Bone defects resulting from oncological surgical resections, congenital facial anomalies, trauma or infection represent a significant and common clinical problem. The present study aimed to evaluate the effects of a commercially-available medicinal plant extract product, Ankaferd BloodStopper (ABS), on bone healing. The present study was performed on 24 female ovariectomized (OVX) rats. A defect on each rat calvarium was created using a trephine burr prior to the rats being divided into two groups. Defects were grafted with a gelatin sponge soaked with normal saline (control group) or plant extract (experimental group). Half of the animals were sacrificed after 2 weeks and the others after 4 weeks. In the control group, the defects were not filled with regenerated bone. By contrast, in the experimental group, all defect areas had an increased amount of regenerated bone and connective tissue. Osteoblastic activity appeared to be greater in the experimental group however, osteoclastic activity was observed to be higher in the control group. At 2 and 4 weeks, there was a significant difference in the amount of newly regenerated bone observed in the experimental group compared with the control group (P<0.05). Therefore, the results of the present study indicated that local ABS application had a positive effect on bone healing in the OVX rat model.
Bone defects resulting from facial trauma, congenital facial anomalies, oncological surgical resections or infection represent a common and significant clinical problem. Bone grafts such as autologous, allogenic or synthetic bone grafts, or prosthetic devices are used to reconstruct these defects (
The formation of an encapsulated protein network representing focal points for vital erythrocyte aggregation, is the basic mechanism of haemostasis (
A total of 24 female Wistar rats were used, with a mean body weight of 250±18 g, aged 3 months. They were obtained from the Bezmialem Vakif University Laboratory Animal Center, Istanbul. The animals were kept in individual plastic cages in an experimental animal room at 22°C with a 12-h light/dark cycle. The animals of the study and control groups were fed a standard laboratory pellet diet and drinking water was available
Surgery was completed under sterile conditions. Rats were anesthetized by intramuscular injection of 5 mg/kg Rompuns™ (Bayer AG, Leverkusen, Germany) and 40 mg/kg Ketalar® (Eczacıbaşı, Holding, Istanbul, Turkey). The abdominal skin of each rat was cleaned with a povidone-iodine solution. A 2-cm midline dorsal skin incision was made and the connective tissue between the skin and muscular layer of the abdominal wall was dissected with a dorsal approach. The ovaries were excised bilaterally following ligation of the uterine horn. The muscle layers and skin were then sutured with coated Vicryl® 4–0 polyglactin 910 (Ethicon; Johnson & Johnson, Somerville, NJ, USA). To prevent postoperative infection, an intramuscular injection of 30 mg/kg ceftriaxon (Roche Diagnostics, Basel, Switzerland) and 4 mg/kg carprofen (Pfizer, Inc., New York, NY, USA) was administered to the animals every 24 h for 3 days starting immediately following the surgery. The oestrous cycle was monitored to confirm the success of the ovariectomy and a vaginal smear was examined for 4–5 days throughout the oestrous cycle in all the rats.
The study was started 12 weeks following OVX surgery. The calvarium defects were formed as described (
Following general anesthesia [intramuscular injection of 3 mg/kg xylazine hydrochloride (Rompuns, Bayer, Leverkusen, Germany) and 35 mg/kg ketamine hydrochloride (10% Ketasol®, Richter Pharma AG, Wels, Austria)], all rat calvariums were shaved and the cutaneous surfaces were disinfected with povidone iodine solution prior to surgery. In order to raise the skin of the calvarium, a skin incision ~2.5 cm long was made over the linea media. A small sharp periosteal elevator was used to raise the cutaneous flap laterally and the periosteum was incised and lifted to expose the calvarium. Following exposure of the calvarium, a defect on the right calvarium of each rat was created with a standard 5-mm diameter trephine bur. Defects in the control groups were grafted with a gelatin sponge mixed with normal saline and the experimental groups were grafted with a gelatin sponge soaked in ABS. In all animals, titanium discs were fixed to the calvarial bone completely over the defects, using Histoacryl® (B. Braun Melsungen AG, Melsungen, Germany;
The area of the original surgical defect and the surrounding tissues were removed
The same histologist, who was blinded to the identity of the samples, performed histological and histomorphometric analyses. The presence of inflammatory infiltrate, connective tissue, resorption and bone regeneration was evaluated and the number of osteoblasts and osteoclasts in the defect area were counted. A digital camera connected to a light microscope with an original magnification of ×40 captured images of the histological sections in all groups and saved them on a computer. For histomorphometric analysis, Clemex Vision-Lite 5.0 software (Clemex Technologies, Inc., Quebec, Canada) was used and the area of newly formed bone within the original surgical defect was calculated using an automated image analysis system (NIS Elements version 4.0, Nikon Corporation, Tokyo, Japan) to generate computer-assisted histomorphometic measurements.
