The effect of phosphate content in soda-lime-phospho-silicate bioactive glass on osteogenesis was investigated for the first time.
Apatite formation and osteogenesis of bioactive glasses can be enhanced by increasing phosphate content.
High phosphate content BG (P5.07) enhanced bone formation in rat calvarial defect at 8 w comparing to phosphate free BG (P0.00).
In vitro bioactivity investigations can potentially be used for the prediction of in vitro and in vivo bone formation ability of bioactive glasses.
High phosphate content was found to significantly increase apatite formation of bioactive glasses (BGs) in vitro . However, there is very limited understanding of the effect of phosphate contents on osteogenesis which is important for clinical applications. The aims of this study were to investigate how phosphate content influences apatite formation ability of bioactive glasses in α-MEM culture medium and whether high phosphate content in bioactive glasses promotes osteogenesis in vitro and in vivo.
Four phosphate containing bioactive glasses were synthesized via a melt-quench method and characterized using X-ray powder Diffraction (XRD), TGA-DSC and Fourier transform infra-red spectroscopy (FTIR). The apatite formation ability in α-MEM culture medium and the in vitro and in vivo osteogenic potential of these bioactive glass were explored.
FTIR spectra confirmed faster apatite formation with an increase in phosphate content. The culture media containing ions released from the BGs showed enhanced cell viability and alkaline phosphatase activity of osteoblasts. Osteoblasts cultured with extracted BGs culture media generally showed increased proliferation, mineralized nodule formation, osteogenic and angiogenic genes expression with an increase in phosphate content in the glass compositions. An in vivo study demonstrated a larger amount of new bone formation in the calvarial defects implanted with high phosphate containing BG granules compared with that of BG without the presence of phosphate at 8 weeks post-surgery.
The presence of higher phosphate content accelerates apatite formation and promotes osteogenesis, indicating that both apatite formation and osteogenesis of bioactive glasses can be tailored by varying phosphate content for specific clinical needs and personalized treatments.
Bone loss and defects caused by oral diseases, for instant periodontitis, peri-implantitis, and maxillofacial defects are very common clinically and reduce the quality of life for patients. Autologous and allogenic bone grafts are routinely applied as a treatment. However, these materials are associated with problems, such as insufficient source and immune rejection [ ]. Therefore, alternative synthetic biomaterials are required, with good biocompatibility and the ability to promote osteogenesis [ ].
The first bioactive glass (BG), 45S5 Bioglass® was developed in 1969 by Hench et al. [ ] to fill voids in the damaged bone without adverse effect from human body. 45S5 Bioglass® comprises in mol% 46.1SiO 2 -24.4Na 2 O-26.9CaO-2.6P 2 O 5, can degrade and lead to rapid release of ions ( e.g. , Si 4+ , Ca 2+ , PO 4 3− and Na + ) and the formation of a hydroxycarbonated apatite (HCA) layer when in contact with simulated body fluid (SBF) [ ]. The released ions were reported to stimulate osteogenic genes associated with the differentiation of osteoblasts [ ] and the formed HCA can bond to collagen fibrils and facilitate the development of a strong interface between the glass and the host bone [ ].
Despite the fact that 45S5 Bioglass® has been widely used in orthopedics and dentistry since 1985 [ ], the apatite formation is relatively slow. Hill and his group designed new BGs with different phosphate contents based on 45S5 Bioglass® and found that high phosphate contents (6.33 mol%) in BG showed superior bioactivity in term of faster apatite formation in SBF in comparison to 45S5 Bioglass® (phosphate content of 2.6 mol%) [ ]. It is believed that glass structure and composition have a strong influence on glass bioactivity, and an enhanced glass degradation can be achieved when a glass moves from a three-dimensional network to a more disrupted linear chain or end members structures [ ]. Edén [ ] proposed that bioactivity can be increased monotonically with an increase in phosphorus content of the bioactive glass providing phosphorus remains predominantly as orthophosphate and the network connectivity (NC) is less than 2.6. O’Donnell et al. [ ] investigated the influence of phosphate content on the bioactivity of a SiO 2 -Na 2 O-CaO-P 2 O 5 system with a constant glass network connectivity and demonstrated faster formation of HCA with an increase in the phosphate content.
