Investigation of the biocompatibility and osteogenic effects of magnesium-doped chloride-containing bioactive glasses
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Abstract
Bioactive glass (BG) degrades in vivo, releasing therapeutic ions and forming an apatite-like phase to repair hard tissues. While many BG-derived materials have been employed clinically, researchers continue to work on improving the physicochemical properties and biological functions of BG due to its inappropriate degradation rate and unsatisfactory bone repair effects. Our previous work revealed that the incorporation of chlorine (Cl) into BG expanded the glass structure, facilitating glass degradation and rapid hydroxyapatite formation.
Keywords
INTRODUCTION
Millions of people worldwide lost their teeth due to periodontitis, caries, trauma, and diabetes[1,2]. Tooth loss can lead to occlusion disorders, reduced chewing ability, psychological burden, and systemic diseases such as hypertension and coronary heart disease[3,4]. Dental implantation is the most effective and common method to restore occlusion and function. However, most patients require bone grafts to maintain the height and width of the alveolar bone, which is essential for the success of dental implants[5]. Generally, autografts are the most ideal option due to their composition. However, their limited availability, risk of rejection, infection, and secondary trauma can restrict therapeutic efficacy and lead to the failure of bone implantation[6].
The commercial anorganic bovine bone matrix (Bio-Oss®) is widely used as a bone substitute in dentistry due to its similar structure and porosity to human bone and its protein-free composition[7]. However, the slow degradation rate of Bio-Oss® may limit the space available for bone regeneration[8]. Additionally,
Incorporating functional elements into BG is an effective approach to modify glass structure, properties and thus its functions[18]. The introduction of strontium (Sr) into BG enhanced the osteogenic ability[19], while increasing phosphorus content reasonably led to more HAP formation and promoted bone formation[20]. Adding copper (Cu) into BG promoted vascularization and antibacterial effects[21]. Chlorine (Cl) is another novel and interesting element that can tune glass properties. Chloride ions, which are abundant in the blood and contribute to two-thirds of the negative charge and one-third of tonicity, are biocompatible and crucial for charge balance and metabolism. Chloride deficiency can lead to metabolic alkalosis and hypokalemia[22]. Chen et al.[23,24] developed the first series of chloride-containing bioactive glasses (GPCls) and revealed that the incorporation of chloride expands BG structure, accelerating glass degradation and HAP formation and making GPCl highly bioactive. Additionally, GPCls have been shown to upregulate the expression of osteogenic and angiogenic genes and proteins and enhance bone formation in vivo[25]. However, the fast degradation of GPCls may cause a mismatch between material degradation and bone formation rates, limiting their bone repair efficacy[26,27].
Magnesium (Mg) has been added into 45S5 and a phosphate glass (Mol%: 20Na2O - 30CaO - 50P2O5), and both were found to postpone glass degradation[28,29]. Mg2+ is the fourth most abundant cation in the human body, primarily stored in bone tissue and involved in hundreds of biochemical reactions[30], making it important for bone formation and biological functions. Studies have shown that Mg is essential for bone health, with Mg deficiency leading to low bone mineral density and bone inflammation[31]. Hohenbild et al.[32] replaced 3 mol% and 6 mol% Ca with Mg in the glass composition ICIE 16 (Mol%: 49.46SiO2-36.27CaO-6.6Na2O-1.07P2O5-6.6K2O), resulting in higher expression levels of osseous extracellular matrix-related genes compared to the ICIE 16-BG group. Dai et al.[33] synthesized Mg-doped bioactive glass cement (Mg-BGC), demonstrating better osteogenesis and immune regulation performance compared to non-Mg BG cement and calcium sulfate cement in vitro. Therefore, given the ability of magnesium to promote BG degradation while facilitating osteogenesis and bone integration, we hypothesized that adding Mg into a GPCl is an effective strategy for tuning its degradation behavior and osteogenic properties, ensuring a balance between material degradation and bone formation.
