Figure 1.
Experimental design and applications.
Figure 2.
Schematic illustration of the study.
Figure 3.
Appearance of the tibia after defect creation (arrow).
Figure 4.
XRD patterns of the MBG and Cu-MBG, Zn-MBG, and Cu–Zn-MBG samples (A) before and (B) after soaking in SBF for 28 d: □, CaCO3, Δ, Ca2SiO4, 
, Ca3(PO4)2, 
, CaSiO3, 
, CuO, 
, Zn2SiO4, 
, ZnCO3, and °, Ca10(PO4)6(OH)2. MBG, mesoporous bioactive glass; SBF, simulated body fluid; XRD, X-ray diffraction.
Figure 5.
FTIR spectra of the MBG, Cu-MBG, Zn-MBG, and Cu–Zn-MBG samples (A) before and (B) after soaking in SBF for 28 d. FTIR, Fourier transformed infrared; MBG, mesoporous bioactive glass; SBF, simulated body fluid.
Figure 6.
Histopathological appearance of the groups on the 15th and 30th days after graft application in the (A) control, (B) MBG, (C) Cu-MBG, (D) Zn-MBG, and (E) Cu–Zn-MBG groups. Defect areas (thin arrows), graft materials (thick arrows), and newly formed bone areas (white arrows), HE, bars = 100 μm. MBG, mesoporous bioactive glass.
Figure 7.
Collagen 1 immunohistochemical expression among the groups on the 15th and 30th days after graft application in the (A) control, (B) MBG, (C) Cu-MBG, (D) Zn-MBG, and (E) Cu–Zn-MBG groups. Positive expressions (arrows) were observed in connective tissue. SBPT, bar = 50 μm. MBG, mesoporous bioactive glass; SBPT, streptavidin–biotin peroxidase technique.
Figure 8.
BMP2 immunohistochemical expression among the groups on the 15th and 30th days after graft application in the (A) control, (B) MBG, (C) Cu-MBG, (D) Zn-MBG, and (E) Cu–Zn-MBG groups. Slight positive reactions (arrows) were observed in some cells. SBPT, bar = 50 μm. MBG, mesoporous bioactive glass; SBPT, streptavidin–biotin peroxidase technique.
Figure 9.
OST immunohistochemical expression among the groups on the 15th and 30th days after graft application in the (A) control, (B) MBG, (C) Cu-MBG, (D) Zn-MBG, and (E) Cu–Zn-MBG groups. Slight-to-moderate immunopositive reactions (arrows) were observed, especially in osteoclastic cells. SBPT, bar = 50 μm. MBG, mesoporous bioactive glass; OST, osteocalcin; SBPT, streptavidin–biotin peroxidase technique.
Table 1.
Statistical analysis results of histomorphic data between the groups
| Groups | 15 days | 30 days |
|---|---|---|
| Total augmentation area (mm2) | ||
| Control | 2.04 ± 0.46 | 2.44 ± 0.31 |
| MBG | 7.04 ± 0.78† | 8.04 ± 1.35† |
| Cu-MBG | 8.84 ± 0.87† | 11.74 ± 1.37‡ |
| Zn-MBG | 10.20 ± 0.34§ | 12.58 ± 1.64‡ |
| Cu/Zn-MBG | 12.54 ± 0.81§ | 14.44 ± 0.89§ |
| Defect closure rate (%) | ||
| Control | 28.20 ± 4.39 | 36.40 ± 4.03 |
| MBG | 44.10 ± 3.90† | 55.20 ± 5.30† |
| Cu-MBG | 59.20 ± 3.91‡ | 75.60 ± 5.08‡ |
| Zn-MBG | 64.50 ± 5.72‡ | 77.20 ± 5.20‡ |
