Effect of doxycycline-bioglass treatment on calvarial bone defect in rats: A histological study

Purpose: To evaluate the osteogenic properties of a bioglass-doxycycline complex for bone regeneration applications in calvarial bone defects in rats. Methods: Three critical-size bone defects were created in each of eight experimental rats using a trephine bur. The experimentally created defects were then filled with bioglass-doxycycline (BG-D group), bioglass alone (BG group), or left unfilled (control group). Four randomly selected rats were sacrificed after 4 weeks while the other four rats were sacrificed after 8 weeks. The amount of newly regenerated bone and the osteoblast and osteoclast counts were calculated using histological analysis. Results: Increase in the amount of regenerated bone was significant in the bioglass-doxycycline group at both 4 and 8 weeks (p < 0.05). Differences between the three groups in the mean number of osteoblasts were also significant at both 4 and 8 weeks (p < 0.05). Comparison of the three groups revealed significant increase in osteogenesis rate and considerable enhancement of the number of osteoblasts in both bioglass and bioglass-doxycycline groups (p < 0.05). Conclusion: The complex of doxycycline and bioglass increased the rate of osteogenesis and number of osteoblasts in rats but decreased the number of osteoclasts. Therefore, bioglass-doxycycline can be considered as a bone graft in bone regeneration for medical applications.


INTRODUCTION
Regeneration of bone defects is a challenging topic in dentistry [1]. Several factors such as type of tissue, defect location and position, number of blood vessels, microbial status, and other local and systemic factors affect the process of wound healing [2,3]. Different types of grafts have been suggested for reconstruction and regeneration of periodontal bone defects including autografts, allografts, and xenografts [4].
At present, biomaterials, including bioglass, have found a pivotal role in bone regeneration applications. Bioglass has represented a growing popularity due to its antimicrobial, osteoinductive, and osteoconductive properties [2,[4][5]. Bioglass is commonly used for coating the orthopedic implants in medicine and as a bone substitute in periodontology and endodontics. It is also used as a scaffolding material in tissue engineering [6][7][8]. Available evidence shows that some medications such as tetracyclines and bisphosphonates and their combination can prevent bone resorption following periodontitis and promote the regeneration of bone defects by inducing osteogenesis.
Doxycycline is a structural isomer of tetracycline with antibacterial, immunosuppressive, antiinflammatory, and anti-collagenase properties. Recently, the topical application of doxycycline has been confirmed to have treating effects on periodontitis and peri-implantitis [9][10][11][12]. Moreover, some recent studies demonstrated that doxycycline inhibits the production of osteoclasts. Since any bone manipulation in surgical procedures is shown to be able of activating osteoclasts, inhibition of their productions by tetracyclines is an advantage [3,4,10].
However, the simultaneous application of tetracyclines with bone grafts has shown controversial results. Some studies reported increased bone regeneration while some others reported no significant advantage for the application of a combination of tetracyclines and bone grafts for osteogenesis compared to bone grafting alone [4,6,13]. Due to the existing controversy concerning the effect of simultaneous application of tetracyclines and bone grafting on the rate of osteogenesis, and considering the gap in the information regarding the effect of applying bioglass-doxycycline complex on osteogenesis, here, the osteogenic properties of bioglass-doxycycline complex was studied for regenerating the calvarial bone defects in rats.

Materials
Bioglass 45S5 (sterile) was purchased from Medzist Company (Tehran, Iran) and doxycycline powder was gifted from Hakim Pharmaceutical Company (Tehran, Iran).

Animals
This animal study was performed on eight adult male Wistar rats of 250 to 300 g weight. All animal experimental protocols were performed according to the Guide for the Care and Use of Laboratory Animals [14]. This study was approved by the Ethical Committee for Animal Research of Islamic Azad University of Tehran, Tehran, Iran (no. IR.IAU.DENTAL.REC.1395,2).
The animals were housed in cages with diurnal lighting on a 12:12 h light-dark cycle and controlled temperature conditions (22 ± 2 °C) and supplied with free access to standard food and water.