In this study, we incorporated the Mg into a sodium-free, high phosphorus and Cl-containing BG (composition in Mol%: 30.3SiO2 - 44.1CaO - 5.0P2O5 - 20.6CaCl2) by replacing CaO with MgO and keeping other components constant to further modify the degradation rate and improve the osteogenic ability of GPCl. Mg-doped Cl-containing BG (GPMgCl) with different amounts of content Mg was synthesized by a melt-quench method. The degradation rate of GPMgCl and HAP formation were evaluated using
MATERIALS AND METHODS
Glass synthesis
Glasses in the SiO2-P2O5-CaCl2-CaO-MgO system were prepared using the melt-quench method. Briefly, analytical grade SiO2, P2O5, CaCl2·2H2O, CaCO3, and MgO were weighed to achieve a total batch size of
The glass composition in mol%
| Glass code | SiO2 | CaO | P2O5 | MgO | CaCl2 |
| GPMg0Cl | 30.3 | 44.1 | 5.0 | 0 | 20.6 |
| GPMg5Cl | 30.3 | 39.1 | 5.0 | 5.0 | 20.6 |
| GPMg10Cl | 30.3 | 34.1 | 5.0 | 10.0 | 20.6 |
| GPMg20Cl | 30.3 | 24.1 | 5.0 | 20.0 | 20.6 |
Bioactivity evaluation in minimum essential medium-α
The glass bioactivity in terms of glass degradation and apatite formation abilities were investigated in Minimum Essential Medium-α (α-MEM). Glass powders with a particle size smaller than 38 µm (75 mg) were dispersed in 50 mL of α-MEM media, resulting in a concentration of
Apatite formation
The apatite formation ability of GPMgCl in α-MEM was evaluated by FTIR (Bruker, Germany) on the collected solids after filtration. The data were collected in the range of 500 to 1,600 cm-1, as previously described by Chen et al.[34].
pH changes and ions release
The pH values of the collected α-MEM were measured using a pH meter (OHAUS, China). Two separate samples from each immersion time point were prepared for all studied glasses. The concentrations of calcium, magnesium, silicon, and phosphorus ions in the α-MEM were quantified using an ICP-OES (PerkinElmer, USA) after dilution by a factor of 2 in deionized water.
Cytotoxicity of glass-conditioned media
The glass-conditioned media was prepared by dispersing 75 mg of glass particles in 50 mL of α-MEM for
Effect on osteoblasts in vitro
ALP staining and activity quantification
After the treatment with glass-conditioned media for 14 days, the cells were washed twice with
Alizarin Red S staining and quantification
After culturing with glass-conditioned media for 14 and 21 days, the mineralized nodule formation ability was assessed by Alizarin Red S (ARS) staining. The cells were washed twice with PBS and fixed in a 4% formaldehyde solution. Subsequently, they were rinsed with ddH2O and stained with 0.5% ARS (pH 4.0) (Sigma, USA) for 30 min. The quantification of mineralized nodules involved incubation with 10% cetylpyridinium chloride solution (Sigma, USA) for 30 minutes, followed by the measurement of absorbances at 570 nm.
Quantitative real-time polymerase chain reaction
After treatment with glass-conditioned media for 7 and 14 days, the expression levels of osteogenic genes osteocalcin (OCN) and bone morphogenetic protein 2 (BMP2), and angiogenic genes vascular endothelial growth factor (VEGF) were evaluated by real-time polymerase chain reaction (RT-qPCR). RNA was reverse-transcribed into cDNA using ReverTra Ace qPCR RT Master Mix reagents (TOYOBO, Japan) following the manufacturer’s instructions. Subsequently, 100 ng of total cDNA was used for RT-qPCR analysis with KOD SYBR® qPCR Mix reagents (TOYOBO, Japan). The relative expression of genes was calculated using the ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous reference. Fold change was determined using 2-ΔΔCt.
Western blotting
After treatment with glass-conditioned media for 7 and 14 days, cells were lysed using radioimmunoprecipitation assay (RIPA) lysate buffer (Beyotime, China) containing protease inhibitors. The protein concentrations were analyzed using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, USA). Equal amounts of protein by weight were subjected to Sodium Dodecyl
In vivo evaluation of bone regeneration ability and biocompatibility
The animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Central South University (Certificate No.CSU-2022-0189). Eight-week-old Sprague-Dawley (SD) rats weighing around 250 g were used in the following in vivo studies.
Surgical procedures
The critical-size calvarial defect model was used to assess the bone formation abilities of GPMgCl and
The bone regeneration ability in vivo
Micro-computed tomography
The evaluation of new bone formation in the harvested skull bones at week 8 post-surgery was conducted using the Hiscan XM Micro-Computed Tomography (Micro-CT) (Suzhou Hiscan Information Technology Co., Ltd, China). The X-ray tube settings were 80 kV and 100 µA, and images were acquired at a resolution of 25 µm. A 0.5° rotation step through a 360° angular range with 50 ms exposure per step was employed. The images were reconstructed using Hiscan Reconstruct software (Version 3.0, Suzhou Hiscan Information Technology Co., Ltd, China), and the bone volume fraction (BV/TV) was analyzed with Hiscan Analyzer software (Version 3.0, Suzhou Hiscan Information Technology Co., Ltd).
