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Research Article

Royal Jelly: A Novel Biological Product in Healing of a Critical Bone Defect Model in Rat

Ahmad Oryan1*, Abdolhamid Meimandi-Parizi2, Emad Sayahi2, Amin Bigham-Sadegh3 d1Pathobiology Department, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.2Clinical Sciences Department, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.3Department of Veterinary Surgery and Radiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.

Citation: Ahmad Oryan., et al. “Royal Jelly: A Novel Biological Product in Healing of a Critical Bone Defect Model in Rat”. EC Orthopaedics 10.4 (2019): 216-227.

*Corresponding Author: Ahmad Oryan, Pathobiology Department, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

Received: January 19, 2018; Published: March 27, 2019

Abstract

This study evaluated the potential role of royal jelly in bone repair along with the chitosan (CS) and demineralized bone matrix (DBM) scaffolds in a nonunion radial bone defect model in rat.Seventy-two bone defects in the radius bones of 36 healthy male rats were created bilaterally and they were divided randomly into 6 groups (n = 12/group). The study groups included defect or untreated group, autograft, CS, DBM, CS-royal jelly (CS-RJ), and DBM-royal jelly (DBM-RJ). The bone defects were evaluated radiographically at 28th, 42nd and 56th postoperative days, and also assessed morphologically, histopathologically, histomorphometrically and biomechanically following euthanasia at the 56th post-operative day.

The CS-RJ and the DBM-RJ groups, showed enhanced biomechanical properties and improved structural characteristics compared to the defect, CS and DBM groups. The defect site in the DBM and untreated groups were mainly restored by fibrous connective tissue while the autograft group was mostly filled by cartilage and a lesser amount of woven bone. The woven bone followed by hyaline cartilage was the main constituent of the newly formed tissues in the RJ treated groups, at the 56th day after injury. The results of this study showed that percutaneous injection of diluted RJ (5 mg/defect) in the bone defect can improve bone formation in critical radial bone defect of rat. Based upon the remarkable superiority of bone formation in the RJ groups over the autograft group, RJ probably has high potentials to replace autologous bone grafting.

Keywords: Bone; Healing; Royal Jelly; DBM; CS; Scaffold

Abbreviations

CS: Chitosan; RJ: Royal Jelly; DBM: Demineralized Bone Matrix

Introduction

Despite the advancements made in the orthopedic methods and surgical procedures, healing of bone fractures are not always satisfying and may led to delayed union or even non-union. Although autograft is the gold standard in bone healing, with the advent of bone graft substitutes, the clinical demand in developing these scaffolds has been increased [1]. The main advantage of these grafts include their insignificant immunogenicity, infinite resources and adjustable physical and chemical properties [2]. Among the natural scaffolds, chitosan (CS) is an interesting material. CS derived from chitin which has many sources in the world [3]. Many properties of CS are related to the degree of de-acetylation and its molecular weight. In addition to physical support, CS increases osteoblasts activity and mineralization, bone regeneration and also enhance collagen production by osteoblasts [4]. Unfortunately, these biological effects are

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not enough in comparison with the autologous bone graft [5] and researchers try to enhance its bone regeneration properties by adding various materials such as growth factors or osteogenic drugs.

Demineralized bone matrix (DBM) is another natural scaffold which has a long history in medicine in treatment of nonunion, osteomyelitis and critical bone defects. The demineralization process is carried out by hydrochloric acid, which leads to destruction of surface antigens and reduced immune reaction to the scaffold [6]. DBM has osteoconductive and osteoinductive properties, but its osteogenic property diminishes by the process of manufacturing [2,7]. DBM could retain drugs and growth factors and slowly release them in the environment; this criterion makes it an interesting choice in bone tissue engineering [7].

Royal jelly (RJ) is a traditional complementary food produced by the mandibular and hypopharyngeal glands of worker bees and is mainly consumed by the queen and larvae. RJ contains water, proteins, carbohydrates, lipids, fatty acids, mineral salts, vitamins and polyphenols [8]. It has been demonstrated that RJ has antibacterial, antioxidant, antitumor, and anti-inflammatory properties [9,10]. Investigations in laboratory animals revealed that RJ results in vasodilation and increases the rate of cellular proliferation and differentiation [11]. It has been demonstrated that apisin, one of the major glycoproteins in RJ, enhances proliferation of neonate skin fibroblasts and result in production of more collagen and other extra cellular matrices. Furthermore, apisin induce MC3T3-E1 cell differentiation to osteoblasts [12]. Regarding the estrogenic effect of RJ and its effect on osteoblasts and collagen production as well as inflammatory modulation, we used RJ along with CS and DBM scaffolds in treatment of critical bone defects. There is no published data regarding the concurrent use of CS or DBM scaffolds with RJ on the healing of critical bone defects. Therefore, the objective of this study was to evaluate the clinical, radiological, macroscopical, histopathological, and biomechanical effects of RJ along with DBM or CS in bone regeneration and healing of critical bone defects.

