Introduction

Bone defect and functional problems have turned into a critical worldwide health and hygiene issue (Grote, Reinhardt, Zhang & Wang, 2019; Van Der Spoel, Van Vliet & Van Heemst, 2019). Moreover, bone restoration is a complex process in closed and compound fractures that involve a bone-tissue loss of more than 3 cm. In comminuted fractures with more than 6 cm of tissue loss, the chances of deformity, shortening, recurrence of fractures due to non-union, and deformity resulting from malunion or asymmetric healing increase significantly (Mitchel, Keating & Robinson, 2010). At the same time, complications spawning from the application of conventional methods have advocated a strong shift towards developing three-dimensional (3D) scaffolds from regenerated biomaterials. The 3D scaffolds from regenerated biomaterials reduce the possibility of nonunion or asymmetric healing by improving cell delivery, elevating cellular support on fracture edges, and enhancing growth and regeneration in the fracture region.

In bone-tissue engineering, scaffold structures must be porous and of a composition purely identical to the bone HA provides stronger and more rapid bone tissue repair and can be considered as a preferable alternative to conventional bone grafting techniques (Angelin Jeba Kala & Asaithambi, 2018; Oberbek et al., 2018).

Avian eggshell, with mineral constituents highly resembling those of marine sponge, has found its way in orthognathic surgery as a potential bone-replacement material. Consequently, in recent years, extensive research has been conducted to improve the functioning power of HA. More recently, several reports have recommended the incorporation of hen or other avian eggshells in microparticle form on local delivery or hydrothermal basis with or without other bone grafting materials. Nevertheless, the findings were contradictory in terms of the quantity and quality of the bone generated (Srisubut et al., 2007; Lozano-Carrascal et al., 2017).

Dupoirieux et al. (2000) compared pericranium and eggshell as void fillers in the guided repair of bones and reported no sign of bone repair in any of the groups in the first 15 days. On day 30, they observed bone regeneration solely in the control group that had received no void filler. On day 90, complete bone regeneration occurred in 3-5 of the cases in the control group. There was no indication of osteogenic activity in the pericranium group, while in the third group, non-resorbable hen eggshell powder failed to express osteoconductive characteristics. In another investigation, Yadao et al. (2004) used ostrich eggshell as an implant and bone replacement graft to treat bone fracture on the floor of rabbit's orbit. They reported positive effects of this material on the healing process. Yadegari et al. (2015) based their study on the radiographic examination of the efficacy of hen eggshell powder on the healing of defected tibia bone in dogs. They reported an increase in bone density on days 30 and 60 that was associated with this material. Alternately, Park et al. (2008) assessed the bone repair process in rat calvaria using hen eggshell and compared the results with allograft transplantation in a cow. Findings underlined the potential characteristic of hen eggshell particles as a bone graft in the treatment of rat calvarial defect. Abdulrahman et al. (2014) perceived the high generating potential of HA and demonstrated how HA extraction from eggshell would prove to be cost-effective in terms of bone-repair costs. This is due to the low costs of extracting calcium carbonate from avian eggshell, its high accessibility, and its biodegradable nature as a primary material for bone grafting. Nevertheless, none of the studies specifically quantified the effect of these particles on improved local bone regeneration.

The natural bone matrix is made of bio-ceramics (i.e. HA) and polymers (i.e. collagen). Consequently, obtaining natural and synthetic biomaterials in the form of natural and synthetic polymers, compounds, and biological ceramic substances to produce bone scaffolds seems to be possible. Preparation of porous nano- and micro-fibers is amongst the most recent procedures (Rezwan et al., 2006). The effectiveness of tissue scaffolds has rendered µ-OES scaffolds worthy of consideration in improving bone healing. Exploring methods to accelerate healing is a crucial demand. With this background in mind, the present study experimentally investigated rat calvarial bone, while comparing the efficacy of µ-OES scaffolds and HA scaffolds in accelerating bone tissue repair.

Materials and Methods

Animals

Forty-five male Wistar rats weighing 250-300 g were used in this study. The rats were housed for two weeks at the facility for acclimatization. All animals were housed individually in light- and temperature-controlled facilities and were fed pellets specifically prepared for laboratory animals. In addition, these rats had free access to water during the study. This study was approved by the Ethics Committee of the School of Veterinary Medicine, Semnan University (approval no. 43-17/7/96).

Surgical Procedure

The µ-OES composite was prepared according to the method described by Sanosh et al. in 2009 (6). The rats were anaesthetized intraperitoneally with 50 mg/kg of 10% ketamine hydrochloride (0.05 mL/100 g) and 5 mg/kg xylazine hydrochloride (0.025 mL/100 g).

The dorsal part of the cranium was shaved and disinfected. Surgical sites were exposed with a skin incision and the periosteum at the midline of the calvaria. After the calvarium was exposed, a 7 mm circular full-thickness bone defect was created using a trephine bur on the midline without damaging the underlying dura mater (Figure 1). All calvarial defects were trephined under irrigation with 0.9% saline solution. The animals were randomly divided into three groups with 15 animals per group for experimental periods of 14, 28, and 42 days.

In the HA and µ-OES groups, the defects were filled with HA and micro-particles of ostrich eggshell, respectively. The rats in the control group received no HA or µ-OES therapy. The soft tissues were sutured with 4-0 monofilament nylon to achieve primary closure. To prevent postoperative infection, cefazoline was administered to the animals as intramuscular injections for 3 days (30 mg/kg). Flunixin (Razak Co. Iran) as an analgesic was injected (2.5 mg/kg) intramuscularly.