Histological analysis of bone
Skull samples after the computed tomography (CT) scan were decalcified by immersing in 10 % ethylenediaminetetraacetic acid (EDTA) decalcifying fluid (Biofroxx, Germany) for 4 weeks. Subsequently, 5 µm-thick sections were prepared from the tissues for histological analysis. Hematoxylin and eosin (H&E) staining and Masson’s Trichrome staining were performed to assess new bone and collagen formation. Immunohistochemistry (IHC) was conducted to analyze the expression levels of BMP2 protein (antibody 1:100) within the defect area.
Biocompatibility evaluation
Blood cell and blood biochemistry analysis
The rats with two defects grafted with different materials are not suitable for investigating the biocompatibility of a single material. Therefore, in this study, rats with one defect grafted with a material and the other left empty were used to evaluate the biocompatibility of the specific material being tested. In order to evaluate the possibility of combining commercially available products with developed bioactive glasses (BGs), the rats grafted with either Bio-Oss® and GPMg0Cl or Bio-Oss® and GPMg20Cl were also studied. Moreover, rats with two defects without grafting materials were used as the control group. Blood samples were collected at 7 days and 8 weeks post-implantation via the caudal vein. The blood was centrifugated at 3000rpm, and the serum was analyzed for level of total protein (TP), albumin (ALB), creatinine (CREA), and blood urea nitrogen (BUN). Whole blood, after anticoagulation, was used to determine levels of white blood cells, red blood cells, and hemoglobin[36].
HE staining of main organs
Upon sacrifice, these organs were fixed in 4% paraformaldehyde for 48 h and embedded in paraffin to prepare 5 µm thick sections. Subsequently, sections were stained with HE to detect any pathological changes.
RESULTS AND DISCUSSION
Bioactivity evaluation in α-MEM
As shown in Supplementary Figure 1, all the studied BGs were largely amorphous. The low-intensity crystalline phase observed in GPMg0Cl and GPMg5Cl is highly likely due to reactions with water during quenching. Figure 1A shows the FTIR spectra of GPMg0Cl after immersion for 6, 24, 72, and 168 h in
Figure 1. Bioactivity evaluation of GPMgCl in α-MEM up to 168 h of immersion: (A-D) FTIR spectra of GPMg0Cl, GPMg5Cl, GPMg10Cl, and GPMg20Cl immersed in α-MEM for 0, 6, 24, 72, and 168 h, respectively; (E-H) Concentration of Ca, Mg, Si, and P ions in α-MEM after GPMg0Cl, GPMg5Cl, GPMg10Cl, and GPMg20Cl immersion for 6, 24, 72, and 168 h; (I) pH variation at 6, 24, 72, and 168 h. The ion concentrations and pH value of 0 h represent the initial ion concentrations and pH of α-MEM. α-MEM: Minimum essential medium-α; FTIR: Fourier-transform infrared spectroscopy; GPMgCl: Mg-doped chloride-containing bioactive glasses.
Supplementary Figure 4 presents the normalized FTIR spectra of collected glass powder after 72 h immersion. It is clear that the intensity of Si-O(NBO) band at about 920 cm-1 was stronger in the glasses with higher Mg content; meanwhile, the Si-O-Si band at 790 cm-1, associated with Si-gel formation, was observed in the spectra of GPMg0Cl and GPMg5Cl but not in GPMg10Cl and GPMg20Cl. These findings indicate that the incorporation of Mg delays glass degradation and probably Si-gel formation.