Materials and Methods

Porous CS scaffold was produced by freeze-drying the CS solution (2% w/v) [13]. For this purpose, the medium molecular weight CS powder (Sigma-Aldrich) was dissolved in 0.5M acetic acid. The final viscous solution was left at 4°C for 24h, then transferred to -20°C for another 24h. In order to create porosity, the scaffold was freeze dried and cross-linked by glutaraldehyde (0.5%). The CS scaffolds was rinsed in distilled water, 4 times every 6h, to eliminate the remaining glutaraldehyde and glycine [14]. The scaffolds were fixed and dehydrated by 96% ethanol. The product was placed in sterile dishes and left at 4ºC before being used in the defects.

The DBM was obtained from the long bones of a healthy 2-year-old Holstein cow slaughtered in a local slaughterhouse. The bones were cut to 1 cm2 pieces, using an iron saw, under normal saline solution irrigation, and after removing the soft tissue remnants, kept at -70°C until further use. After thawing in ethanol (100%) and air drying, the bone pieces were placed in 0.5M hydrochloric acid (Merck) at 4ºC under daily examination to be appropriately demineralized [15]. Radiography of bone pieces showed soft tissue opacity after 16 days, confirming sufficient demineralization. The demineralized bone matrices were rinsed with deionized distilled water until the pH meter confirmed their neutrality. The demineralized bone matrices were immersed in absolute ethanol for 24h until the alcohol was completely evaporated and then stored at -20°C aseptically. The fresh RJ was bought from a local honey bee farm. The pure RJ was freeze-dried just after delivery; this led to a white fine powder which was kept away from light and moisture until use. RJ powder was sterilized at the Cobalt 60 source with 20 kG gamma radiation for 18 hours.

Animals and surgical operations

A total of 39, eight-weeks-old, weighing 200 - 250g, male Wistar rats were purchased from a certified licensed local animal house. Appropriate human nursing was provided for all the rats in accordance with the animal use and care guidance published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). All regulations related to the animal study was approved by the Local Ethics Committee “Regulations for the Use of Animals in Scientific Methods” in the Veterinary Faculty of our university.

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After anesthetizing the animals (using ketamine (10%, 75 mg/kg BW) and xylazine (2%, 10 mg/kg BW) (both from Alfasan Co., Woerden, Holland), the midshaft of the radius bone of both fore limbs were exposed and a 5 mm bone piece was harvested from the radius to create a critical non-union bone defect model (Bigham-Sadegh and Oryan). Three rats (n = 6; number of radial bones) were randomly selected and considered as normal bone group to compare the biomechanical properties of intact radius and ulna bone complexes with other groups. Based on the implanted material, the radial bone defects were randomly divided into six groups (n = 12 radial bone /group). The bone defects remained empty (untreated or the defect group) or filled with extracted bone pieces from the opposite forelimb of the same rat (autograft group). Groups 3 - 6 were the CS, DBM, CS-RJ, and DBM-RJ groups, in which the bone defects were implanted with the CS or DBM scaffolds with similar size of the harvested radial bone segments (dimensions = 2 × 2 × 5 mm3). After engrafting the scaffolds and suturing the incision, in the CS-RJ and DBM-RJ, 0.1 ml of the diluted RJ solution (50 mg/ml) was injected percutaneously into the defect site. An injection of the RJ was repeated similarly in the third postoperative day. Dosage of RJ in each defect was chosen according to the previous in vitro and in vivo studies [16-20]. RJ was confined to the defect area by sutured muscles and fascia. Analgesic and antibiotic therapy were provided with an intramuscular injection of enrofloxacin (Enrofan 5%) and Flunixin meglumine (2.5 mg/kg) for 5 days. The animals were euthanized at the 56th postoperative day.

Clinical examinations

The clinical behavior, physical activities, and weight bearing capacity on the basis of the surgical site in terms of the pain expression at the digital touch and presence of inflammatory symptoms such as hyperemia, swelling, and edema, were evaluated blindly throughout the experiment. The animals were euthanized at the 56th day after injury. For this purpose, the animals were anesthetized deeply as mentioned previously and cardiac arrest was induced by rapid intra cardiac injection of potassium chloride [21]. The radius and ulna complex was extracted and the healing area of the radius was evaluated macroscopically.

Evaluation of the diagnostic imaging

Lateral radiography from the injured forelimbs in the anesthetized animals was performed at the 0th, 28th, 42nd and 56th post-surgical days. Blind evaluation of the radiographs was performed by two orthopedic surgeons and scored for bone formation, union and remodeling by lane and Sindhu system which has been described in table 1.

Bone formation

No evidence of bone formation 0Bone formation occupying 25% of the defect 1Bone formation occupying 50% of the defect 2Bone formation occupying 75% of the defect 3

Bone formation occupying 100% of the defect 4Union (proximal and distal evaluated separately)

No union 0Possible union 1

Radiographic union 2Remodeling

No evidence of remodeling 0Remodeling of medullary canal 1

Full remodeling of cortex 2Total point possible per category

Bone formation 4Proximal union 2

Distal union 2Remodeling 2

Maximum score 10

Table 1: Modified Lane and Sandhu radiological scoring system.