Specimen Preparation and Histological Evaluation

Animals were euthanized with a lethal dose of thiopental (150 mg/kg), at post-operative periods of 14, 28, and 42 days. The skin was dissected and the area of the original surgical defect was removed en bloc with the surrounding tissues from the animals’ calvarium bone and was immediately submerged in 10% neutral buffered formalin for 48 h. Afterwards, it was rinsed with water and demineralized in 10% formic acid. Following decalcification, each specimen was divided longitudinally into two blocks in the sagittal direction and the blocks were embedded in paraffin. Serial sections were cut longitudinally beginning at the center of the surgical defect. The sections were stained with hematoxylin and eosin for analysis. In the histopathologic evaluation, the progression of bone defect healing was investigated in three regions, including two edges and the center of the defect in each sample. The levels of granulation tissue formation, fibrosis tissue, immature bone tissue, and adult ossification were evaluated to obtain healing rates in different samples. For each sample, three slides were prepared and each slide was investigated by two pathologists who were blind about the samples. Two edges and the central region of calvarial slides were studied by ×100 magnification of light microscope in three different fields for each region. Bone healing was scored according to Allen’s grading system (Deniza et al., 2015).

Figure 1. Photomicrograph of all groups at day 14 post operation. A: in the control group, a granulation tissue that is accompanied with fresh vascularization, edema, and bone-matrix formation, develops on the edge of defect. B: in the HA group, the immature bone tissue was seen. C: a slight immature-bone formation occurs on the µ-OES group

µ-OES: Micro ostrich eggshell, HA: Hydroxyapatite, Co: Control, Wo: Wowen, L: Lamellar, H: Hematoma, NB: new bone ossification, V: Vessel, GT: granular tissue, F: Fibrous (H&E, ×100).

Measurement of Alkaline Phosphatase Activity

Blood samples were taken from the heart of animals 14, 28, and 42 days post-operation, as well as before euthanasia. After separating serum, alkaline phosphatase (ALP) activity was assessed using a colorimetric ALP activity assay kit (Pars Azmun, Iran).

Statistical Analysis

Statistical analysis was performed by the Kruskal-Wallis test using the SPSS software version 16 (SPSS Inc, Chicago, Ill., USA). Descriptive statistics of the data are expressed as mean ± standard deviation (SD). Differences were considered significant when P-value<0.05.

Results

Table 1 presents the results of histopathological studies. Based on the results of day 14, a significant difference was observed in the edge and center of the defect between the two experimental groups, compared to the control group (P≤0.05). However, the difference between the two test groups was not significant in this regard. In the control group, a granulation tissue accompanied by fresh vascularization, edema, and bone matrix formation developed on the edge of the defect. In the HA group, contrary to the control group, a slight immature bone formation occurred on the same day in the same area. A similar pattern was observed on a larger scale in the µ-OES group (Figure 1).

Table 1. The Mean ± SD of the histopathological changes in all groups.

There was no significant difference between the same letters in each row, but significant difference was observed between the non-identical letters.

µ-OES: Micro ostrich eggshell, HA: hydroxyapatite.

On day 28, no significant difference was observed in the edge and center of the defect in either group. At this time, bone matrix and immature bone formation persisted in the control group. In the HA group, bone matrix formation was distinguishable from the immature bone formation as the bone regeneration continued. At the same time, in the µ-OES group, bone regeneration and the formation of parallel arrays of mature lamellar bone were observed. In this group, in the center of bone defect, the granulation tissue advanced towards fibrosis on day 14. Bone matrix deposition on day 28 became distinct, compared to the other two groups (Figure 2).

Figure 2. Photomicrograph of all groups at day 28 post operation. A: The bone-matrix and immature-bone formation persisted in the control group. B: In the HA group, bone-matrix formation was distinguishable from immature-bone formation as the bone regeneration continued. C: in the µ-OES group, bone regeneration and formation of parallel arrays of mature lamellar bone pursued.

µ-OES: Micro ostrich eggshell, HA: Hydroxyapatite, Co: Control, Wo: Wowen, L: Lamellar, H: Hematoma, NB: new bone ossification, V: Vessel, GT: granular tissue, F: Fibrous (H&E, ×100).

On day 42, no other significant difference was revealed in the edge and center of the defect in either group. In the control group, repair progress towards dense fibrosis was accompanied by the formation of thick collagen fibers. In addition to immature bone tissue, trace mature parallel bone was distinguished on the edge of the defect. On the edge of the defect in the HA group, immature bone completely regenerated and the extent of development of mature parallel bone arrays on this day exceeded that of the control group. As for the µ-OES group, the formation of parallel arrays of bone tissue was observed and reported. In the center of the defect, progress from fibrous tissue to the deposition of underlying bone material and formation of new immature bone was significantly greater in the µ-OES group, compared to the other two groups (Figure 3).

Figure 3. Photomicrograph of all groups at day 42 post operation. A: The progression of defect healing was seen towards dense fibrosis along with thickened collagen fibers and a small amount of lamellar mature bone in the control group. B: In the HA group, the immature and irregular bone was formed on the edge of defect; and the parallel and blade mature bone formation was seen. C: In the N-OES group, both edges of defect site were reaching together by a new bone tissue.

µ-OES: Micro ostrich eggshell, HA: Hydroxyapatite, Co: Control, Wo: Wowen, L: Lamellar, H: Hematoma, NB: new bone ossification, V: Vessel, GT: granular tissue, F: Fibrous (H&E, ×100).

In terms of ALP activity, the results of the present study indicated that the applied treatments increased the serum levels of ALP indicating an improvement in healing trends in the tested groups. A reduction was reported exclusively in the HA group on day 28, in comparison with day 14. This difference was not significant and could be due to a type II statistical error, small sample size, and higher standard deviation in the data of this group (Table 2).

Table 2. The Mean ± SD of the ALP changes in all groups.

 µ-OES: Micro ostrich eggshell, HA: hydroxyapatite

Discussion