Comparison of Ostene® and Bone wax on Bone Healing: A Comparative experimental study in rabbits

Corresponding author: Huda A. Salim. Mosul 41002, Iraq. E-mail: hudaakram81@yahoo.com Abstract: Introduction: Ostene® is a new synthetic bone hemostatic waxlike inert and biocompatible material that dissolves within two days after application. Bone wax is a wellknown topical hemostatic agent, easy to use, and its application is very simple. Wound healing is a complex biological process; Bone is a dynamic tissue that is continuously resorbed, renewed, and remodeled. Materials and methods: Twenty domestic rabbits were divided into four groups (day 1, day 3, day 7, and day 14). Each rabbit was anaesthetized and three holes were drilled in the mandible: one was filled with Ostene®, another with bone wax, and the other was left unfilled as control. Sites of intervention were assessed by histopathology. Results and Discussion: Ostene® and bone wax showed osteoinductive property in bone healing with no inf lammatory reaction. Our study revealed new bone formation within 14 days in Ostene® group. After histopathological analysis and scoring was finished, analysis by SPSS 14 software showed a significant difference between the use of Ostene® and bone wax. Conclusion: Ostene® showed superiority over bone wax in bone healing, and it can be used in the same way as bone wax with no interference with bone healing and osteogenesis. Ostene® has no side effects following application.


INTRODUCTION.
Bone is a biologically highly dynamic vascularized living tissue, constant undergoing adaptation to the mechanical and metabolic demand of the body by means of bone regeneration and repair, as a result of the balance between the activity of osteoblasts and osteoclasts. [1][2][3][4] Bones are largely made up of an organic matrix (osteoid) and the calcium hydroxyapatite mineral, which gives the bones strength and hardness. It is continuously resorbed, renewed, and remodeled. These processes are carried out by several different types of bone cells that are regulated by a number of transcription factors, cytokines, and growth factors. 5 Osteoblasts are mature cells that are actively mobile, located on the surface of bones. These cells synthesize, transport, and arrange many proteins of matrix and bone forming cells. 5, 6 Osteoclasts are multinucleated cells which are responsible for bone resorption. They are larger than osteoblasts and osteocytes. 5,7 The healing process of bone has three overlapping stages: inflam-mation, bone production, and bone remodeling. Regeneration can occur by three different mechanisms: osteogenesis, which is formation of new bone from bone forming cells; osteoconduction, the physical property of the graft to serve as a scaffold for new bone growth; and osteoinduction, which is a process whereby one tissue, or product derived from it, causes a second undifferentiated tissue to differentiate into bone. Bone healing processes depend mostly on osteoinduction. 8- 10 Ostene® is a new synthetic bone hemostatic wax like material made of water-soluble alkylene oxide copolymers (AOC). It is composed of a sterile mixture of hydrophilic, biocompatible copolymers derived from ethylene oxide and propylene oxide. It is an inert material and dissolves from the bone within 48 hours after application and it is excreted from the body unchanged through renal clearance safely without metabolism. [11][12][13][14][15][16] Ostene® is applied in a similar fashion as bone wax and to a thickness of 1-2mm; it does not cause infection, nor induce inflammatory reactions, and it does not interfere with coagulation, nor with osseous union. 14- 18 AOC have a long history of use in the medical and pharmaceutical field, its approved by the U.S Food and Drug Administration, it dissolves from the site of application faster than other materials, and it is also hypothesized that AOC permits faster bone healing than other resorbable hemostatic agents. 19,20 Ostene® may be a better alternative to bone wax due to its water solubility. 13, 21 Bone wax is a well-known topical hemostatic agent, composed of cera alba (beeswax) (88%) and isopropyl palmitate (12%), a palm oil-based emollient, moisturizer, and thickening and anti-static agent. [22][23][24][25] Bone wax is relatively easy to use, and its application is very simple. It is a non-absorbable hemostatic sealant and it functions as a mechanical tamponade to stop bone bleeding in patients undergoing thoracic, orthopedic, craniofacial and neurosurgery. 26,27 This study aims to evaluate and compare the effects of Ostene® and bone wax in the healing process of the mandibular bone of the rabbit.

Study design
Twenty domestic rabbits were selected and divided into four groups according to sacrificing day: (day 1, day 3, day 7, and day 14).
Three holes were drilled in each rabbit´s mandible, these holes filled with different materials: one filled with Ostene®, the other filled with bone wax, and the last one left unfilled, as a control.