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Histopathological evaluations

The extracted radius and ulna complexes (n = 6/group) were fixed in 10% neutral buffered formalin solution. They were decalcified by nitric acid solution (0.5%). Hematoxylin and eosin staining was performed on the 5 µm in thickness tissue sections. Blind assessments of tissue sections were performed by two experienced pathologists. The cells including fibroblasts/fibrocytes, chondroblasts/chondrocytes, osteoblasts/osteocytes, osteoclasts and also inflammatory polymorphonuclear and mononuclear cells as well as blood vessels were counted on three different microscopic field in each tissue sections (×400, magnification), using a digital camera (Olympus, Tokyo, Japan) attached to an ordinary light microscope (Olympus, Tokyo, Japan). The density of fibrous connective tissue (DFT), cartilaginous tissue (DCT) and osseous tissue (DOT) were also assessed in the same magnification.

Biomechanical evaluations

The samples intended for biomechanical assessments (n = 6, in each group) were wrapped by PBS-soaked sterile gauze and kept at -20°C. The three-point bending test was performed by a universal tensile testing machine (Instron, London, UK). After measuring the diameter of each bone specimen, it was placed horizontally on two supporting bars with 20 mm distance. A third rod with the pressure of 10 mm/min was lowered at the healing area (middle of the defect) until fracturing. Simultaneously the load-deformation curve was generated and the maximum load, maximum stress, yield load, bending stiffness, ultimate strain, and yield strain were measured and calculated. All the extracted data from the load-deformation curve were presented as Mean ± SD, as previously described [22].

Statistical analysis

The quantitative data such as the biomechanical and histomorphometric results were presented as mean ± SD and were analyzed statistically by one-way ANOVA and followed by Tukey post-hoc test. The qualitative or scored data were presented as mean (min-max) and subjected to Kruskal-Wallis, non-parametric ANOVA, and subsequent Mann-Whitney U test. P-values lower than 0.05 were considered statistically significant. The whole statistical analysis was done by SPSS software, version 19.0 (SPSS, Inc., Chicago, USA).

Results

Findings of diagnostic imaging

There was no significant difference in radiographic evaluation between the DBM-RJ and CS-RJ groups. Whereas the CS, DBM, CS-RJ and DBM-RJ groups showed significant differences with the autograft group at the 28th postoperative day (p = 0.000, p = 0.000, p = 0.007 and p = 0.019 respectively). The DBM-RJ group showed a significant superiority over the CS and DBM groups (p = 0.036 and p = 0.031) at this time point. There was no significant difference between other groups at day 28 post-injury. The radiographic results are shown in figure 1 and table 2.

Figure 1: Macroscopic and radiographic findings of the radial defects at 56 days after injury. It was demonstrated by gross evaluation that the defects in the autograft group were filled by hard connective tissue such as cartilage and bone

which were connected to the edges of the radius and the ulnar periosteum. In the chitosan-RJ and DBM-RJ groups, the healing areas were filled by remaining of the scaffolds associated with bone and cartilage tissues. In the untreated defect group, the defect areas were mostly filled by loose soft connective tissue. The autograft group demonstrated

significant superiority over the untreated defect group at the 56th post-operative day (p < 0.05). The untreated defect, chitosan, and DBM groups demonstrated significantly inferior bone formation in comparison to the autograft,

chitosan-RJ and DBM-RJ groups at the 56th post-operative day (p < 0.05). There were no statistically significant differences between the radiographic scores of the autograft and royal jelly treated groups (chitosan-RJ and DBM-RJ).

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Radiographic evaluation did not show any significant difference between the DBM-RJ, CS-RJ and autograft groups at the 42nd post-operative day. There was no statistical difference between the CS and CS-RJ despite the superiority of CS-RJ (p = 0.051), while superiority of the DBM-RJ over DBM group was statistically significant (p = 0.002) at the 42nd post-operative day. There was a significant statistical difference between the defect and DBM-RJ (p = 0.003) at this stage whereas the CS-RJ was not significantly different with the defect group. There was no significant difference among other groups at day 42 post-injury.

Radiographic evaluation at the 56th day post-operation did not demonstrate significant differences between the CS-RJ, DBM-RJ and autograft groups. There were significant differences between the CS-RJ and DBM-RJ with the defect group (p = 0.000 and p = 0.004 respectively). Radiographic evaluations demonstrated significant differences between the CS-RJ and CS group (p = 0.000) and between the DBM-RJ and DBM group (p = 0.038) at this stage. There were also significant differences between the autograft group and CS and DBM groups (p = 0.008 and p = 0.031, respectively). There was no significant difference between other groups at the 56th day after surgery.