The changes in ion concentration and pH of α-MEM were measured to further investigate the bioactivity of the GPMgCl. Figure 1E-H shows the concentrations of Ca, Mg, Si, and P in α-MEM after BG immersion for 6, 24, 72, and 168 h. The data at 0 hours represent the original ion concentrations in the α-MEM. The concentration of Ca2+ [Figure 1E] in all BGs increased rapidly from 0 to 6 h, suggesting that the rate of degradation was faster than the HAP formation. From 6 to 24 h, the concentration of Ca2+ decreased, indicating that the HAP formation rate was faster than the BG degradation rate. The concentration of Ca2+ in GPMg0Cl and GPMg5Cl increased from 24 to 72 h, then decreased from 72 to 168 h. However, the trend of Ca2+ concentration in GPMg10Cl was contrary to those of GPMg0Cl and GPMg5Cl, while GPMg20Cl showed an unchanged Ca2+ concentration from 24 to 168 h. Within 72 h of immersion, the percentage of Ca released decreased with increasing Mg content in the glass compositions
It is apparent that the Si4+ concentration [Figure 1G] had a positive correlation with the Mg content of BGs at 6 h, suggesting that high Mg2+ concentration may affect glass degradation and the Si-gel formation. This is also evident in the FTIR spectrum of GPMg20Cl [Figure 1D], which shows the highest peak intensity at
The decrease in P5+ concentration is attributed to the consumption of PO43- for HAP formation. Moreover, the phosphate concentration in high Mg-containing BGs is higher than that in lower ones, implying high Mg content significantly delayed HAP formation. However, the HAP formation in GPMg5Cl was not significantly affected by the relatively low concentration of Mg2+. Mg released from BG could potentially be incorporated into the reactive apatite layer, given the remarkable ability of apatites to accommodate a wide range of substitutional ions. The use of 31P magic angle spinning-nuclear magnetic resonance (MAS-NMR) and 25Mg MAS-NMR would indeed be valuable in investigating such substitutions. However, it is worth noting that obtaining a good signal for 25Mg MAS-NMR can be particularly challenging, especially when the level of Mg substitution is low.
As the BGs degraded, the pH of α-MEM increased at 6 hours in all groups and continued to increase from 24 to 72 h, while the pH in GPMg20Cl remained stable after 6 h [Figure 1I]. The increase in pH was caused by the BG degradation, which led to the exchange of metal cations (Ca2+ and Mg2+) from BGs with H+ in the media, resulting in an increase in OH- concentration[37]. The formation of HAP consumes OH-, causing a decrease in pH. The rapid increase in pH indicated fast degradation of BGs, while the stable pH in GPMg20Cl suggested a slow glass degradation and apatite formation process, and a relatively balance between the glass degradation rate and HAP formation rate. From 72 to 168 h, the pH in GPMg0Cl, GPMg5Cl, and GPMg10Cl decreased, with GPMg0Cl showing the lowest pH value, indicating the most HAP formation in the GPMg0Cl group during this period. This result is also evident from the most significant Ca consumption [Figure 1E] in GPMg0Cl from 72 to 168 h. Since abundant ions are present in
Cell culture and cytotoxicity of glass-conditioned culture media
As shown in Supplementary Figure 7, none of the glass-conditioned culture media were cytotoxic to the MC3T3-E1 cells compared to the control group. Although on day 1, the viability of MC3T3-E1 cells in the GPMg20Cl group decreased slightly compared to the other experimental groups, there was no significant difference compared with the control. This may be due to the low cell density on day 1 and the highest concentration of Mg in the GPMg20Cl group, as seen in Figure 1F, resulting in low viability[38]. However, as the cells continued to proliferate, no differences were observed among the groups.
In vitro osteogenic effects
ALP activity
As a marker of osteogenic differentiation, the activity level of ALP represents osteogenic differentiation ability[39]. Figure 2A shows that GPMg5Cl, GPMg10Cl, and GPMg20Cl significantly enhanced the ALP activity of MC3T3-E1 cells, whereas GPMg0Cl did not show a distinct enhancement in the ALP activity compared to the control group on day 14 [Figure 2B]. This result indicates that the environment created by GPMgCl degradation, likely due to the released Mg2+, upregulated the expression of ALP, thereby promoting osteoblast differentiation. However, there was no significant difference in ALP activity among GPMg5Cl, GPMg10Cl, and GPMg20Cl. Zhong et al.[40] synthesized a series of Mg-doped BGs by replacing 6, 20, and 36 mol% Ca with Mg in a glass composition of 60.0 SiO2 - 4.0 P2O5 - 36.0 CaO (Mol%) to analyze the effect of the Mg-BG on mineralization and odontogenesis ability of human dental pulp stem cells (hDPSCs). The Mg-BG significantly upregulated the expression of ALP on days 7 and 14. There was no significant difference among the different Mg content groups on day 14, which is consistent with our results.