In vitro degradation and apatite formation in SBF have been used as an indicator for the formation of an intimate bond to host bone in vivo, however, there is a lack of evidence on the reliability of estimating BG bioactivity at cytology, animal and human levels based on bioactivity studies in SBF. More importantly, there is no study on evaluating the effect of phosphate contents in BG on osteogenesis in vitro and in vivo yet.
In order to better inform and predict the clinical success of soda-lime-phospho-silicate BGs, understanding of their influence on osteogenesis, how well do they perform in animal models, and the correlation between the in vitro and in vivo results are vital. Here, it was hypothesized that high phosphate content in bioactive glasses promotes apatite formation in culture media and facilitates osteogenesis in vitro and in vivo . The inﬂuence of phosphate on glass bioactivity in α-MEM was evaluated. More importantly, in vitro response of osteoblast cell MC3T3-E1 and in vivo response of calvarial defects to the bioactive glasses with different phosphate contents were investigated.
Materials and methods
Bioactive glass fabrication
BGs in the system of SiO 2 -P 2 O 5 -CaO-Na 2 O designed by O’Donnell et al. [ ] ( Table 1 ) were synthesized by a melt-quench method. Briefly, a mixture of glass reagents was melted in a platinum/rhodium crucible at 1395 °C for 1 h and quenched into water to prevent crystallization. The glass frits were dried, ground and sieved. The collected glass powders with a particle size less than 38 μm were used for further bioactivity and in vitro studies according to [ ] and the unified in vitro evaluation method for glass bioactivity recommended by Technical Committee 4 (TC04) of the International Commission on Glass (ICG) [ ]. Glass granules with a particle size between 63 and 90 μm were applied for animal study based on our preliminary results as larger glass granules with significantly reduced surface areas are more appropriate for a balanced glass degradation and new bone formation.
|Glass Code||SiO 2||CaO||Na 2 O||P 2 O 5|
Bioactive glass characterization
The amorphous structure of the glass was confirmed by X-ray Diffraction (XRD) using Philips X’Pert PRO theta-theta PW3050/60 diffractometer with PW3064 sample spinner and X’Celerator (2.122° active length) 1D-detector in Bragg-Brentano geometry employing a Copper Line Focus X-ray tube with Ni kβ absorber (0.02 mm; Kβ = 1.392250 Å) Kα radiation (Kα1 = 1.540598 Å, Kα2 = 1.544426 Å, Kα ratio 0.5, Kαav = 1.541874 Å). Data was collected at 5−70° 2 theta with a step size of 0.033° and a step time of 200 s/step. The sample was mounted on a silicon low background holder. Fourier Transform Infrared Spectroscopy (FTIR, ALPHA II, Bruker UK) was used to collect spectra in the range of 500 to 1600 cm −1 of wavenumber at a resolution of 4 cm −1 .
The glass transition temperature (T g ) was determined using TGA-DSC (TGA/DSC 3+, Switzerland). Glass frit (40 mg) was heated at a rate of 20 °C/min under nitrogen (a flow a rate of 60 mL/min) from 25 to 1100 °C in a Pt crucible. T g was extracted from the DSC traces with an accuracy of ±5 °C and compared with data reported previously by O’Donnell et al. [ ].
Glass powders (75 mg each) were dispersed in 50 mL α-MEM cell culture medium, corresponding to a concentration of 1.5 g/L [ , ]. All samples were placed in an orbital shaking incubator (IKA®KS 4000i control, Germany) at 37 °C with an agitation rate of 60 rpm for 4, 12, 24 and 72 h and then filtered to separate BGs conditioned-culture media and solids. The pH of the extracted BGs culture media was measured using a pH meter (OHAUS, China). Two separate samples from each immersion point of interest were prepared for all studied glasses.