Gross and histopathological findings

Throughout the term of study, there were no wound complications, toxemia or local foreign body reactions. There was no animal death and all animals were normally active and their weight increased at the end of experiment. The defect area in both the CS-RJ and DBM-RJ groups resulted in a moderate inflammatory reaction in the first 2 weeks which was subsided in the third week. The inflammatory reaction subsided in the third week, in the autograft group, and was similar to the RJ treated groups at this time point. The defect sites in the RJ treated groups as well as the autograft group were filled with a relatively firm tissue and palpating the bone edges were difficult at the 56th post-operative day. While in the DBM and CS groups there was a high inflammatory reaction which subsided at the end of experiment. The healing area of CS and DBM group was filled with a semi-firm tissue, some movable parts could be palpated and the bone edges were palpable after 56 days of injury. A severe inflammatory response was observed in the defect group which subsided over the 56 days of experiment and at the end of the study the defects were filled with soft fibrous connective tissues and the bone edges could be palpated easily.

Histopathologic evaluation at the 56th post-operative day revealed that autologous bone graft yet could be observed in the defect site. The distance of autograft and bone edges were filled with woven bone and hyaline cartilage. While these two hard connective tissues made the most portion of the regenerated tissue, some dense fibrous connective tissue was also visible in the middle of the defect. The least regeneration and osteosynthesis was observed in the untreated defects and most parts of the defect area was filled with loose areolar connective tissue; however, some cartilage and minimum amounts of bone tissue was observed near the cut edges of the radial bone.

The scaffold was partly resorbed and fibrous connective tissue enveloped the scaffold remnants, in the case of CS scaffold. The fibrous tissue changed to cartilaginous tissue mostly in areas near ulnar periosteum and ultimately become osseous in some limited extent near the old radial bone edges. The DBM treated defects showed bone and hyaline cartilage formation. Around the DBM in the middle part of defect, most of the reconstructed tissues included cartilage, and bone tissues. There was no bridging bone tissue between the bone edges and DBM. The whole gap, except the peripheral area of the defect, in the CS-RJ group, was filled by woven bone. Remnants of CS scaffold could be found on the surface of the regenerated tissue. Appropriate blood supply and initial states of remodeling were evident in the lesions of animals of this group. The scaffold’s borders in the DBM-RJ group were replaced with cartilage and spongy bone. Bone regeneration begun in the middle of the defect, leaving only a narrow boundary of the hyaline cartilage between the woven bones. The histopathologic results are shown in figure 2 and 3 and table 3.

pa

Mean (min - max)Postoperative

days DBM-RJ (6) (n = 12)

Chitosan-RJ (5) (n = 12)

DBM (4)(n = 12)

Chitosan (3)(n = 12)

Defect (2)(n = 12)

Autograft (1) (n= 12)

0.0003.0 (0 - 8)2.54 (0 - 6)1.0 (0 - 2)e1.2 (0 - 4)d1.58 (1 - 2)c5.27 (3 - 8)b280.0315.54 (1 - 9)4.18 (1 - 8)1.81 (0 - 4)i2.5 (1 - 4)h2.91 (1 - 4)g5.90 (4 - 10)f420.0136.09 (2 - 9)6.72 (4 - 9)3.54 (1 - 5)m3.7 (2 - 5)l3.00 (1 - 5)k6.36 (4 - 10)j56

Table 2: The sum of radiographically scored bone healing at determined at different postoperative intervals.

DBM (demineralized bone matrix)/DBM-RJ (demineralized bone matrix and royal jelly)/chitosan-RJ (chitosan and royal jelly). aKruskal-Wallis non-parametric ANOVA. bp < 0.05 (1 vs. 2, 3, 4, 5, 6). cp = 0.036 (3 vs. 6). dp = 0.031 (4 vs. 6).

ep < 0.05 (1 vs. 2, 3, 4). fp = 0.003 (2 vs. 6). gp = 0.002 (3 vs. 6). hp = 0.002 (4 vs. 6). ip < 0.05 (1 vs. 2, 3, 4). jp = 0.000, p = 0.004 (2 vs. 5, 6). kp = 0.000, p = 0.024 (3 vs. 5, 6). lp = 0.006, p = 0.038 (4 vs. 5, 6).

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Figure 2: Tissue sections from the radial bone defects at the 56th post-operative day. Cartilage and woven bone filled the distance between the radial bone edges and the autologous bone graft, in the autograft group. The healing area, in the untreated group,

is filled by loose areolar connective tissue, and minimum amounts of bone and cartilage tissue which are observed in vicinity of the radial bone edges. Residual of the scaffolds have still been preserved, in the healing area of the chitosan and DBM groups

after 56 day of implantation. The remnants of chitosan scaffold have been encapsulated by fibrous connective tissue and in some regions the fibrous tissue is modified to cartilaginous tissue. Some limited extent of woven bone could be found near the radial bone edges and ulnar periosteum, in the chitosan group. In the chitosan and DBM groups which were treated by royal jelly, a remarkable

