Bone 63 (2014) 53–60

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Original Full Length Article

Effects of osteogenic medium on healing of the experimental critical bone defect in a rabbit model Ahmad Oryan a, Amin Bigham-Sadegh b,⁎, Fatemeh Abbasi-Teshnizi a a b

Department of Veterinary Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran Faculty of Veterinary Medicine, Shahrekord, Iran

a r t i c l e

i n f o

Article history: Received 21 November 2013 Revised 13 February 2014 Accepted 18 February 2014 Available online 25 February 2014 Edited by: David Fyhrie Keywords: Osteogenic medium Maintenance medium Bone healing Rabbit model

a b s t r a c t Today, finding an ideal biomaterial to treat the large bone defects, delayed unions and non-unions remains a challenge for orthopedic surgeons and researchers. Several studies have been carried out on the subject of bone regeneration, each having its own advantages. At the same time, a variety of disadvantages still remain. The present study has been designed in vivo to evaluate the effects of osteogenic medium on healing of experimental critical bone defect in a rabbit model. Twenty New Zealand albino rabbits, 12 months old, of both sexes, weighing 2.0 ± 0.5 kg were used in this study. An approximately 10 mm segmental defect was created in the mid portion of each radius as a critical size bone defect. In the osteogenic medium group (n = 5) 1 ml osteogenic medium, in the maintenance medium group (n = 5) 1 ml maintenance medium, and in the normal saline group (n = 5) 1 ml normal saline were injected in the defected area while the defects of the rabbits of the control group (n = 5) were left empty. Radiological evaluation was done on the 1st day and then at the 2nd, 4th, 6th and 8th weeks post injury. Biomechanical and histopathological evaluations were done 8 weeks post injury. The radiological, histological and biomechanical findings of the present study indicated a superior bone healing capability in the osteogenic and maintenance medium groups, by the end of 8 weeks post-surgery, in comparison to the normal saline and control groups. In conclusion, this study demonstrated that the osteogenic medium and maintenance medium could promote bone regeneration in long bone defects better than the control group in rabbit model. © 2014 Elsevier Inc. All rights reserved.

Introduction The number of patients suffering from bone tumor resections, fracture defects, or chronic infection is rapidly increasing and more than 1 million bone graft operations are performed in the United States every year [1]. Therefore, it is still a challenge for orthopedic surgeons and researchers to find an ideal biomaterial for treatment of the large bone defects, delayed unions and non-unions. Following surgical procedures or trauma to the bone tissue the healing cascade starts with acute inflammation which is associated with polymorphonuclear cell infiltration, edema, fibrin and blood clot accumulation and hyperemia. This acute inflammatory phase usually lasts for about 4 days [2] and is then followed by the chronic stage of inflammation which is coincident with infiltration of macrophages, lymphocytes and plasma cells. At acute and chronic inflammatory phases, the metalloproteinases, growth factors and vasoactive substances gradually accumulate in the injured area, particularly in the blood clot, to participate in proliferation of the osteoblasts and ⁎ Corresponding author at: Department of Veterinary Surgery and Radiology, School of Veterinary Medicine, Shahrekord University, Shahrekord, Iran. E-mail address: [email protected] (A. Bigham-Sadegh).

http://dx.doi.org/10.1016/j.bone.2014.02.010 8756-3282/© 2014 Elsevier Inc. All rights reserved.

endothelial cells and to control production of the bone matrix by osteoblasts in the next phase of fracture healing which is fibroplasia or proliferative phase of healing. Collagen type III, glycosaminoglycans, and proteoglycans are secreted by the osteoblasts at the fibroplasia stage of fracture healing. This phase is then followed by the remodeling or maturation phase which is a long standing stage and may even last for several years [2]. In treatment of nonunion and bone defects, autograft is the gold standard for bone repair. However, there are some disadvantages associated with the autografts, such as the limited abundance in supply, new nerve damage, persistent pain and new fractures. Allografts have been used successfully in the orthopedic operations owing to its excellent osteoconductivity and abundance in supply. However, allografts have the potential risk of infection, disease transmission and immune response. On the other hand, allografts are inferior in promoting bone regeneration compared to the autografts, because they require processing, sterilization steps, and preservation before they are used [3–5]. To date, several studies have been conducted to promote bone regeneration. Some of these studies include: application of bone marrow with static magnetic field [6], coral with human platelet rich plasma [7], hydroxyapatite with human platelet rich plasma [8], omentum with adipose tissue stem cells [9], demineralized bone matrix [10,11], nano-hydroxyapatite/collagen,