Figure 2. Osteogenesis ability evaluation after culturing in glass-conditioned culture media for different time points. (A) ALP staining quantification of MC3T3-E1 at 14 days (n = 4); (B) Quantification of ALP activity at 14 days; (C) Alizarin Red S staining at 14 and 21 days (n = 3); (D) Quantification of mineralized nodule formation at 14 and 21 days. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001, compared with the control group. #p < 0.05, ##P < 0.01, ###P < 0.005, ####P < 0.0001, compared with each experimental group. The bar represents 250 µm, and the values were normalized by comparison with the control group. ALP: Alkaline phosphatase.
ARS staining
The formation of mineralized nodules represents osteogenic and mineralized abilities[41]. As shown in Figure 2C and D, the formation of mineralized nodules was significantly promoted in all the BG groups compared to the control after 14 and 21 days. On day 14, the formation of mineralized nodules followed the decreasing order: GPMg10Cl, GPMg5Cl, GPMg20Cl, GPMg0Cl, and control. The trend on day 21 was generally the same as on day 14. However, the mineralized nodules in GPMg0Cl were more numerous than in GPMg20Cl on day 21. This implies that a high concentration of Mg may negatively impact bone mineralization and osteogenesis. The GPMg10Cl group showed the best mineralization at each time point of interest. Although the mineralized nodule formation in GPMg20Cl was less than in GPMg0Cl on day 21, it was still better than the control group. Chu et al.[42] reported that a high level of Mg can stimulate ALP activity but disrupt bone mineralization.
Osteogenesis-related gene expression
To further evaluate the effect of GPMgCl on osteogenesis, RT-qPCR was conducted to assess the expression levels of osteogenesis-related genes in MC3TE-E1 cells treated with BG-conditioned culture media. On day 7, the expression of OCN was significantly upregulated in all the BG groups in the following decreasing order: GPMg5Cl, GPMg10Cl, GPMg20Cl, GPMg0Cl, and control [Figure 3A]. On day 14, the highest OCN gene expression was observed in the GPMg10Cl group, followed by GPMg5Cl, GPMg0Cl, GPMg20Cl, and control. OCN is a key gene involved in bone mineralization and alignment of HAP, enhancing bone density and strength. Incorporating Mg into GPCl significantly promoted OCN expression, indicating that Mg is important for bone mineralization. The expression level of the runt-related transcription factor 2 (RUNX2) gene is shown in Figure 3B. RUNX2 was upregulated in the GPMg0Cl, GPMg5Cl, and GPMg10Cl groups on day 7, with no significant difference between GPMg20Cl and the control. On day 14, all the BG groups had lower RUNX2 levels compared to the control. This is due to the fact that RUNX2 is a key transcription factor in the early stages of osteoblast differentiation, and its expression decreases in mature osteoblasts[43,44]. VEGF is a key gene involved in angiogenesis, significantly promoting angiogenesis and providing adequate nutrition for bone formation. Additionally, VEGF was found to directly promote osteoblast differentiation and bone repair[45]. Our previous study found that Cl-containing BG promoted VEGF expression to stimulate osteogenesis[25]. In this study, VEGF gene expression was upregulated in GPMg0Cl, GPMg5Cl, and GPMg10Cl groups compared to the control group on day 7 [Figure 3C]. However, on day 14, there was no difference in VEGF gene expression among all the groups.
Figure 3. Osteogenesis-related gene and protein expression levels: (A-C) Gene expression of OCN, RUNX2, and VEGF in MC3T3-E1 cells following treatment with BG-conditioned culture media for 7 and 14 days, respectively (n = 3); (D) and (F) Representative images of BMP2 and VEGF protein expression in MC3T3-E1 cells on days 7 and 14; (E) and (G) Quantification of BMP2 and VEGF protein expression levels by ImageJ. Data are presented as the mean ± SD (n = 3). *P< 0.05, **P < 0.01, ***P< 0.005, ****P< 0.0001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.005, ####P < 0.0001, compared with each experimental group. OCN: Osteogenic genes osteocalcin; BMP2: Bone morphogenetic protein 2; VEGF: Vascular endothelial growth factor; BG: Bioactive glass; RUNX2: Runt-related transcription factor 2.
Osteogenesis-related protein expression
The expression and quantification of BMP2 protein are shown in Figure 3D and E, respectively. On day 7, BMP2 protein expression was upregulated in the GPMg5Cl and GPMg10Cl groups. On day 14, compared to the control group, the expression level of BMP2 protein was further increased, with the GPMg5Cl group showing the most significant upregulation. On day 7, VEGF protein expression was promoted in the GPMg5Cl and GPMg10Cl groups [Figure 3F and G], while there was no significant difference among the groups on day 14. The GPMg20Cl group showed no significant upregulation effect on BMP2 and VEGF protein expression, indicating that the large amount of Mg generated by dissolving GPMg20Cl in α-MEM may have an adverse effect on osteogenesis. Li et al.[46] incorporated Mg into nanoporous titanium and found that the presence of Mg coating significantly increased mineralized nodule formation. However, as the Mg content increased, mineralized nodule formation was inhibited by excessive Mg. This suggests that a too-high concentration of Mg may harm the growth of osteoblasts.