superiority in bone formation and a significant decrease in cartilage and fibrous connective tissue is evident compared to the untreated, chitosan and DBM groups. The scaffold in the DBM-RJ group more effectively participated in the healing compared

to the DBM group after 56 days of injury. New bone formation, in the DBM-RJ group, didn’t lead to a bridging bone between the radial bone edges. In the chitosan-RJ group, the woven bone filled the distance between the radial bone edges and the remnants

of the chitosan scaffold were rarely visible at the 56th day after implantation. Primary osteons were present very often in the newly formed osseous tissue which indicates early stages of remodeling. More fibrous and cartilage tissue in the DBM-RJ group

in comparison to the chitosan-RJ group indicates a slower rate of the healing process in the DBM-RJ group (H & E staining).

Abbreviations: LACT: Loose Areolar Connective Tissue; FCT: Fibrous Connective Tissue; RBE: Radial Bone Edge; WB: Woven Bone; BV: Blood Vessel; CCT: Calcified Cartilaginous Tissue; HC: Hyaline Cartilage; DCT: Dense Connective Tissue;

BM: Bone Marrow; R: Remnants of the Scaffold; IR: Inflammatory Reaction; UB: Ulnar Bone; PO: Primary Osteon.

paDBM- RJ (6)Chitosan-RJ (5)DBM (4)Chitosan (3)Defect (2)Autograft (1)

Volume (number)Mean ± SDMean ± SDMean ± SDMean ± SDMean ± SDMean ± SD

0.0005.8 ± 2.166.0 ± 1.812.4 ± 4.03e9.71 ± 3.35d10.86 ± 4.48c7.0 ± 2.05bInflammatory cells0.00013.4 ± 2.711.2 ± 2.2734.4 ± 8.05i54.14 ± 5.49h65.8 ± 15.57g18.6 ± 7.35fFibroblast+ fibrocyte

0.03234.0 ± 5.4724.7 ± 4.38n73.2 ± 12.79m41.14 ± 10.12l26.14 ± 6.98k52.4 ± 10.54jChondroblast+ chondrocyte

0.00155.88 ± 6.0154.5 ± 7.9223.2 ± 6.61r34.57 ± 7.85q7.86 ± 2.26p46.9 ± 7.78oOsteoblast+ osteocyte0.5313.2 ± 1.33.56 ± 1.132.2 ± 1.31.29 ± 0.75u0t4.2 ± 1.54sOsteoclast0.0006.2 ± 2.385.33 ± 1.227.6 ± 2.42.57 ± 1.27x10.0 ± 2.30w3.9 ± 1.72 vBlood vessels0.2604.4 ± 2.305.89 ± 1.694.2 ± 1.32.43 ± 1.07ab0.86 ± 0.69z2.1 ± 1.37 yOsteons

0.00011.4 ± 2.3010.78 ± 1.8522.4 ± 2.3af37.71 ± 4.23ae54.14 ± 9.77ad13.50 ± 4.76acDensity of fibrous tissue (%)

0.00028.6 ± 3.1323.56 ± 3.39ak48.2 ± 5.93aj28.71 ± 6.52ai22.0 ± 6.75ah39.5 ± 6.60agDensity of cartilage tissue (%)

0.00046.8 ± 4.0851.67 ± 3.8715.4 ± 4.03ao24.14 ± 4.56an6.57 ± 2.14am35.6 ± 6.96alDensity of osseous tissue (%)

Table 3: Histomorphometric findings in the bone defects at 56 days after injury.aOne-way ANOVA followed by Tukey post-hoc test; b p = 0.037 (1 vs. 4).cp = 0.029 (2 vs. 5, 6). dp = 0.028, p = 0.034 (3 vs. 5, 6). ep = 0.020,

p = 0.018 (4 vs. 5, 6). fp < 0.05 (1 vs. 2, 3, 4, 5). gp < 0.05 (2 vs. 4, 5, 6). hp < 0.05 (3 vs. 4, 5, 6). ip = 0.002, p = 0.003 (4 vs. 5, 6). jp < 0.05 (1 vs. 2, 3, 4, 5, 6). kp = 0.008, p = 0.000 (2 vs. 3, 4). lp = 0.002, p = 0.004 (3 vs. 4, 5). mp = 0.001 (4 vs. 5, 6). np = 0.015 (5 vs. 6).

op < 0.05 (1 vs. 2, 3, 4, 5, 6). pp < 0.05 (2 vs. 3, 4, 5, 6). qp < 0.05 (3 vs. 4, 5, 6). rp = 0.000 (4 vs. 5, 6). sp < 0.05 (1 vs. 2, 3, 4). tp < 0.05 (2 vs. 3, 4, 5, 6). up = 0.000, p = 0.026 (3 vs 5, 6). vp < 0.05 (1 vs. 2, 4). wp < 0.05 (2 vs. 3, 5, 6). xp < 0.05 (3 vs. 4, 5, 6). yp < 0.05 (1 vs. 2, 4, 5).

