Materials
Carboxyl NDs and 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM) were purchased from Tokyo Chemical Industry Co., Ltd. (TCI, Tokyo, Japan). Alendronate (Alen) was provided by Samjin Pharm. Co. Ltd. (Seoul, Korea). Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan, Korea). Formaldehyde solution (4%) was purchased from T&I (Chuncheon, Korea). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories Co. Ltd. (Kumamoto, Japan).
Synthesis of Alen-NDs
Alen-NDs were synthesized by forming an amide bond using DMTMM as a coupling reagent. ND powders (200 mg) were dispersed in 50 mL of deionized water. For conjugation of Alen onto the surface of NDs, DMTMM (10 mg) and Alen (100 mg) were added into the aqueous NDs dispersion, and stirred for 24 h at 60 °C, followed by centrifugation for purification with distilled water three times. The sample was then finally freeze-dried. To characterize ND-based nanoparticles, the dynamic light scattering method (Zeta-sizer Nano ZS, Malvern Instruments Ltd., Malvern, UK) was used to measure their sizes and zeta-potentials. Scanning electron microscopy (SEM, S-4800, Hitachi, Tokyo, Japan) was used to analyze their morphologies.
Cell viability and proliferation
The cell viability of mouse fibroblast (NIH/3T3) and mouse calvaria-derived preosteoblast (MC3T3-E1) cells were evaluated after the treatment of NDs and Alen-NDs. Aqueous dispersions of NDs (100 μg/mL) and Alen-NDs (10, 50, and 100 μg/mL) were added to each well containing 2 × 104 cells/mL in a 96-well plate and incubated in a humidified atmosphere containing 5% CO2 at 37 °C. The culture medium consisted of DMEM supplemented with heat-inactivated 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). The number of cells was evaluated using the CCK-8 assay with respect to time. The CCK-8 solution (20 μL) was added to each well of the 96-well plates and maintained in an incubator for 1 h [26]. The sample extracts were transferred to 96-well plates, and their absorbances at 450 nm were measured by a microplate reader (Spectra Max Plus 384, Molecular Devices, Co. Ltd., Philadelphia, USA) [27]. The cell viability (%) was determined as the percentage of the absorbance of the treated group divided by the absorbance of the non-treated group (control) under the same condition.
Cell proliferation was measured using the CCK-8 assay at (1, 3, 5, and 7) days after cell seeding (2 × 104 cells/mL). Aqueous dispersions of NDs (100 μg/mL) and Alen-NDs (only DMEM, 10, 50, and 100 μg/mL) were added to each well and cell proliferation was analyzed by the same method as in cell viability analysis.
Cell morphology and differentiation
To confirm the morphological change of MC3T3-E1 cells, the cells (2 × 104 cells/mL) were cultured in an 8-well Cell Culture Slide (SPL Life Sciences, Pocheon, Korea) containing 100 μL of the culture medium in an incubator. Aqueous dispersions of the only DMEM (control), NDs, and Alen-NDs (100 μg/mL) were added to each well and allowed to grow for 7 days. The culture slides were gently washed with PBS, fixed with formaldehyde solution (4%) for 15 min at room temperature, and rinsed 3 times with PBS. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min and stained with rhodamine for 1 h in dark. The fluorescence was visualized by confocal microscopy (LSM710, Carl Zeiss, Oberkochen, Germany), and the cellular dimensions were analyzed using ImageJ® software (National Institutes of Health, Bethesda, USA).
The osteogenic differentiation was confirmed by fluorescence-activated cell sorting analysis (FACS, FACS Canto II, BD Biosciences, San Jose, USA). MC3T3-E1 cells (1 × 105 cells/mL) were cultured after treatment by NDs and Alen-NDs (100 μg/mL) in the medium for 7 days. Then, anti-CD44, anti-CD51, and anti-CD45 antibodies (eBioscience) were treated to each well for 30 min on ice. After staining, cells were fixed with paraformaldehyde (2%) and analyzed with a FACS. Data were analyzed with FlowJo software (v. 10.1, FlowJo LLC, USA).