In vivo osteogenic effects
Micro-CT analysis of new bone formation
Figure 4A shows the representative images of bone defect after 8 weeks of implantation. It is clear that the control group exhibited the largest defect size. The size of defects grafted with either Bio-Oss® or BGs reduced significantly. Generally, a small amount of residual BG granules was present in the defects at week 8, particularly in the GPMg20Cl group. In the Bio-Oss® group, a significant amount of residual Bio-Oss® granules and limited integration were observed within the defect, with the interface between Bio-Oss® and bone remaining visible. These observations are attributed to the limited degradation and osteoinduction abilities of Bio-Oss®. All GPMgCl groups showed varying but desirable degrees of degradation and integration with host bone, with the GPMg10Cl group exhibiting complete closure of the defect. In addition, the interface between the glass and the host bone was barely identifiable, indicating excellent osseointegration, which is necessary for successful implantation[47]. Figure 4B presents the quantitative analysis of newly formed bone. The volume of newly formed bone in the Bio-Oss® group was higher than in the control, but lower than in the GPCls. The volume of newly formed bone increased with the Mg content in the glass from 0 to 10 mol%. Although the BV/TV value in GPMg20Cl was lower than that in the GPMg10Cl, it was still higher than in the control group. Cheng et al.[48] also found that grafting Bio-Oss® in a rat mandible defect showed some new bone formation, but no direct connection between the granules and the surrounding bone, suggesting that a lack of osteoinduction impairs osseointegration.
Figure 4. Micro-CT evaluates the bone formation ability of GPMgCl and Bio-Oss® (A) The representative images of calvarial defects in rats implanted with GPMgCl or Bio-Oss® for 8 weeks; (B) The value of BV/TV in every group at week (n = 5). Micro-CT:
Histology analysis
The two critical-size skull defect model is a well-established method for studying bone defects and regeneration, as well as potential interactions between the two defects when using different graft materials. To minimize such interactions, we ensured that the histological analysis (including HE, Masson, and IHC staining) focused on bone tissues located far from the midline between the two defects. This approach was intended to avoid any contamination from the adjacent defect site. Figure 5A shows the HE staining results of the interface between bone and grafting materials after 8 weeks. In the control group, there was a small amount of new bone formation, and the defect was mainly filled with disorder and funicular fibers. Bio-Oss® promoted a small amount of new bone formation in the defect area. The GPMgCl group, especially in the GPMg10Cl and GPMg5Cl, exhibited a large amount of compact and lamellar bone formation. Additionally, some pores were observed in the tissue of the defect area, likely caused by the material granules. When the granules were implanted into the defect, cells clustered and adhered to the granules, forming the fibers and collagen. As cell proliferation, differentiation, and mineralized matrix secretion occurred, new bone tissue grew more extensively. This process requires the material to degrade to provide more space for bone formation, indicating that an appropriate degradation rate is necessary for bone regeneration[49]. The largest pores, surrounded by fibrous connective tissue, were observed in the Bio-Oss® group due to its slowest degradation rate[50]. To further analyze the composition of the tissue in the defect area, Masson staining was conducted. As shown in Figure 5B, a large amount of the red-stained connective fiber tissue was present in the control and Bio-Oss® groups. In contrast, the defect area in the GPMgCl groups was composed of
Figure 5. (A) Representative images of HE staining and (B) Masson staining of the interface between the host bone and the materials; (B) Representative immunohistochemistry images of BMP2 between the material granules and tissue. The bar represents 500 µm. HE: Hematoxylin and eosin; BMP2: Bone morphogenetic protein 2.
BMP2 is a crucial growth factor for bone formation[53]. Figure 5C shows the BMP2 expression level in the defect area. In the control and Bio-Oss® groups, immune-complex deposition was barely detectable, indicating low levels of BMP2. In contrast, the GPMg0Cl, GPMg5Cl, and GPMg10Cl groups exhibited a large amount of BMP2. Sahin et al.[54] found that Mg2+ promotes BMP2 levels in high-fat-fed rats by regulating the BMP2/RUNX2 signaling pathway. However, the GPMg20Cl group showed low BMP2 expression levels, consistent with the western blot results. Some researchers have also reported the negative effects of sustained high doses of Mg release on bone repair[42,46,55,56]. According to these and our data, although Mg is a well-accepted osteogenesis-promoting element, the release dose and content of Mg in bone regeneration materials should be appropriate.