Animal surgery
The rats were anesthetized using a mixture of 60 mg/kg ketamine and 16 mg/kg xylazine. Their head fur was shaved and the exposed scalp was disinfected using povidone-iodine. After preparation, a longitudinal incision was created in the scalp and a flap was elevated to expose the calvaria. Then, three defects with the critical size of 5 mm in diameter were created in the calvaria bone of each rat using a trephine 5 mm diameter bur ( Figure 1). The defects were located in the right, left, and center of the calvaria two of which were filled with bioglass-doxycycline (BG-D group) or standard bioglass (BG group) alone, and one was left unfilled (control group). The scalp was then sutured using 3-0 sutures. After 4 weeks, four rats were randomly chosen and sacrificed while the remaining four were sacrificed after 8 weeks. The rats were sacrificed in a CO2 chamber. Thereupon, the resected rats' calvaria pieces were fixed in 10% formalin. They were then decalcified, embedded in paraffin, and sectioned into 5 µm slices at the center of defects. Staining the tissue sections was performed using Masson's trichrome and Hematoxylin-Eosin protocols [15][16][17][18][19][20][21]. The stained tissue sections were inspected under a light microscope (Leica, Germany) at × 40 magnification. The newly formed bone was evaluated in each group using ImageJ software [5] and reported.

Statistical analysis
Data were analyzed using SPSS version 22 (SPSS Inc, IL, USA). The groups were compared using repeated-measures ANOVA. The significance of differences was analyzed by a paired t-test and the level of significance was set at p < 0.05.

Qualitative histological findings
The margins of the calvarial bone and the defect site were evident at 4 weeks in the control group and no newly regenerated bone was observed in the calvarial bone tissue. However, the defect site was covered with a fibrous membrane in this group. In the BG group (that received the standard bioglass alone), the distance between the defect margins was filled with bioglass granules and the space between the granules was filled with connective tissue. A newly regenerated bone was observed around the granules and calvarial bone after 4 weeks. In the BG-D group (that received the bioglassdoxycycline), the defect margins were filled with granules and the connective tissue between the granules was more organized and the blood vessels were seen as well. The newly regenerated bone could be observed around the granules, and the bone trabecula had penetrated between the granules at 4 weeks ( Figure 2).
In the 8-week rats, the control group represented an evident calvarial bone margin and defect site and the newly regenerated bone was seen in the calvarial bone tissue. The defect site was covered with a thicker fibrous membrane. In the BG group, the defect was filled with bioglass granules. The space between the granules was filled with a more organized connective tissue compared to the 4-week group. The newly regenerated bone was detected around the granules and adjacent to the calvarial bone. Bone trabecula was formed and penetrated through the granules and blood vessels were observed around the granules within the connective tissue. Compared to other groups, the rate of osteogenesis was higher in the BG-D group. Also, an organized connective tissue containing blood vessels and bone trabeculae filled the space of defect ( Figure 2).

Newly regenerated bone
The repeated measures ANOVA showed the rate of the new bone formation to be significantly different between the three groups at 4 (p = 0.004) and 8 weeks (p = 0.004). The maximum mean percentage of the new bone formation was seen in the BG-D group followed by the standard BG group and the control group at either 4 or 8 weeks (Table 1).

Number of osteoblasts
The three groups showed a significant difference in the number of osteoblasts at 4 (P = 0.007) and 8 weeks (p = 0.001). The maximum number of osteoblasts was considerable in the BG-D group compared to the BG and control groups at both 4 and 8 weeks (Table 2).

Number of osteoclasts
Repeated measures ANOVA revealed that the three groups were not significantly different in terms of the number of osteoclasts at 4 (p = 0.057) or 8 weeks (p = 0.189). At 4 weeks, the maximum mean number of osteoblasts was higher in the BG-D group, followed by the BG group and the control group (Table 3), respectively. At 8 weeks, the mean number of osteoclasts showed more decrease in the BG-D group compared to the BG group.