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synthetic poly (glycolic-co-lactic acid) polymer [12] and true bone ceramics or sintered bovine bone [13,14]. Each has its own advantages, at the same time various disadvantages still remain. For example, ceramic and polymer-based bone graft substitutes are mostly osteoconductive but are not potentially osteoinductive. Other problems may include unsuitable degradation rates and inferior mechanical properties. In addition, protein- or growth factor based bone graft substitutes usually require addition of an osteoconductive scaffold for structural support [15,16]. Thus far, bone morphogenic proteins (BMPs) have been used in clinical trials to enhance bone healing properties [17–19]. It has been stated that the BMPs are able to stimulate the local undifferentiated mesenchymal cells to transform into osteoblasts (osteoinduction), leading to early bone formation [20–23]. However, critical views on the use of BMPs have recently surfaced due to their short half-lives, high cost and ineffectiveness [24–26]. In a recent study, Bigham-Sadegh et al. [9] inadvertently showed that maintenance medium (MM) with omentum had superior osteogenic properties in comparison to the omentum alone and the control groups. Osteogenic medium (OM) supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and β-glycerophosphate (β-GP) has been commonly used for the osteogenic differentiation of the mesenchymal stem cells (MSCs) in vitro [26–28]. In other studies, osteogenic medium has been added to the adipose derived stem cells (ASC) to induce the differentiation of these cells to osteoblasts; the osteogenic medium induced-ASC was then used to evaluate the healing of the bone defect. These studies have shown a significant enhanced bone healing with the osteogenic medium-induced ASCs compared to the non-induced ASCs [29–31]. In addition, a more recent study showed that, osteogenic medium enhances differentiation of the human adipose derived stem cells (hASC) towards bone-forming cells significantly more than growth factors in a tri-dimensional (3D) culture [32]. Therefore, the present study has been designed to evaluate the effects of the osteogenic medium on healing of an experimental critical bone defect in a rabbit model. Materials and methods Animals and operative procedures Twenty New Zealand albino rabbits, 12 months old, of both sexes, weighing 2.0 ± 0.5 kg, were kept in separate cages, fed a standard diet and allowed to move freely during the experimental period. The animals were randomly divided into four equal groups such as osteogenic medium (OM) group (n = 5) [osteogenic medium is a combination of the maintenance medium with L-ascorbic acid 2-phosphate, dexamethasone and β-glycerophosphate that has been used for the osteogenic differentiation of MSCs in vitro], maintenance medium [Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY), containing 10% fetal bovine serum (FBS, Gibco), 100 μg/ml streptomycin and 100 U/ml penicillin] (MM) group (n = 5), normal saline (NS) group (n = 5) and empty defect group (n = 5, control group). All the animals were anesthetized by intramuscular administration of 40 mg/kg ketamine hydrochloride and 5 mg/kg xylazine. The right forelimb of all the animals was prepared aseptically for operation. A 5 cm incision was made craniomedially in the skin of the fore limb and the radius was exposed by dissecting the surrounding muscles. An osteoperiosteal segmental defect was then created on the middle portion of each radius at least twice as long as the diameter of the diaphysis for creation of a nonunion model [33]. As the diameter of the radius of the adult New Zealand albino rabbits is about 5–6 mm, the radial defect was 10–12 mm long. Subcuticular and skin incisions were closed routinely. In the OM group 1 ml osteogenic medium, in the MM group 1 ml maintenance medium, and in the NS group 1 ml normal saline were injected in the defected area 4 days after operation, while the defects in the rabbits of the control group were left empty. The animals were housed in compliance with our institution's guiding principles “in the care and