The apatite formation ability of studied bioactive glasses in α-MEM cell culture medium was evaluated using FTIR ALPHA II (Bruker, Germany). The extracted cell culture media containing ions released from BGs were diluted by a factor of 10 in deionized water for the ion concentration quantification of calcium, silicon and phosphorus using an inductively coupled plasma optical emission spectrometer (ICP-OES, PerkinElmer, USA). The ions concentration of each BGs conditioned-culture medium was measured in triplicate, the Mean ± SD of the 6 measurements were presented as measured ion concentration for the BG conditioned-culture medium at the immersion point of interest.
The investigation of osteogenesis of osteoblasts in vitro
The filtered BG conditioned α-MEM media were sterilized by passing through a 0.2 μm filter and supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin [ ]. The prepared BGs conditioned culture media were used for the following in vitro investigation of osteogenic and angiogenic effects.
Pre-osteoblasts (MC3T3-E1) cells purchased from Shanghai cell bank (ATCC) were incubated in α-MEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin [ ] in a humidified CO 2 incubator at 37 °C. Culture medium was changed and replenished with fresh medium every two days till cells grew to 80%–90% confluence.
Cell viability of MC3T3-E1cells
The MC3T3-E1 cells were seeded into 96-well plates at a density of 1 × 10 3 cells/well and incubated at 37 °C overnight. Cells were treated with extract BGs conditioned culture media and the control group (α-MEM with supplements). After 1, 3 and 7 d incubation, cell viability was assessed by the cell counting kit-8 (CCK-8) test according to the manufacturers’ instructions (Meilunbio®, China), and the absorbance was quantified at 450 nm using a UV-Vis Absorption Spectroscopy (Bio Terk, USA).
Quantitative alkaline phosphatase activity (ALP)
ALP activity was determined by enzyme histochemical assay [ ]. After 7, 10 and 14 d culture, the cells were lysed by freezing and thawing, and then reacted with 2.5 mg/mL of 4-nitrophenyl phosphate disodium hexahydrate containing 1 mM MgCl 2 (pH = 9.5) for 1 h. The reaction was finally stopped with 0.5 M NaOH and the absorbance was quantified at 405 nm.
The mineralization abilities of BGs with different phosphorus contents (0–5.07 mol%) were assessed by Alizarin Red S staining. Cells were cultured in extracted BG conditioned culture media with 50 μg/mL L-ascorbic acid and 5 mM β-glycerophosphate for 14 to 28 d. Plates were then stained with 2% Alizarin Red S (Solarbio, China) accordingly [ ] and the absorbance of the resulting solution was measured at 570 nm.
Quantitative real-time polymerase chain reaction (RT-qPCR)
After 4, 7, 14 and 21 d culture, the expression of osteogenic genes (OPN, OCN and RUNX2) and angiogenic genes (VEGF) were evaluated by RT-qPCR as described by Liu et al. [ ]. Briefly, according to the manufacturer’s instruction, ReverTra Ace qPCR RT Master Mix reagents (TOYOBO, Japan) were used to reverse RNA into cDNA. Subsequently, 100 ng of total cDNA was applied for RT-qPCR analysis using KOD SYBR® qPCR Mix reagents (TOYOBO, Japan). Finally, the relative expression was calculated using ΔΔCt method against endogenous reference, GAPDH. The fold change was determined from 2 −ΔΔCt . All primer sequences were presented in Table S1. Note that the genes expression of control group at different culturing days was set as 1 respectively. The expression level of the BGs-contained culture media were normalized and compared against the control with the same culturing days.