zp < 0.05 (2 vs. 3, 4, 5, 6). abp = 0.045, p = 0.000 (3 vs. 4, 5). acp < 0.05 (1 vs. 2, 3, 4). adp < 0.05 (2 vs. 3, 4, 5, 6). aep < 0.05 (3 vs 4, 5, 6). afp = 0.000 (4 vs. 5, 6). agp < 0.05 (1 vs 2, 3, 4, 5, 6). ahp = 0.000 (2 vs. 4). aip = 0.000, p = 0.029 (3 vs. 4).ajp = 0.000, p = 0.001 (4 vs. 5, 6).

akp = 0.021 (5 vs. 6). alp < 0.05 (1 vs. 2, 3, 4, 5, 6). amp < 0.05 (2 vs. 3, 4, 5, 6). anp < 0.05 (3 vs. 4, 5, 6). aop = 0.000 (4 vs. 5, 6).

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Figure 3: Density of fibrous, cartilage and osseous tissues in the regenerated tissue of different groups at 56 days post-injury. The autograft had the highest DCT and the defect demonstrated the highest DFT. The Chitosan-RJ and DBM-RJ had the highest

DOT (p < 0.05). The least amount of DOT belonged to the Defect group while the lowest DFT contributed to the chitosan-RJ group (p < 0.05). There was no significant difference between the Chitosan-royal jelly and DBM-royal jelly in the DFT, DCT and DOT levels.

The least healing was observed in the defect group and the main regenerated tissues, in the lesions of the animals of this group, was fibrous connective tissue (fibroblast + fibrocyte + collagen) and the highest density of fibrous tissue (DFT) was observed in the defect of the animals of this group (p < 0.05). The Density of cartilaginous tissue in the defect group was significantly inferior compared to the autograft group (p = 0.000). The number of osteocytes + osteoblasts and the density of osseous tissue (DOT) was significantly inferior, in the defect group, to other groups (p < 0.05). There was a significant superiority in the number of osteocytes + osteoblasts and DOT in the autograft group, over the DBM (p = 0.000 and p = 0.000 respectively) and the CS groups (p = 0.007 and p = 0.001 respectively), while the CS-RJ and DBM-RJ groups showed significantly higher number of osteocytes + osteoblasts (p = 0.049 and p = 0.034, respectively) and DOT (p = 0.000 and p = 0.002, respectively) compared to the autograft group. There was no significant difference between the CS-RJ and DBM-RJ groups in the number of osteocytes+ osteoblasts and DOT criteria. There was also significantly higher number of osteoblasts + osteocytes and DOT in the CS-RJ group than the CS group (p = 0.000). The primary osteons count in the DBM-RJ and CS-RJ was significantly higher than the untreated defect group (p = 0.024, p = 0.000, respectively). The Defect and DBM groups led to a more inflammatory reaction in comparison to other groups in the 56th post-operative day.

Biomechanical performance

The normal group showed significantly higher maximum load, maximum stress, yield load, and bending stiffness and significantly lower ultimate strain and yield strain compared to the autograft, defect, CS and DBM groups (p < 0.05). There was no significant difference in biomechanical parameters between the CS-RJ and DBM-RJ with the normal group except for the maximum stress (p = 0.009, p = 0.004, respectively). There was also no significant difference between the autograft group and the DBM-RJ group in any biomechanical factors except for yield strain (p = 0.045) and bending stiffness (p = 0.004) which were higher and lower in the autograft group, respectively. The CS-RJ group was significantly superior to the autograft group in the yield strength (p = 0.030) and bending stiffness (p = 0.001). The biomechanical properties of CS-RJ group were statistically higher than the CS group in the maximum load (p = 0.026), yield load (p = 0.010), and bending stiffness (p = 0.000). There was no significant difference between the CS-RJ and CS groups in the maximum stress (p = 0.232). The ultimate strain and yield strain in the CS-RJ group were significantly lower than the CS group (p = 0.027 and p = 0.025, respectively). The DBM-RJ group showed significant superiority to the DBM group in maximum load (p = 0.044), maximum stress (p = 0.008), yield load (p = 0.030), and bending stiffness (p = 0.000). The yield strain (p = 0.014) and ultimate strain (p = 0.008) were higher in the DBM group than the DBM-RJ group. The biomechanical findings are shown in figure 4 and table 4.