Biocompatibility evaluation
Blood cell and blood biochemistry analysis
Blood collected via the caudal vein was used to further assess the biocompatibility of GPMgCl. Data from day 0 were collected before the surgical procedures and implantation. After 7 days of implantation, white cell counts had slightly increased in all groups, indicating mild inflammation caused by the surgical operation [Table 2]. The control group showed the most significant increase, likely due to the trauma from the bone defect operation. The implantation of GPMgCl did not cause notable inflammation. By 8 weeks, white cell counts had decreased to normal levels. Red cell and hemoglobin levels showed no significant differences compared to the control at all time points. These results suggest that GPMgCl did not impair the hemopoietic system. Table 3 shows the biochemical indices of rats at different time points. The TP, ALB, BUN, and CREA data also suggest that GPMgCl did not affect liver and kidney functions[57,58]. Combining these data with the HE staining of organs, it is apparent that the GPMgCl is biocompatible.
Blood cell test of rats before surgery (day 0) and after implanted with control, Bio-Oss®, GPMg0Cl, GPMg5Cl, GPMg10Cl, GPMg20Cl, and mixed groups on day 7 and at week 8
| Items | Blood cells | ||||||||
| White cell (109/L) | Red cell (1012/L) | Hemoglobin (g/L) | |||||||
| Groups | 0 d | 7 d | 8 w | 0 d | 7 d | 8 w | 0 d | 7 d | 8 w |
| Control | 7.44 | 13.84 | 14.12 | 8.71 | 7.66 | 8.88 | 167 | 145 | 177 |
| Bio-Oss® | 17.07 | 17.04 | 6.01 | 8.44 | 7.98 | 5.35 | 160 | 149 | 108 |
| GPMg0Cl | 9.53 | 10.99 | 10.61 | 8.52 | 8.91 | 8.25 | 159 | 163 | 162 |
| GPMg5Cl | 18.00 | 13.69 | 10.03 | 8.83 | 8.78 | 9.35 | 158 | 153 | 172 |
| GPMg10Cl | 11.05 | 13.79 | 9.98 | 8.88 | 8.22 | 7.63 | 165 | 148 | 144 |
| GPMg20Cl | 10.31 | 14.69 | 13.46 | 7.62 | 7.7 | 8.18 | 142 | 141 | 157 |
| Bio-Oss® and GPMg0Cl | 10.05 | 8.35 | 9.49 | 6.61 | 6.41 | 9.09 | 123 | 117 | 172 |
| Bio-Oss® and GPMg20Cl | 15.43 | 11.39 | 20.19 | 8.51 | 7.64 | 8.73 | 159 | 142 | 167 |
The biochemical indices TP, ALB, BUN, and CREA level among groups of rats before surgery (day 0) and after implanted with control, Bio-Oss®, GPMg0Cl, GPMg5Cl, GPMg10Cl, GPMg20Cl, and mixed groups on day 7 and at week 8
| Items | Blood biochemistry | |||||||||||
| TP (g/dL) | ALB (g/dL) | BUN (mg/dL) | CREA (mg/dL) | |||||||||
| Groups | 0 d | 7 d | 8 w | 0 d | 7 d | 8 w | 0 d | 7 d | 8 w | 0 d | 7 d | 8 w |
| Control | 8.7 | 6.5 | 7.7 | 5 | 3.2 | 3.7 | 18 | 9 | 17 | 0.1 | 0.5 | 0.5 |
| Bio-Oss® | 7.3 | 6.6 | ＞12 | 4.4 | 3.4 | 4.7 | 18 | 16 | 16 | 0.5 | 0.5 | 0.7 |
| GPMg0Cl | 7.3 | 6.7 | 10.7 | 4.4 | 3.9 | 3.2 | 22 | 14 | 17 | 0.2 | 0.2 | 0.5 |
| GPMg5Cl | 8.2 | 7.4 | 10.1 | 4.8 | 4.2 | 4.3 | 17 | 13 | 16 | 0.1 | 0.2 | 0.5 |
| GPMg10Cl | 7.1 | 6.6 | 9.6 | 4.4 | 3.4 | 4.5 | 17 | 13 | 20 | 0.5 | 0.5 | 0.5 |
| GPMg20Cl | 6.6 | 6.7 | 7.7 | 4 | 3.3 | 5.7 | 19 | 18 | 21 | 0.5 | 0.5 | 0.6 |
| Bio-Oss® and GPMg0Cl | 7.8 | 9 | 6 | 4.8 | 5.2 | 5.3 | 19 | 15 | 17 | 0.1 | 0.1 | 0.8 |
| Bio-Oss® and GPMg20Cl | 7.1 | 3.4 | 7.7 | 4.4 | 3.4 | 3.9 | 19 | 15 | 20 | 0.4 | 0.4 | 0.6 |
HE staining of main organs
To investigate the biocompatibility of GPMgCl, we collected the cerebrum, heart, liver, lung, and kidney from rats after 8 weeks of material implantation [Figure 6]. There were no signs of pathology in the organs. In addition, the structure of the cerebrum in the GPMgCl groups was similar to that in the control and