MC3T3-E1 cells were treated with extracted BGs conditioned culture media for 7, 14 and 21 d. At the end of the treatment, cells were collected and lysed with RIPA lysate buffer (Beyotime, China) containing protease inhibitor. The BCA protein assay kit (Thermo Scientific, USA) was used to analyze the protein concentrations. Same amount of protein by weight was subjected to Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were blocked in 3% BSA and incubated with primary antibody (Abcam, UK) at 4 °C overnight and then HRP-conjugated secondary antibody (Abcam, UK) at room temperature for 1 h. Protein bands were detected with a Super Sensitive ECL Luminescence Reagent (Meilunbio, China) and visualized on an enhanced chemiluminescence (ECL) detection system (Tanon, China). The optical densities of the bands were measured by ImageJ software. GAPDH was used as a loading control. The protein secretion level of control group at different culturing day was set as 1 respectively, while the protein concentration of the BGs were normalized and compare against the control with the same culturing days.
After 7 and 14 d in culture, cells were fixed with 4% paraformaldehyde (PFA) for 15 min, blocked with 3% BSA in PBST at 4 °C overnight and then incubated with OPN antibody for 1 h at room temperature. Cells were washed with PBST for 3 times and incubated with Alexa Fluor® 455 (green) goat anti-rabbit IgG (H + L) antibody for 1 h at room temperature. After further washing with PBST for 3 times, cells were labelled with 4, 6-diamino-2-phenyl indole (DAPI) in PBS for 5 min. Finally, the fluorescent images were photographed by a fluorescent microscope (Carl Zeiss, Germany).
In vivo investigation of new bone formation in rat critical-sized calvarial defects
The animal experiment was approved by the Animal Research Ethics Committee of the Third Xiang Ya Hospital of Central South University (Certificate No 2019SYDW0154). Six male Sprague-Dawley (SD) rats (220–250 g in weight, 8–10 w old) were randomly divided into 2 groups (P0.00 group and P5.07 group) for an eight-week in vivo study. We followed the surgical procedures as described previously [ , ]. Briefly, Rats were anesthetized with intraperitoneal injection of 1% sodium pentobarbital (3.5 mg/100 g), around 1.5 cm sagittal incision was made in the scalp and the calvarium was exposed by blunt dissection. Two critical size calvarial defects (5 mm in diameter) with distance of 4–5 mm in the central area of each parietal bone were created using a dental bone drill. BG granules were implanted in the created defects accordingly with and the soft tissues were sutured. After surgery, 80,000 units of penicillin were injected intramuscularly to prevent infection. The rats were sacrificed at 8 w post-surgery and the parietal bones with BG granules were harvested and fixed in 4% paraformaldehyde solution for further micro-CT scan.
Micro-computed tomography (micro-CT)
The evaluation of new bone formation in rat calvarial defects after 8 w post-surgery was conducted using a micro-CT (SCANCO MEDICAL AG/vivaCT80, Switzerland). Brifely, the settings of X-ray tube were 55 kV and 145 μA. The harvested bone specimens were scanned at a resolution of 11.4 μm and a 5.0 mm in diameter region of the interest (ROI) was defined to evaluate the extent of bone formation. After 3D reconstruction, the new bone volume to total bone volume (BV/TV) were analyzed using the SkyScan software, accordingly [ ].
Data were presented as Mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to perform statistical analysis. A value of p < 0.05 was considered as statistically significant.
Material characterization and apatite formation
XRD patterns ( Fig. 1 a) show the characteristic amorphous halo with no signs of crystallization peaks for all studied BGs indicating the glassy nature of the materials. FTIR also demonstrate typical glassy spectra (Fig. S1). This is in agreement with the previous studies of O’Donnell et al. [ ]. Glass transition temperature (T g ) extracted from DSC traces were plotted against phosphate content in the BGs as shown in Fig. 1 b. It is clear that T g decreases with an increase in phosphate content which are also in good agreement with those measured by O’Donnell et al. [ ], suggesting that the synthesized glasses are the same as what O’Donnell achieved so that the results obtained on this work can be compared with the in vitro study carried out previously [ ]. The slight difference in values could be attributed to the use of different DSC instruments.