use of animals”. The local Ethics Committee for Animal Experiments approved the design of the experiment. Post operative evaluations Radiological evaluation To evaluate bone formation, union and remodeling of the defect, radiographs of each forelimb were taken postoperatively on the 1st day and then at the 2nd, 4th, 6th and 8th weeks post injury. The results were scored using the modified Lane and Sandhu scoring system [34] (Table 1). Biomechanical evaluation Eight weeks after operation the rabbits were euthanized for histopathological and biomechanical evaluation. All rabbits were euthanized and the radius attached to the ulna was harvested. The connective tissue was removed, and only the bony structure was kept. The radius and ulna were not separated. Biomechanical test was conducted on the fused radius and ulna in the defected area of each rabbit. The tests were performed using a universal tensile testing machine (Instron, London, UK). The three-point bending test was performed to determine the mechanical properties of bones. The bones were placed horizontally on two rounded supporting bars located at a distance of 30 mm, and were loaded at the midpoint of the diaphysis by lowering the third bar so that the defect was in the middle and had an equal distance from each grip. The force was first received by the ulna and then delivered to the healed defected area of radius. The bones were loaded at a rate of 10 mm/min until fracturing occurred. Deformation (delta w) and ultimate (maximum) load were detected from the graph sketched by the machine. The bending stiffness was derived using the following equation: Bending stiffness (or bending rigidity) S = EI in N mm2 is the product of the Elastic modulus E and the axial second moment of inertia I. This is calculated by the formula: S = EI = (L3 / 48) × (delta F / delta w), where L is the distance between the supporting bars, F is the force, and w is the deformation. Delta F / delta w is taken from the (most) linear part of the load–deformation curve [7]. The data derived from the load– deformation curves, such as ultimate load and bending stiffness, were expressed as Mean ± SD for each group. Histopathological evaluation Immediately after biomechanical testing, the specimens were referred for histopathological evaluation. Histopathological evaluation was carried out in the injured area of five rabbits of each group. Sagittal sections containing the defect were cut with a slow speed saw. Each slice was then fixed in 10% neutral buffered formalin. The formalinTable 1 Radiographical findings for healing of the bone defect. Bone formation No evidence of bone formation Bone formation occupying 25% of the defect Bone formation occupying 50% of the defect Bone formation occupying 75% of the defect Bone formation occupying 100% of the defect Union (proximal and distal ends were evaluated separately) No union Possible union Radiographic union Remodeling No evidence of remodeling Remodeling of medullary canal Full remodeling of cortex Total possible points per category Bone formation Proximal union Distal union Remodeling Maximum score

0 1 2 3 4 0 1 2 0 1 2 4 2 2 2 10

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fixed bone samples were decalcified in 15% buffered formic acid solution and processed for routine histological examination. Two 5 μm thick sections were cut from the center of each specimen and were stained with Hematoxylin and Eosin. The sections were blindly evaluated and scored by two pathologists according to the Heiple's scoring system [35] (Table 2).

Statistical analysis The radiological and histopathological data were compared by Kruskal–Wallis, non-parametric ANOVA, when P-values were found to be less than 0.05, then pair wise group comparisons were performed by Mann–Whitney U test. The biomechanical data were compared by One way ANOVA test (Bonferroni's method for multiple testing) used for biomechanical analysis between the treated bones of the groups (SPSS version 18 for windows, SPSS Inc., Chicago, USA).

Results There was no intra-operative and postoperative death during the study. None of the rabbits sustained an ulna bone fracture at the radial bone defect. Fig. 1. Radiographs of day 0, immediately after the operation, A) control group, B) normal saline group, C) maintenance medium group and D) osteogenic medium group.