Figure 6. HE staining of the cerebrum, heart, liver, lung, and kidney of the control, Bio-Oss®, GPMg0Cl, GPMg5Cl, GPMg10Cl, and GPMg20Cl groups after 8 weeks of implantation. The bar represents 100 µm. HE: Hematoxylin and eosin; GPMgCl: Mg-doped chloride-containing bioactive glasses; Bio-Oss®: Anorganic bovine bone matrix.
CONCLUSIONS
In this study, we synthesized the GPMgCl with Mg content ranging from 0 to 20 %mol. The incorporation of Mg delayed the HAP formation and degradation rate of GPMgCl, while all the GPMgCl exhibited good bioactivity in α-MEM. The in vitro osteogenesis results revealed that an appropriate amount of Mg can promote osteogenic differentiation and mineralized nodule formation, whereas excessively high Mg concentrations weaken the osteogenesis effect. The in vivo study demonstrates the superior bone regeneration ability of GPMgCl than Bio-Oss®, with GPMg10Cl showing the best bone formation effect. Moreover, biocompatibility investigations reveal that Mg-doped GPCls are biocompatible. GPMg10Cl is a promising bone substitute that addresses the high demand for bone regeneration. Future studies should focus on elucidating the mechanisms by which Mg-doped GPCls promote bone formation and systematically evaluating their biocompatibility in large animals.
DECLARATIONS
Acknowledgments
This study was supported by the Changsha Prominent Young Innovators Program (kq2305026), the Science and Technology Talent Lifting Project of Hunan Province (2023TJ-N18), Huxiang Youth Talent Program of Hunan Province of China (2020RC3064), and Furong Scholar of the Hunan Provincial Department of Education (202058).
Authors’ contributions
Experiment implementation, data acquisition and interpretation, statistical analyses, manuscript writing: Ouyang, Z.
In vivo study: Li, P.; Ru, X.; Liu, L.
Glass fabrication: Shah, P.
Study design, manuscript review and editing: Liu, O.
Glass design, discussion, manuscript revision: Hill, R.
Glass design, data interpretation, conceptualization, manuscript review and editing: Chen, X. (Xiaohui Chen)
Conceptualization, methodology, data interpretation, manuscript writing, review and editing, supervision, and funding acquisition: Chen, X. (Xiaojing Chen)
Availability of data and materials
The rata data supporting the findings of this study are available within this Article and its Supplementary Materials. Further data are available from the corresponding authors upon reasonable request.
Financial support and sponsorship
This study was supported by the Changsha Prominent Young Innovators Program (kq2305026), the Science and Technology Talent Lifting Project of Hunan Province (2023TJ-N18), Huxiang Youth Talent Program of Hunan Province of China (2020RC3064), and Furong Scholar of the Hunan Provincial Department of Education (202058).
Conflicts of interest
All authors declared that there are no conflicts of interest.
Ethical approval and consent to participate
The animal experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Central South University (Certificate No.CSU-2022-0189).
Consent for publication
Not applicable.
Copyright
© The Author(s) 2025.
Supplementary Materials
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Ouyang, Z.; Li, P.; Ru, X.; Liu, L.; Shah, P.; Liu, O.; Hill, R.; Chen, X.; Chen, X. Investigation of the biocompatibility and osteogenic effects of magnesium-doped chloride-containing bioactive glasses. Microstructures 2025, 5, 2025013. http://dx.doi.org/10.20517/microstructures.2024.54
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