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paDBM-RJ (7)Chitosan-RJ (6)DBM (5)Chitosan (4)Normal (3)Autograft (2)Defect (1)Value

(number) Mean ± SDMean ± SDMean ± SDMean ± SDMean ± SDMean ± SDMean ± SD0.00144.4 ± 8.549.23 ± 10.035.3 ± 5.2f35.8 ± 6.3e47.1 ± 7.1d39.0 ± 3.98c29.9 ± 2.8bMaximum load

0.0007.2 ± 1.297.12 ± 1.84.9 ± 1.1j5.5 ± 2.110.1 ± 1.07i8.1 ± 1.36h4.3 ± 0.78gMaximum stress

0.00039.6 ± 7.6144.1 ± 9.0130.4 ± 5.0o29.3 ± 5.9n39.7 ± 5.7m33.2 ± 2.3l24.4 ± 2.2kYield load

0.00049.7 ± 7.4551.87 ± 6.6923.28 ± 5.48t22.73 ± 6.09s45.83 ± 7.13r32.5 ± 5.2q19.4 ± 3pBending

stiffness0.0004.05 ± 1.444.49 ± 1.87.32 ± 1.8y7.54 ± 1.9x3.79 ± 1.01w5.7 ± 1.0v8.5 ± 2.1uUltimate strain0.0003.13 ± 1.573.4 ± 1.66.1 ± 1.9ae6.1 ± 1.5ad2.53 ± 1.3ac5.04 ± 0.44ab6.68 ± 1.4zYield strain

Table 4: The results of three-points bending biomechanical test on the radius-ulna complex 56 days after injury.

Normal (intact radius-ulna bone complex of rat)/ DBM (demineralized bone matrix)/ chitosan-RJ (chitosan-royal jelly)/DBM-RJ (DBM-royal jelly). aOne-way ANOVA followed by Tukey post-hoc test. bp < 0.05 (1 vs. 2, 3, 4, 5, 6, 7); cp = 0.043 (2 vs. 3); dp = 0.047,

p = 0.017 (3 vs. 4, 5); ep = 0.026 (4 vs. 6); fp = 0.018, p = 0.044 (5 vs. 6, 7); gp < 0.05 (1 vs. 2, 3, 6, 7); hp < 0.05 (2 vs. 3, 4, 5); ip < 0.05 (3 vs. 4, 5, 6, 7) ; jp = 0.034, p = 0.008 (5 vs. 6, 7); kp < 0.05 (1 vs. 2, 3, 4, 5, 6, 7). lp = 0.044, p = 0.030 (2 vs. 3, 6).

mp = 0.039, p = 0.032 (1 vs. 4, 5). np = 0.010, p = 0.046 (4 vs. 6, 7). op = 0.012, p = 0.030 (5 vs. 6, 7). pp < 0.05 (1 vs. 2, 3, 6, 7). qp < 0.05 (2 vs. 3, 4, 5, 6, 7). rp = 0.000 (3 vs. 4, 5). sp = 0.000 (4 vs. 6, 7). tp = 0.000 (5 vs. 6, 7). up < 0.05 (1 vs. 2, 3, 6, 7).

vp = 0.010 (2 vs. 3). wp = 0.008, p = 0.001 (3 vs. 4, 5). xp = 0.027, p = 0.013 (4 vs. 6, 7). yp = 0.019, p = 0.008 (5 vs. 6, 7). zp < 0.05 (1 vs. 3, 6,7). abp = 0.003, p = 0.045 (2 vs. 3, 7). acp = 0.003, p = 0.002 (3 vs. 4, 5). adp = 0.025, p = 0.012 (4 vs. 6, 7). aep = 0.023, p = 0.014 (5 vs. 6, 7).

Figure 4: Biomechanical properties of the radius and ulnar bone complex, 56 days after injury. Maximum stress (a), Maximum load (b), bending stiffness (c), yield load (d), yield strain (e),

and ultimate strain (f) included for all the untreated and treatment groups.

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Discussion

In the present study, in order to achieve nonunion, a critical radial bone defect model was created in rat [23]. This model does not need any external or internal fixator devices which could interfere with the healing processes [24]. The control defect group had no appreciable healing. The radiographic, histopathologic and biomechanical results indicated enhancement and improvement in bone healing after using RJ with CS or DBM scaffolds. This is the first time that RJ has been locally applied along with DBM scaffold in bone healing. The results of the present study confirmed the positive role of RJ in bone healing and since there were no significant differences between the CS-RJ and DBM-RJ groups, higher chondroblast and chondrocyte contents in DBM-RJ group make it a more promising combination in longer duration studies.

There is no evidence on the osteogenic potential of the CS and DBM scaffolds, but it has well been stablished that DBM has osteoinductive properties [7]. Both scaffolds have mostly been used because of their osteoconductive properties. The results of the present study showed that RJ along with CS and DBM scaffolds led to a favorable response in bone repair. The radiographic results showed better healing in the RJ treated groups compared to the untreated, CS and DBM groups (p < 0.05). The histopathological results with primary osteons formation indicate that the remodeling phase had started in the RJ treated groups. However, the animals in the defect group could not pass the fibroplasia state after 56 days of injury. The biomechanical data also confirmed these results. DBM has the osteoinductivity property resulting in enhanced biomechanical performance as it has been shown in in vivo studies [25]. In the defects which were filled by DBM, cartilage, fibrous and fibrocartilage tissues filled the defect area after 56 days of injury induction. Some bone formation also happened but was not enough to result in bone union.