Radiological findings In the 2nd week post-op, no significant differences were noted in the radiographs between all groups. However, in the 4th, 6th and 8th weeks post-op the radiographs showed significant differences between the groups (P b 0.05) (Figs. 1 to 5, Table 3). The osteogenic and maintenance medium groups were superior to the normal saline and control groups. There were no significant differences between the osteogenic and maintenance medium groups at any postoperative day (Figs. 1 to 5, Table 3).

Table 2 Histopathological findings for healing of the bone defect. Union (proximal and distal portions were evaluated separately) No evidence of union Fibrous union Osteochondral union Bone union Complete organization of shaft Cancellous bone No osseous cellular activity Early apposition of new bone Active apposition of new bone Reorganizing cancellous bone Completely reorganization of cancellous bone Cortical bone None Early appearance Formation under way Mostly reorganized Completely formed Marrow None in the resected area Beginning to appear Present in more than half of the defect Complete colonization by red marrow Mature fatty marrow Total possible points per category Proximal union Distal union Cancellous bone Cortical bone Marrow Maximum score

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 4 4 4 4 4 20

Fig. 2. Radiographs of the 2nd postoperative week, A) control group, B) normal saline group, C) maintenance medium group and D) osteogenic medium group.

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Fig. 3. Radiographs of the 4th postoperative week, A) control group, B) normal saline group, C) maintenance medium group and D) osteogenic medium group.

Fig. 4. Radiographs of the 6th postoperative week, A) control group, B) normal saline group, C) maintenance medium group and D) osteogenic medium group.

Discussion Histopathological findings In all the histopathological criterium, with the exception of the cancellous bone criterium, the osteogenic medium group was significantly (P b 0.05) superior to the normal saline and control groups. There were no significant differences between the osteogenic and maintenance medium groups (P N 0.05) (Fig. 6, Table 4). As shown in Figs. 6C and D, by the end of the 8th week post-surgery, histological examination demonstrated presence of the regenerated bone with the typical structure of trabecular bone in the defect site of the osteogenic and maintenance medium groups, while the normal saline and control groups showed weak osteogenesis activity and were, in most instances, filled with fibrous and fibrocartilage tissues (Figs. 6A and D).

Biomechanical performance All the biomechanical data passed normal distribution test and Bonferroni's method, used for multiple testing, and the results are presented as Means ± SD. In the ultimate load criterion the osteogenic medium group was significantly (P = 0.02) superior to the normal saline and control groups, and there were no significant differences (P N 0.05) between the osteogenic and maintenance medium groups. In the bending stiffness criterion, the osteogenic medium group was significantly (P = 0.02) superior to the control group. However, there were no significant differences between the osteogenic, maintenance medium and normal saline groups in the bending stiffness criterion (P N 0.05) (Table 5).

In this study, both the osteogenic and maintenance medium groups demonstrated superior osteogenic potential in healing of the radial bone defect in a rabbit model. The radiological, histological and biomechanical findings indicate a superior bone healing capability in the osteogenic and maintenance medium groups, by the end of the 8th week post-surgery, in comparison to the normal saline and control groups. A rabbit model was used for critical defect of long bone healing in the present study. Rabbits are commonly used as animal models in approximately 35% of the musculoskeletal researches in medical investigation [36]. Radial, fibular and calvarial bones of rabbits have been reported to be suitable because there is no need for external or internal fixation of the defect site after experimental induction of the bone defect model [37–39]. In the present study the three point bending test was performed for biomechanical evaluation. This test is simple and straightforward, but has the disadvantage of creating a high shear stress near the middle section of the specimen. The four-point bending yields pure bending in the middle portion of the specimen between the two loading points, while the transverse shear stresses are absent in this situation. However, it requires that the force at each loading point be equal, and the specimen's length be sufficient to accommodate the two loads. These requirements are simple to achieve for regularly shaped specimens, but somewhat difficult for testing the whole bone. Thus, the three-point bending test is used more often to measure the biomechanical properties of whole bones [40,41]. Bending causes tensile stresses on one side of the specimen and compressive stresses on the other side. Because the bone is weaker in tension than compression, failure usually occurs on the

