dispersant was added to 240 mL of deionized water to make slurry. The slurry was premixed for 1.5 h and then placed in a grind chamber with 500 g of 0.1 mm zirconium particles for 1.0 h at 2000 rpm. After grinding, the slurry was passed through a 200 mesh sieve to remove the larger particles. The slurry was then dried by freeze-drying at 218 K under vacuum. The original morphologies of the specimens were characterized by scanning electron microscopy (SEM) as shown in Figure
TEM image of ground pearl powders.
Differential thermal and thermogravimetric analyses (DT/TGA) were conducted on powder samples in 50 mg quantities at heating rates of 5°C/min. Samples were heated from 25°C to 1200°C in air. Crystalline phases were identified using X-ray diffraction (XRD) analysis. XRD was performed using an X-ray diffractometer with Cu K
The mineralization of the pearl powders was assessed in a simulated body fluid (SBF) similar to human plasma [
The cytotoxicities of the MBGs were tested by filtration and culture of the fibroblast cells (NIH 3T3, abbreviated 3T3). The cells were provided by the National Institute of Health (NIH) in Taiwan. The 3T3 cells were derived from newborn mouse fibroblasts and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen Taiwan Ltd., MD) containing 10% bovine serum (BS) (Biological Industries, Haemek, Israel). An XTT Cell Viability Assay Kit provided a simple method to count live cells using an absorbance reader. The cells’ adhesive and reproductive abilities were measured at two early stages: 24 h and 72 h. After the cultured time, the cells on the sample surface were washed with phosphate-buffered saline (PBS) and transferred to 200
E1 cells were cultured on pearl disks placed in a 96-well culture plate at an initial density of 1 × 105 cells/cm2. The cells were incubated for 1, 24, 72, and 168 h. The pearl disks were then fixed in a 25% glutaraldehyde, 4% paraformaldehyde matrix for 1 h at 4°C. The cells were washed in wash buffer containing 4% sucrose in PBS and postfixed in 1% osmium tetroxide in PBS for 1 h at 4°C. The samples were then dehydrated sequentially in graded ethanol (30%, 50%, 70%, 95%, and 100% ethanol). The specimens were dried in hexamethyldisilazane (HMDS) for 3 min before they were coated with gold for SEM analysis. The morphological characteristics of the cells attached to the pearl disks were determined using field-emission SEM. To quantitatively evaluate the E1 cells adhered to the pearl disks over time, an alamarBlue assay kit (AbD Serotec, Oxford, UK) was used. After 1, 24, 72, and 168 h, the tested samples were washed with PBS and moved to a new plate. Then, 500 mL of culture medium and 50 mL of alamarBlue were added to the samples, and the samples were incubated for 2 h. After incubation, 100 mL of the reactants were extracted and measured using the ASYS UVM 340 microplate reader. The absorbance values of the reactants (
The ALP and TRAP activities were detected using a TRACP and ALP Double-Staining Kit (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Elevation in ALP activity in D1 cells reflects those osteogenic cells that were undergoing terminal differentiation. D1 cells were seeded at 1 × 105 cell/well in a 48-well plate. Cells were cultured for 1, 24, 72, and 168 h. D1 cells were harvested and washed with PBS. After washing, solutions of 500
The morphology of the milled pearl crystallites was characterized by transmission electron microscopy (TEM) as shown in Figure
DTA/TGA curves of pearl powders at a heating rate of 5°C/min in air.
One significant characteristic of bioactive materials is their ability to bond with living tissue, in this case bone. Bonding occurs through the formation of an HA interface layer on the surface, both in vitro and in vivo [
FE-SEM micrograph spectra of pearl powders before (a) and after being immersed in SBF for (b) 4 h, (c) 6 h, and (d) 24 h.
XRD patterns of pearl powders before (a) and after being immersed in SBF for 4 h (b), 6 h (c), and 24 h (d).
FTIR spectra of pearl powders (a) before and after being immersed in SBF for (b) 4 h, (c) 6 h, and (d) 24 h.
The in vitro cell response was assessed by evaluating the cytotoxicity of these materials against the mouse embryonic fibroblast cell line. Cell proliferation in the presence of nanograde pearl crystallites was evaluated using an XTT assay. Cells were assessed in both the absence and presence of nanograde pearl crystallites as a function of culture time. NIH-3T3 mouse embryonic fibroblast cells were cultured in media containing pearl crystallites for 24 and 72 h, as depicted in Figure
Viability of NIH-3T3 cells for various durations cultured for 72 h and immersed in the media. (
The cells were cultured for 1, 24, 72, and 168 h each to determine their adhesive and initial proliferative abilities. Figure
SEM images of the attachment of MC3T3-E1 cultured on pearls: (a) initial, (b) 1 h, (c) 24 h, (d) 72 h and (e) 168 h.
MC3T3-E1 cell numbers were counted after (a) 1 h (b) 24 h (c) 72 h, and (d) 168 h. (
The ALP activities were significantly larger over time when compared with the initial time points. The examination of ALP quantities via staining confirmed this phenomenon. A longer incubation time greatly increased the level of ALP (Figure
Light photos of the pearl promoted the ALP activity of cell line D1 at (a) initial (b) 24 h (c) 72 h and (d) 168 h.
Pearl promoted the ALP activity of cell line D1 at 24 h, 72 h and 168 h. (
The main objective of this study is to characterize the morphology, mineralization, and osteoblast cells response of pearl nano-crystallites. Results are summarized as follows. DTA/TG heating showed an endothermic peak at about 307°C accompanied by a 2% weight loss ascribed to the decomposition of the organic species of the materials included in the pearl powder. The exothermic peak at 439°C is due to the aragonite to calcite transformation of the CaCO3 nanoparticles. During the heating from 580 to 750°C, the weight loss and exothermic peak around 741°C are ascribed to the decomposition of CO2. X-ray diffraction (XRD), Fourier transform infrared (FTIR) absorption spectra, and analysis showed further evidence that pearls can induce the formation of an HA layer on their surface in SBF, even for short soaking periods. Nanograde pearl powders induced the formation of apatite layer on their surface after soaking in SBF for 4 h, which demonstrates the excellent in vitro bone forming bioactivity of nanograde pearls. The cytotoxicities of pearls were tested by the filtration and culture of NIH-3T3 mouse embryonic fibroblast cells. The viability of the cultured cells in media containing pearl crystallites for 24 and 72 h is greater than 90%. Bone cells adhered to the pearl disks, as evidenced by scanning electron micrographs. Specifically, these micrographs of E1 cells were adhered to pearl disks taken over a culture interval of 1 h. The osteoblastic cells shown in these micrographs are elongated, with their alignment predominately along the specimen. The cells shown in these images also appear well attached and cover the surface almost completely after 1 h. It is therefore believed that pearls can facilitate bone cell adhesion. The pearl nanocrystallites had a positive effect on the osteogenic ability of ALP activity, and this promoted the osteogenic differentiation of MSCs significantly at explanations.
J.-C. Chen and J.-C. Kung contributed equally to this work.
This work was financially support of a Grant from the National Science Council of Taiwan (NSC 98-2622-E-037-001-CC3). The authors also acknowledge the supported by Grants from the Kaohsiung Medical University Hospital (KMUH100-0 M25, KMUH100-0 M51) and Grants from the Kaohsiung Medical University Research Foundation (KMU-M110008, KMU-M102006).