Rubus coreanus Enhances Peri-Implant Bone Healing and Biomineralization in Ovariectomized and Healthy Rats

Abstract

Estrogen deficiency contributes to osteoporosis and can therefore compromise the peri-implant bone. Hence, this study evaluated peri-implant bone healing when Rubus coreanus (RC) was administered orally in ovariectomized and healthy rats. Forty 4-month-old female rats were divided into four groups: SHAM (healthy rats), SHAM/RC (healthy rats treated with RC), OVX (ovariectomized rats), and OVX/RC (ovariectomized rats treated with RC). The oral administration of RC started thirty days after ovariectomy, and implant placement into the rat tibia occurred ninety days after the ovariectomy. Euthanasia occurred sixty days after implantation. The analyses performed included removal torque, RT-PCR, confocal microscopy, and immunolabeling. A significance level of p < 0.05 was considered for all tests. The highest reverse torque values were observed in the SHAM/RC group, followed by the OVX/RC group. Confocal microscopy showed the greatest bone biomineralization in the SHAM/RC group, followed by the OVX/RC group. RT-PCR data indicated that RC decreased the RANKL/OPG ratio in both conditions. Immunohistochemistry demonstrated a balance between bone formation and resorption in all groups, especially stimulating osteoblastogenesis in both treated groups. In conclusion, RC enhanced peri-implant bone healing and biomineralization in both healthy and ovariectomized rats, with stronger effects in healthy rats, suggesting that estrogen may enhance its efficacy. These findings support RC's potential as a prophylactic and therapeutic agent.

Keywords: Rubus coreanus; biomineralization; bone; osseointegration; osteoporosis.

Figure 1

The fluorochrome application timeline for assessing bone mineralization. Animals received an initial injection of calcein (20 mg/kg) 14 days after implantation to label older bone, followed by alizarin red (20 mg/kg) 42 days post-implantation to mark new bone formation. Both fluorochromes bind to bone minerals, allowing the measurement of bone mineralization.

Figure 2

Biomechanical analysis of removal torque. The dot–bar graph shows the mean results, and the error bars indicate the standard deviation for the removal torque value from experimental groups followed by SHAM, SHAM/RC, OVX, and OVX/RC at 60 days after implantation. The p < 0.05 indicates statistical differences among the groups.

Figure 3

Molecular analysis for each gene expression (A–E). The dot–bar graph shows the mean results, and the error bars indicate the standard deviation for relative gene expression from the real-time PCR in peri-implant bone healing from the SHAM, SHAM/RC, OVX, and OVX/RC groups after 60 days of implantation. In RT-PCR, the genes evaluated were OPG, RANKL, OCN, and ALP. p < 0.05 indicates statistical differences between the groups.

Figure 4

The confocal microscopy results analyzed bone dynamics: the dot–bar graph shows the mean results, and the error bars indicate the standard deviations for the fluorochromes of calcein and alizarin precipitations at 14 and 42 days after implantation, respectively. The calcein is evidence of an older bone, while the alizarin indicates a new bone at 42 days. Different values of p indicate statistical differences between the groups; p < 0.05.

Figure 5

Mineral apposition rate (MAR). The MAR was measured to evaluate the rate of bone formation in response to RC treatment. Data are expressed as the distance between two fluorochrome labels (calcein and alizarin red) divided by the time interval between the injections. Results are presented as the mean ± SEM for each group. Statistical comparisons were made between groups, with a significant difference indicated by a corresponding p value of p < 0.05.

Figure 6

Bone dynamics. A representative image of bone dynamics formed by overlapping the fluorochromes of calcein and alizarin in the peri-implant bone of the tibia in each group for the SHAM, SHAM/RC, OVX, and OVX/RC groups after 60 days of implantation using to ImageJ software (version 1.52v, National Institutes of Health, Bethesda, MD, USA).

Figure 7

Immunolabeling in peri-implant bone healing for the SHAM, SHAM/RC, OVX, and OVX/RC groups after 60 days of implantation. The red arrows indicate positive immunostaining for the proteins Runx2, ALP, OPN, OCN, OPG, RANKL, and TRAP. Scale bar = 100 µm. Original magnification: 20×.