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Fig. 5. Radiographs of the 8th postoperative week, A) control group, B) normal saline group, C) maintenance medium group and D) osteogenic medium group.

tensile side of the bone in bending tests [42]. According to this phenomenon, in the present study, the force loaded first on ulna and then shifted to the defected area of the radius bone; that is, the radius bone in the side of the tensile stress. The major problem with the mechanical testing in our study was the phenomenon of the covering of the new bones on both the radial and ulnar sides during the osteogenic process in the defected area. Therefore, it was impossible to measure the mechanical strength of the new bone in the radius only. Of course, this method of mechanical testing has previously been performed in bone defect of animal models in many studies [2,7,8,11,39,43,44]. The acute or inflammatory phase of fracture healing usually lasts for 4 days and the repair stage begins after it [2]. Therefore, we injected the osteogenic, maintenance or normal saline in the organized hematoma in the injured area to exert its effect in a longer period of fibroplasia phase of healing, as it has been reported that the hematoma and fibrin clot in the fracture site act as a natural scaffold [2]. Injection of the osteogenic medium in the bone defect, in the present study, led to superior osteogenesis in comparison with the normal saline and control groups as was confirmed by radiological, histopathological and biomechanical

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findings. The osteogenic medium used for the osteogenic differentiation of MSCs in vitro is a combination of maintenance medium with L-ascorbic acid 2-phosphate, dexamethasone and β-glycerophosphate [26–28]. In a previous study it has been proved that L-ascorbic acid 2-phosphate stimulates collagen accumulation and cell proliferation by skin fibroblasts, and leads to formation of a tri-dimensional tissue like substance [45]. It has also been shown that L-ascorbic acid 2-phosphate stimulates differentiation of human osteoblast-like cells [46]. Mori et al. [47] showed that dexamethasone, another osteogenic medium, enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells. In addition, it has been shown that β-glycerophosphate, another osteogenic medium, accelerates calcification in cultured bovine vascular smooth muscle cells [48]. We propose that these properties of L-ascorbic acid 2-phosphate, dexamethasone and β-glycerophosphate in the osteogenic medium groups resulted in proliferation and differentiation of the host cells and accelerated the calcification process in the injured area. The maintenance medium is a combination of DMEM/F-12, 10% FBS, 1% L-glutamine, and 1% antibiotics [32]. In our study the maintenance medium, such as the osteogenic medium, resulted in acceleration of the osteogenesis in the injected area. The positive effects of the maintenance medium on osteogenesis have previously been reported in a long bone defect in a dog model [9]. However, the investigators of the study did not explain the possible mechanism of action but hypothesized that the maintenance culture medium provides the necessary nutrient environment for the migrated host cells at the earlier stage of healing and leads to superior bone formation [9]. Interestingly, intravenous injection of L-glutamine has been shown to speed up bone healing in a rat model [49]. FBS in the maintenance medium and also in the osteogenic medium contains a large number of growth factors and extracellular matrix molecules that enhance cell proliferation and differentiation [50,51]. Therefore, these biomaterials in the maintenance and osteogenic medium groups could lead to enhancement of the bone repair in the present study. In our study statistical analysis did not reveal any significant differences between the results of the osteogenic and maintenance medium groups, however it seems that the quality of healing in the osteogenic medium group was more advanced than the maintenance medium. Such beneficial effect could be related to the presence of stimulating factors (L-ascorbic acid 2-phosphate, dexamethasone and β-glycerophosphate) in the osteogenic medium in comparison to the maintenance medium. Based on the radiological, histopathological and biomechanical findings of the present study, bone formation was inferior in the normal saline and control groups compared to the osteogenic and maintenance medium groups. The defect area in the normal saline and control groups was filled with fibrous connective tissues and rarely with cartilage instead of osseous tissue. This unfavorable bone healing in such a critical sized bone defect, in the absence of healing stimulating factors, was expected in the normal saline and control groups. Barnes et al. indicated that the chondrocytes derived from the mesenchymal progenitors proliferate and synthesize cartilaginous matrix until all the fibrous granulation tissues are replaced by cartilage. Where cartilage production is deficient, the fibroblasts replace the region with generalized fibrous