All statistical analyses were performed using SPSS, version 20.0 (IBM SPSS, Armonk, NY, USA). The data were subjected to statistical analysis with the Mann-Whitney U test followed by the Kruskal-Wallis test for intergroup comparison and the Wilcoxon signed-rank test for comparisons between the 2- and 4-week groups. P<0.05 was considered to represent a statistically significant difference.
No damage to the dura mater was observed in any of the specimens No inflammatory processes were noted in any of the groups. Connective tissue with collagen fibres parallel to the defect surface and a moderate number of fibroblasts were detected in all groups.
In the control groups, similar histological results were observed in the 2- and 4-week groups; no defects were completely filled with regenerated bone. No ossification was identified in the defect area of the 2-week control group (
In the 2-week experimental group, all defect areas were mostly filled with regenerated bone and connective tissue. The presence of dense fibroblast connective tissue, dense ossification, osteoblast and osteoclast cells in the defect area were notable (
In a comparison of all groups, osteoblastic activity was observed to be greater in the experimental groups than in the control groups (P<0.01;
Animal models serve a critical role in bone-related studies, particularly in the assessment of the biological characteristics of bone metabolism, in physiological and pathological conditions. Additionally, experimental animals have been used extensively to model human pathological states, including osteoporosis (
In the present study, a rat calvarial defect model was used to evaluate bone regeneration. This model is considered comparatively locatable, simple and reproducible as in this defect, spontaneous healing does not occur. Furthermore, the model was observed to be effective at evaluating the potential for bone regeneration. Finally, this model has some compressive force similar to intraoral conditions (
Osteoporosis is associated with a decreased regenerative capacity of bone, which may account for the clinical impairment observed with bone-regenerative approaches in osteoporotic models (
Bone regeneration is a complex biological process with three key components: A morphogenetic signal, a convenient carrier matrix that acts as a scaffold for bone regeneration and responding host cells that are able to differentiate into bone cells (
ABS is a commercially-available plant extract product, which has been used as a haemostatic agent in Anatolia, Turkey. Its usage has been approved in Turkey for use in the management of dental surgery bleeding and external haemorrhage (
A number of studies have focused on bone healing in the calvarial defect (
In conclusion, the present study presented certain limitations, including the fact that it was an animal study with a small sample size. However, the local application of ABS had a positive effect on bone healing in the OVX rat model. Further experimental and clinical studies focused on ABS may be required for determining the therapeutic dose of ABS.
Surgical procedure in control and experimental mice. (A) A defect on the calvarium was created with a diameter trephine burr. (B) In the control group the defect area was grafted with gelatin sponge mixed with saline solution and the experimental group was grafted with gelatin sponge mixed with Ankaferd BloodStopper. (C) Titanium disc fixed to the calvarial bone completely over the defect. (D) Periosteum and then the skin were sutured.
Histological assessment. (A) No defect was completely filled in the 2-week control group; the histological image is observed at original magnification (hematoxylin and eosin, ×40) (B) Loose and thin connective tissues in the defect areas and regenerated bone areas were observed in the 4-week control group. Red arrows indicate the presence of the thin connective tissue in the defect area. Minimal ossification was detected originating from the defect margins (hematoxylin and eosin, ×100). Scale bar, 0.5 mm. Rb, regenerated bone.
Histological assessment for the 2-week experimental and control groups. (A) Regenerated bone areas with fibrocellular connective tissue and host bone area boundaries are observed (hematoxylin and eosin, ×100). (B) The presence of dense fibrocellular connective tissue, dense ossification, osteoblasts, and osteoclast cells are indicated (hematoxylin and eosin, ×400). Arrow, osteoblast; o, osteoclast; f, fibrocellular connective tissue; hb, host bone; rb, regenerated bone; line (−—), border of host bone tissue and regenerated tissues; ABS, Ankaferd BloodStopper.
Histological assessment for the 4-week experimental and control groups. Bone growth was complete, formed of lb and bm (hematoxylin and eosin, ×200). Scale bar, 0.5 mm. lb, lamellar bone; bm, bone marrow.
Histomorphometric analysis of osteoblastic activity. Experimental groups exhibited increased osteoblastic activity compared with control groups. **P<0.01, ***P<0.01 vs. control.
Histomorphometric analysis of osteoclastic activity was higher in the control groups compared with the experimental groups *P<0.05, ** P<0.05 vs. experimental groups.
A comparison of 2 and 4-week control and experimental groups. A statistically significant difference was determined between the control and experimental groups at 2 and 4 weeks with regard to histomorphometric analysis of regenerated bone.**P<0.05, ***P<0.05 vs. control.