ALP activity
To evaluate early osteogenic differentiation, MC3T3-E1 cells were seeded on a 24-well culture plate at a concentration of 1 × 105 cells/mL, and incubated in culture medium (C.M., DMEM supplemented with 10% FBS and 1% antibiotics), osteogenic medium (O.M., C.M. supplemented with 50 μg/mL ascorbic acid, 10 nM dexamethasone, and 10 mM 𝛽-glycerophosphate), and culture medium containing NDs (100 μg/mL) or Alen-NDs (25, 50, and 100 μg/mL). The cells were obtained at predetermined time points of 3, 7, and 10 days. The cells were lysed using 1× RIPA (radioimmunoprecipitation assay) buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% Tergitol-type-40 (NP-40), and 1 mM ethylenediaminetetraacetic acid (EDTA) including protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin)]. The cell lysates were centrifuged at 13,500 rpm for 3 min at 4 °C. The supernatants were incubated with p-nitrophenyl phosphate solution for 30 min at 37 °C. The reaction was stopped by the addition of 500 μL of 1 N NaOH. ALP activity was determined by measuring the conversion of p-nitrophenyl phosphate to p-nitrophenol [28]. Optical density was determined by a microplate reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 405 nm.
Calcium contents
To evaluate late osteogenic differentiation, MC3T3-E1 cells were seeded at a concentration of 1 × 105 cells/mL on a 24-well culture plate. The cells were incubated in C.M., O.M., and culture medium containing NDs. After 21 days of culture, the cells were washed with PBS, treated with 0.5 N HCl, and centrifuged at 13,500 rpm for 1 min. The resulting supernatant was used for calcium deposition measurement using a QuantiChrom Calcium Assay Kit (DICA-500, BioAssay Systems, Hayward, CA, USA), according to the manufacturer’s instructions. The amount of calcium produced was determined at 612 nm by a microplate reader.
Gene expression
To evaluate the mRNA expression of the osteogenic differentiation markers (Runx-2, osteocalcin, and osteopontin), we performed a real-time polymerase chain reaction (RT-PCR). MC3T3-E1 cells (1 × 105 cells/mL) were incubated in C.M., O.M., and culture medium containing NDs (100 μg/mL) and Alen-NDs (25, 50, and 100 μg/mL) in a 24-well culture plate. After 7 and 21 days of culture, cDNA was synthesized with 1 μg total RNA and oligo (dT) primer using the Superscript First-Strand Synthesis System (Bioneer Inc., Daejeon, Korea), according to the manufacturer’s instructions. The following oligonucleotide primers were used for RT-PCR: Runx-2, (F) 5′-ATG GCA TCA AAC AGC CTC TTC AGC A-3′, (R) 5′-CGT GGG TTC TGA GGC GGG ACA CC-3′; ALP, (F) 5′-GTG GAA GGA GGC AGA ATT GAC CA-3′, (R) 5′-AGG CCC ATT GCC ATA CAG GAT GG-3′; OCN, (F) 5′-TGA GAG CCC TCA CAC TCC TC 3′, (R) 5′-ACC TTTGCT GGA CTC TGC AC-3′; OPN, (F) 5′-GAG GGC TTG GTT GTC AGC-3′, (R) 5′-CAA TTC TCA TGG TAG TGA GTT TTC C-3′; GAPDH, (F) 5′-ACT TTG TCA AGC TCA TTT CC-3′, and (R) 5′-TGC AGC GAA CTT TAT TGA TG-3′. PCR amplification and detection were carried out on an ABI7300 Real-Time Thermal Cycler (Applied Biosystems, Foster, CA, USA) with the DyNAmo SYBR Green qPCR Kit (Finnzymes, Espoo, Finland). The relative mRNA expression levels of Runx-2, osteocalcin, and osteopontin were normalized to that of GAPDH. All results were confirmed by repeating the experiment three times.
Statics
Quantitative data are presented as the mean ± standard deviation and comparisons were carried out using one-way ANOVA (Systat Software Inc., Chicago, IL, USA). Differences were considered statistically significant at 𝑃 < 0.05 (∗∗𝑃 < 0.01, ∗∗∗𝑃 < 0.001).