Table 3 Radiographical findings for healing of the bone defect (sum of the radiological scores) at various post-operative intervals. Pa

Med (min–max) Postoperative weeks

OM group (n = 5)

MM group (n = 5)

NS group (n = 5)

Control group (n = 5)

2 4 6 8

1(2–3) 7(4–10)b 8(6–10)b 9(8–10)b

1(1–3) 6(3–9)b 7(5–9)b 8(5–10)b

1(0–3) 2(3–5) 4(2–6) 5(3–7)

1(0–3) 3(0–4) 4(1–5) 5(2–5)

Significant P-values are presented in bold face. Osteogenic medium (OM), Maintenance medium (MM), Normal saline (NS). a Kruskal–Wallis non-parametric ANOVA. b Compared with NS group and control group by Mann–Whitney U test. OM and MM groups were significantly (P b 0.05) superior to NS and control groups.

0.2 0.02 0.04 0.04

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Fig. 6. Micrographs of the injured bones after 8 weeks. The histological examination demonstrated fibrous and fibrocartilage tissue in the control (A: 10×), and normal saline (B: 40×) groups. Regenerated bone with typical structure of the trabecular bone is seen in the defect of the osteogenic medium (C: 10×) and maintenance medium (D: 10×) groups (H&E staining).

tissue. Discrete cartilaginous regions progressively grow and merge to produce a central fibrocartilaginous plug between the fractured fragments that splints the fracture [52]. The limitations in autograft and allograft restricted their clinical application. Alternatively, tissue engineering approach may offer a new solution to producing bone substitutes for clinical use. Over the last twenty years, tissue engineering of the bone has made remarkable progress, although the problems of translating it into clinical application still remain. Various types of stem cells have been used to form mineralized bone in vitro. In contrast, fewer studies have focused on the healing efficacy and its potential side effects. One main barrier is the complicated in vivo environment, which has profound interactions with the implanted cell types. This is especially true for the allogeneic cells, where the host immune reaction is likely to play a very important role, with the macrophage system currently being intensely studied [53,54]. In our study OM or MM was used successfully in enhancing the healing criteria of the bone defect and did not induce any signs of acute or chronic immune reaction as seen by the histopathological evaluation after 8 weeks post-surgery. Bone morphogenic proteins are members of the transforming growth factor β superfamily. The major biological effect of BMP lies in their ability to stimulate the aggregation, proliferation and differentiation of mesenchymal cells, which results in acceleration of bone and

cartilage formation. Among all the BMP families, BMP-2 has the strongest biological activity [16]. However, an important issue to consider in the use of BMPs is their side-effects [55]. BMPs are reported to be safe if they are used appropriately and the side effects include heterotopic ossification, local erythema and swelling, and immune response. Critical issues include the potential risk that BMPs will induce heterotopic bone formation, especially when implanted near to neural tissues. An unforeseen issue is their role in osteoclast activation and formation. Osteoclasts are formed before osteoblasts which may lead to a wave of resorption that precedes the appearance and effect of osteoblasts [56]. rhBMP-2 or other growth factors alone do not achieve the expected efficacy of bone or cartilage formation due to the short retention of protein in vivo, thus an ideal scaffold material as a delivery carrier is necessary [57]. As a delivery carrier for growth factors, it should have two primary functions: first, it should maintain growth factors' bioactivity and optimal release amount at the implantation site to maximize the osteogenic effect of growth factor; second, it should be an osteoconducitve scaffold with suitable pore structure for vascularization and new bone formation [58]. Nowadays, a large number of natural and synthetic biomaterials have been fabricated for the economical delivery of rhBMP-2 [16,59]. However, critical views on the use of BMPs have recently surfaced due to their short half-lives, high cost and ineffectiveness [24–26].