| Cu/Zn-MBG | 74.80 ± 4.77§ | 93.70 ± 2.75§ |
| New bone area (mm2) | ||
| Control | 0.19 ± 0.03 | 1.54 ± 0.43 |
| MBG | 0.31 ± 0.03 | 1.77 ± 0.47† |
| Cu-MBG | 1.29 ± 0.08† | 2.61 ± 0.37‡ |
| Zn-MBG | 1.24 ± 0.13† | 2.35 ± 0.15‡ |
| Cu/Zn-MBG | 1.91 ± 0.10§ | 3.80 ± 0.14§ |
| Residual material area (mm2) | ||
| Control | 0.00 ± 0.00 | 0.00 ± 0.00 |
| MBG | 17.80 ± 0.78† | 7.60 ± 0.96† |
| Cu-MBG | 17.30 ± 1.41† | 7.30 ± 1.41† |
| Zn-MBG | 15.80 ± 2.44‡ | 8.60 ± 1.42† |
| Cu/Zn-MBG | 12.90 ± 3.54§ | 5.50 ± 1.26§ |
| Osteoclast number | ||
| Control | 6.30 ± 1.41 | 9.00 ± 1.49 |
| MBG | 12.30 ± 1.70† | 16.80 ± 1.61† |
| Cu-MBG | 16.60 ± 2.06‡ | 20.60 ± 1.57‡ |
| Zn-MBG | 17.10 ± 1.59‡ | 23.30 ± 2.40‡ |
| Cu/Zn-MBG | 19.70 ± 1.05§ | 29.20 ± 2.89§ |
| Osteoblast number | ||
| Control | 10.10 ± 0.99 | 20.40 ± 1.17 |
| MBG | 13.70 ± 2.66† | 25.20 ± 2.27† |
| Cu-MBG | 16.90 ± 1.66‡ | 29.20 ± 1.75‡ |
| Zn-MBG | 16.90 ± 2.23‡ | 27.10 ± 2.07‡ |
| Cu/Zn-MBG | 24.30 ± 1.88§ | 33.00 ± 1.56§ |
Data presented by mean ± standard deviation (SD). One-way ANOVA Duncan test was used in statistical analysis. The difference of mean between group were tested by one-way ANOVA (with a Duncan post hoc test).
Table 2.
Statistical analysis results of immunohistochemical scores between the groups
| Col-1 | BMP-2 | OST | VEGF | |
|---|---|---|---|---|
| 15 days | ||||
| Control | 0.50 ± 0.16 | 0.60 ± 0.51 | 0.20 ± 0.13 | 1.20 ± 0.42 |
| MBG | 1.10 ± 0.56† | 1.20 ± 0.63† | 1.20 ± 0.63† | 1.50 ± 0.52 |
| Cu-MBG | 1.50 ± 0.52†, ¥ | 1.30 ± 0.48† | 1.30 ± 0.48† | 1.80 ± 0.42† |
| Zn-MBG | 1.20 ± 0.42† | 1.60 ± 0.51§ | 1.30 ± 0.48† | 1.60 ± 0.51 |
| Cu/Zn-MBG | 2.30±0.48§, ф, ψ, δ | 1.60 ± 0.69§ | 1.80 ± 0.91§, #, ψ, ф | 2.30 ± 0.82§, ¥, ψ, ф |
| 30 days | ||||
| Control | 0.70 ± 0.48 | 1.00 ± 0.47 | 0.80 ± 0.42 | 1.50 ± 0.52 |
| MBG | 1.40 ± 0.51† | 1.80 ± 0.63† | 1.60 ± 0.51† | 2.10 ± 0.56† |
| Cu-MBG | 1.90 ± 0.31§, ¥ | 2.20 ± 0.91‡ | 1.90 ± 0.56† | 2.00 ± 0.47† |
| Zn-MBG | 1.60 ± 0.51† | 2.20 ± 0.63‡ | 1.80 ± 0.78† | 2.20 ± 0.63† |
| Cu/Zn-MBG | 2.50 ± 0.52§, ф | 2.40 ± 0.84§ | 2.30 ± 0.67§ | 2.80 ± 0.42§, ψ |
Data presented by mean ± standard deviation (SD). Kruskal-Wallis test was used in statistical analysis. The difference of mean between group were tested by Dwass-Steel-Critchlow-Flinger pairwise comparisons test.
Figure 10.
VEGF immunohistochemical expression among the groups on the 15th and 30th days after graft application in the (A) control, (B) MBG, (C) Cu-MBG, (D) Zn-MBG, and (E) Cu–Zn-MBG groups. Slight-to-moderate immunopositive reactions (arrows) were observed, especially in osteoclastic cells. SBPT, bar = 50 μm. MBG, mesoporous bioactive glass; SBPT, streptavidin–biotin peroxidase technique; VEGF, vascular endothelial growth factor.