Royal jelly contains water, proteins, amino acids, lipids, vitamins, sugars and little amounts of steroids [26]. The proteins of RJ include the family of major RJ proteins (MRJP1-9), consisting approximately 90% of its whole proteins, and apisimin, royalisin, and jelleins [27-30]. Apisin is a unique, major protein complex in RJ that consists of MRJP1 and apisimin combination [31]. The major lipid in RJ is 10-hydroxy-trans-2-decanoic acid which is also known as “Royal jelly acid”. This fatty acid and its saturated form, 10-hydroxydecanoic acid, compose 75% of the lipid contents in RJ [32-34].

It has been reported that MRJPs have an important role in some of the RJ bioactivities such as antibacterial and regulation of immune responses [35-37]. Royal jelly inhibits bone loss in post-menopausal time and in a comparative study its effect was evaluated as 17β estradiol [16]. This effect is not related to RJ proteins and may be related to its lipid part. It has also been demonstrated that RJ improves bone formation both at in vitro and in vivo conditions. It enhances osteoblast proliferation and also upregulates collagen synthesis in these cells as happened in the present study [17,20]. An in vitro study on the effects of RJ on MC3T3-E1 cell line, demonstrated that RJ could stimulate their proliferation; this effect was disappeared by administering estrogen receptor antagonist ICI182,780 [17]. In the present study, there was significant differences between the RJ treated groups with others in the number of osteoblasts + osteocytes at the 56th day after injury. In another study oral administration of RJ in rats led to a significant increase in ash contents of the tibia after 9 weeks. DNA microarray analysis revealed that expression of extracellular matrix related genes, had significantly increased in the RJ treated group [17]. Izua and colleagues reported that RJ suppresses the VEGF-induced angiogenesis by 10H2DA action as an anti-tumor activity [38]. It has been reported that treatment with RJ was associated with an increase in Transforming Growth Factor-β (TGF-β) expression [39]. This growth factor plays a significant role in the cellular growth and differentiation and also as a chemotaxis for inflammatory cells. Induction of collagen gene expression in fibroblasts depends on this factor [40,41].

When bone fracture happens, the traumatized tissues release an abundant number of free radicals. One of the sequels of the excessive release of free radicals could be an increase in bone resorption and osteoclast activity, leading to overproduction of Reactive Oxygen Species (ROS), which in turn could result in elevation of malondialdehyde (MDA) in serum. There are evidences suggesting that ROS significantly affect survival and production of osteoblasts, osteocytes, and osteoclasts [42]. Ozan and colleagues investigated the effects of oral administration of RJ on bone regeneration in maxillary bone in rats [43]. They concluded that oral administration of RJ improves the quality of bone regeneration due to its antioxidant activity. The results of the present study confirm the improvement of osteogenesis in the treated groups with RJ. In the present study radiological assessments revealed superiority of radiological scores of DBM-RJ group over

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CS-RJ group at the 28th and 42nd days after surgery. Despite that these results have not statistically significant differences but we can track these superiorities in histopathologic and histomorphometric results where the number of chondroblasts + chondrocytes and density of cartilage tissue in DBM-RJ group were significantly higher than the CS-RJ group [44-49].

Conclusion

The present study well established that local administration of RJ along with CS and DBM scaffolds induce more bone formation in the healing of a critical bone defect model in rat. The gold standard autograft group heals with nonunion and a distal syndesmosis, both are not ideal when translated to a clinical outcome. Concurrent application of RJ with CS or DBM scaffold enhances the integration of implanted scaffold with the new regenerated bone in the healing area. Radiographic, histopathologic and biomechanical findings indicate a marked improvement in quality and quantity of the newly regenerated bone in the RJ treated groups compared to the defect group. In this study, the osteogenesis mechanism of RJ was not studied. Also, our study did not include in vitro or in vivo gene expression criteria for a better understanding of the molecular aspect of bone healing after RJ administration. The results of the present study demonstrated that treatment with RJ enhanced the bone healing potential of CS and DBM scaffolds. There were no significant differences between the CS-RJ and DBM-RJ groups in most estimated criteria. Yet the insignificant superiority of DBM-RJ group over CS-RJ group at the 28th and 42nd days after surgery and significantly higher cartilage tissue in DBM-RJ than CS-RJ group, suggest that the healing process of bone defects in the DBM-RJ group have gotten delayed at the last 14 days of the study, but there are many more chondrocytes and chondroblasts to transform to osteoblasts and osteocytes in the DBM-RJ group than CS-RJ which should not be ignored. Although administration of RJ in clinical bone defects needs more investigations, the results of our study represent the potential role of RJ in enhancing bone healing processes without the drawbacks of the autogenous bone graft.

Acknowledgements

The authors would like to thank the authorities of Veterinary School, Shiraz University for providing facilities and supports. They are also thankful to Iran National Science Foundation (INSF) for financial support (Grant #96006039).

Conflict of Interest

There is no conflict of interest.

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