Table 4 Histopathological findings according to the Heiple scoring system. Pa

Med (min–max) Histopathological criterium

OM group (n = 5)

MM group (n = 5)

NS group (n = 5)

Control group (n = 5)

Union Cancellous bone Cortical bone Marrow

1(1–1)b 2(2–3)b 3(3–4)b 3(2–3)b

2(2–3) 2(1–3) 3(2–3) 2(2–3)

2(1–3) 1(1–2) 2(2–2) 1(1–2)

1(1–2) 1(1–2) 1(1–2) 1(1–2)

0.01 0.08 0.004 0.01

Significant P-values are presented in bold face. Osteogenic medium (OM), maintenance medium (MM), normal saline (NS). a Kruskal–Wallis non-parametric ANOVA. b Compared with the NS group (P = 0.02) and the control group (P = 0.02) by Mann–Whitney U test. The OM group was superior to the NS and control groups in all histopathological criterium except that of the cancellous bone criterium.

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Table 5 Biomechanical findings on the 8th postoperative week. Mean ± SD Three point bending test criteria Ultimate load (N) Bending stiffness (N mm2)

OM group (n = 5) a

(93.6 ± 11.8) (81,837 ± 7911)b

MM group (n = 5)

NS group (n = 5)

Control group (n = 5)

(80.0 ± 5.0) (68,617 ± 8759)

(70.0 ± 8.6) (69,374 ± 9193)

(70.0 ± 5.0) (65,274 ± 6653)

Osteogenic medium (OM), maintenance medium (MM), normal saline (NS). a P = 0.02 (OM was significantly superior to the NS and control groups). b P = 0.02 (OM was significantly superior to the control group).

Since in the present study no experiments were performed for comparison of OM and BMP effects on bone defect healing, the results of the present study cannot be compared with the results of BMP effects on bone defect healing, however, some mentioned side effects of BMPs such as local erythema and swelling, and immune response were not observed in the OM group. In addition, OM has some advantages such as availability and inexpensiveness in comparison with BMPs. Tirkkonen et al. [32] in an in vivo study demonstrated that OM had superior capacity to induce osteogenic differentiation and proliferation of hASCs than the growth factors and showed increased viability and cell number by the OM group when compared with growth factor groups. In addition, OM induced significantly higher alkaline phosphatase (ALP) activity than BMP-2 (1.7-fold) and BMP-7 (4.6-fold) tested in their study. The use of biomaterials and development of scaffolds are especially important for engineering bone grafts, because they need to provide mechanical support and bioactive aspect for bone formation during bone repair. In order to obtain optimal mechanical properties and high biocompatibility, numerous composite materials have been designed to acquire integrated properties from the individual components. The achievements in engineering bone tissue so far are encouraging, while new challenges and opportunities highlight the potential of bone tissue engineering in clinical application [60]. In our study a new kind of injectable biomaterial was developed by introducing the osteogenic medium with high capacity of promoter for bone regeneration; however, as it has only osteoinductive properties it should be used concurrent to an osteoconductive material to obtain optimal mechanical properties in the large bone defect area; this could be one of the limitations in clinical application of the present OM. Another limitation in usage in future clinical application of OM may be related to the presence of FBS in this osteogenic medium because there is a risk of contamination of bovine infectious agents and their possible transmission to patients [50,51].

Conclusion In conclusion, this study demonstrated that the osteogenic and maintenance media could promote bone regeneration in the long bone defects better than the control group in the rabbit model. This finding introduces the osteogenic medium as an attractive alternative for reconstruction of the major diaphyseal defects in the long bones in large animal models and the favorable results of our study may be useful for human clinical application in future.

Conflict of interest All authors declare that there is no conflict of interest.

Acknowledgments The authors would like to thank the authorities of the Veterinary School, Shiraz University for their financial support and cooperation.

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