Surgical protocol
All surgery was done under general anesthesia, each animal was injected intramuscularly in the thigh muscle with 40 mg/kg ketamine and 4mg/kg xylazine. 26,28, 29 Ten minute after anesthesia, shaving and cleansing of the skin was performed, then under aseptic conditions, a 1 cm incision was made under the mandible, and the mandibular bone was exposed.
Three holes were drilled with a 2mm spherical dental burr to assure a standardized defect with equal depth and the drilling was done under irrigation with normal saline, two holes in the right side the nearest one to the midline filled with Ostene® and the second one filled with bone wax, the other hole in the left side was left unfilled, as a control. Suturing was then performed using sterile synthetic absorbable, Vicryl suture size 2-0.
Histopathological study At the day of sacrificing, each mandible was dissected and fixed in 10% neutral buffered formalin, followed by decalcification of the bone to complete processing for the preparation of slides and staining with hematoxylin eosin. The histopathological scoring used was that of Sultana

RESULTS.
After all animals were sacrificed as scheduled, analysis of the differences between the three experimental groups with Friedman test was performed. (Tables 1, 2, 3, 4 and 5) In the Ostene® group enhance bone healing was apparent at 14 days illustrated by woven bone during this period. Abundance of bone trabeculae can be seen in histopathological section with a great amount of granulation tissue. ( Figure 1A, Table 2) For the control group, normal bone healing occurred as expected during the healing period. Regarding the bone wax group, healing occurred to a lesser degree than in Ostene® group, granulation tissue and bone trabeculae was found in histopathological section but in lesser amounts compared to the Ostene® group. (Table 2) DISCUSSION.
Ostene® appears to have many advantages over bone wax as it did not cause inflammation in excess while bone wax did. Furthermore Ostene® is removed from the body unchanged while bone wax is not. Our results are in agreement with Gurcan et al., 23 who showed that the advantages of Ostene® over bone wax is that Ostene® is absorbed within two days, in addition to that it does not have adverse effects on healing and is excreted unchanged by the body. Nooh et al., 27 informed a foreign body reaction to bone wax in the form of granulomatous inflammation and fibrous encapsulation with the inhibition of bone healing and osteogenesis. In addition, Vestergaard et al., 32 showed that bone wax significantly inhibits bone healing and induces chronic inflammation in pigs whereas Ostene® did not.
About granulation tissue, there is a significant difference in most of the groups but it is more marked in the Ostene® group, which may be due to Ostene® dissolving from the site of application within two days, allowing the early phases of bone healing to occur. This is in agreement with Wellisz et al., 11 who showed that the polymer material dissolved from the site of application within 24 to 48 hours allowing early bone healing to occur.
It is reported in Wellisz et al., 18 that bone wax increased infection rates and impaired the ability of bone to clear bacteria. Also this study revealed that bone wax remain as a foreign body at the site of application, and osteoblasts in bone wax group have been shown to be absent in the presence of a thin layer of bone wax. Our study revealed new bone formation (woven bone)  11 who showed that new bone grew within 10 days in a rat femur defect treated with polymer and untreated controls. Bone wax is insoluble and therefore not resorbable, and it interferes with bone healing at the site of application, it may aid in the development of infection by decreasing bacterial clearance in the cancellous bone and inhibits osteoblasts from reaching bony defects, leading to impaired bone healing, whereas Ostene® does not cause infection, inflammatory reactions, or interference in the osseous union. 14, 21 Both Wellisz et al., 18 and Wellisz et al., 20 found that, although bone wax was effective in stopping bone healing, it remains at the site of application and has a number of adverse effects, forming a physical barrier to bone healing. All the defects filled with Ostene® showed newly woven bone rich in cellular activity, with no evidence of fibrotic scar tissue, indicating that good healing occurred with an absence of fibrotic scar tissue. This revealed that Ostene® has an anti-fibrosis effect in agreement with results from other studies. 19, 23 Regarding the histopathological analysis, abundant, poorly organized bony trabeculae with maturation of granulation tissue into the connective tissue was observed the control group during 2 nd week; while at the same period in the Ostene® group more organized granulation tissue with bone trabeculae which increased in number, thickness and organization was visible, in contrast to bone wax group who displayed less bone trabeculae in a disorganized manner, a phenomenon corroborated by other studies. 12