DISCUSSION
This study assessed the osteogenic properties of the bioglass-doxycycline complex in bone regeneration. Histological findings showed that the percentage of newly regenerated bone in defects filled with bioglass-doxycycline complex was significantly higher than that in other groups at either 4 or 8 weeks. Accordingly, Silva et al [4] assessed the efficacy of topical application of doxycycline gel for regeneration of bone defects in rats' calvaria. They found that at the end of week 8, the new bone formation rate in the BG-D group was significantly higher than in other groups. They concluded that doxycycline gel can effectively enhance bone formation in calvarial bone defects of rats. In the present study, the bioglass-doxycycline complex decreased the number of osteoclasts after 8 weeks more efficiently than 4 weeks.
Evidence shows that doxycycline inhibits the production of osteoclasts by decreasing RANKL [11,22]. It also prevents bone resorption due to its anti-collagenolytic and anti-proteolytic properties. It increases the chemotaxis of osteoblasts and leads to bone regeneration [23]. Soufdoost et al researched the novel Tadalafil/β-TCP/collagen scaffold for bone regeneration and they found that Tadalafil/β-TCP/collagen scaffold had better results in in vitro and in vivo tests compared to β-TCP/Collagen scaffold [5]. In an animal study, Gomes et al [24] evaluated the systemic doxycycline impact on the alveolar bone regeneration and the number of osteoblasts and osteoclasts. They found a complete tissue remodeling and bone formation after 14 days in the doxycycline-treated group. Moreover, they reported a reduction in the number of osteoclasts and an increase in the number of osteoblasts in the groups treated with doxycycline after 7 and 14 days.
In the present study, the maximum number of osteoblasts was detected in the bioglassdoxycycline group at both 4 and 8 weeks that complied with their results. However, no reduction in the number of osteoclasts at 4 weeks was detected in the bioglass-doxycycline group compared to the bioglass and control groups. After 8 weeks, the mean number of osteoclasts showed more decline in the bioglassdoxycycline group than the bioglass group alone. The different results of the two studies can be possibly due to different assessment time points and different methods of doxycycline application.
Furthermore, Agarwal et al [23] compared the efficacy of decalcified freeze-dried bone allografts with and without topical doxycycline for regeneration of 16 infrabony defects in patients suffering from moderate to severe chronic periodontitis. They reported that the topical application of doxycycline accompanied by allografts did not show any greater efficacy for the regeneration of bone defects than using allograft alone. The difference in the results of the two studies may be due to differences in the study design, type of used graft materials, and type of bone defects.
Al-Ali et al [13] evaluated the effect of topical application of doxycycline with/without tri-calcium phosphate on the regeneration potential of periodontal defects in the alveolar bone of dogs. They reported an increase in the formation of new bone, cementum, and periodontal ligament, and a reduction in crystal bone resorption in doxycycline and doxycycline plus tricalcium phosphate received groups. The rate of regeneration was greater in doxycycline plus tricalcium phosphate group. They concluded that the combination of doxycycline with tricalcium phosphate results in faster and easier attachment of tricalcium phosphate to bone [13].
Also, positive effects have been confirmed for systemic administration of low-dose doxycycline on bone resorption. In an animal study, Yagan et al [10] demonstrated that the systemic administration of low-dose doxycycline for 21 days significantly decreased bone resorption compared to the no-medication received group (control). Zhou et al [11] evaluated the efficacy of tetracycline for preventing osteoclasts from formation and activity. They showed that the number of osteoclasts in tetracycline and oxytetracycline groups significantly decreased in comparison with other groups. In the present study, the number of osteoclasts was reduced in the bioglass-doxycycline complex group; however, the difference between the three groups in this respect was not statistically significant. The difference between present results and foregoing studies may be due to the different methodologies and staining methods for detection of osteoclasts [11].
Osteogenesis enhancement by bioglassdoxycycline complex is hypothesized to be due to the osteoinductive and osteoconductive properties of bioglass, the bactericidal, inflammation reductive, immunosuppressive, and anti-collagenase properties of the doxycycline, osteoclastogenesis inhibition by BG-D, and increasing the chemotaxis of osteoblasts [4,23]. Further studies are required to better determine the contribution of each aforementioned parameters. In the present study, bone regeneration was higher in the bioglassdoxycycline group compared to the bioglass group. Further studies containing a larger sample size are required to assess the doxycycline efficacy in combination with bone grafts. The efficacy of doxycycline in combination with bioglass for regeneration of bone defects should be also tested in clinical trials.

CONCLUSION
The results obtained show that doxycycline increases the number of osteoblasts and decreases the number of osteoclasts when it used in combination with bioglass to enhance new bone formation in rats. Thus, bioglassdoxycycline complex may be a suitable graft in bone regeneration for medical applications.

Acknowledgement
The authors are deeply thankful to all colleagues for their kind support.

Conflicts of interest
No conflict of interest is associated with this work. No financial support was received for this study

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. Mona Mokhtarian contributed to the proposal preparation, materials order, and surgical assessments. Nasrin Esfahanizadeh contributed to the proposal preparation, analysis of the data, project management, and manuscript writing. Mohammad Reza Nourani contributed to composing the main idea, designing the research plan, analyzing the histological slides, and finalizing the manuscript preparation.

Open Access
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