Antigen-extracted xenogeneic cancellous bone graft with recombinant human bone morphogenetic protein-2 enhances bone regeneration in repair of mandibular defect in rabbits

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ORIGINAL ARTICLE

Antigen-extracted xenogeneic cancellous bone

graft with recombinant human bone

morphogenetic protein-2 enhances bone

regeneration in repair of mandibular defect in

rabbits

Ze-jian Li

a

, Chun-ting Lu

b

, Zhi-qiang Feng

a

, Qing-tong Zhao

a

,

Zhi-ying Zhou

a

, Ren-fa Lai

a,

*

a

Medical Centre of Stomatology, First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province, China

b

Department of Endocrinology and Metabolism, First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province, China

Received 22 April 2014; accepted 11 September 2014 Available online 21 November 2014

KEYWORDS Alveolar bone grafting; Antigen-extracted xenogeneic cancellous bone; Osteogenesis; Recombinant human bone morphogenetic protein-2

Abstract The effects of a large-piece of xenogeneic bone that was separated from healthy pigs as a scaffold for the repair of a mandibular defect was investigated, and the applicability of antigen-extracted xenogeneic cancellous bone (AXCB) soaked with recombinant human bone morphogenetic protein-2 (rhBMP-2) in bone defect repair was assessed. Mandibular de-fects were created in 48 New Zealand rabbits, and the animals were randomly divided into four groups, in which the mandibular defects were grafted with AXCB, AXCB soaked with rhBMP-2, and autograft bone, or left blank. An equal number of animals from each group were classified into three time points (4, 8, and 12 weeks) after surgery for gross pathological observation, hematoxylin and eosin (H and E) staining, radiographic examination, and bone density mea-surement. H and E staining revealed that the area percentage of bone regeneration in the group of the AXCB/rhBMP-2 graft was 27.724.68, 53.9021.92, and 77.359.83 at 4 weeks, 8 weeks, and 12 weeks, respectively. These results were better than those of the autogenous bone graft, suggesting that the group of the AXCB/rhBMP-2 graft achieved a good osteogenic effect. With regard to the AXCB graft without rhBMP-2, the area percentage of bone regeneration was only 14.035.02, 28.4911.35, and 53.9021.92. Therefore, the osteogenic effect of the AXCB/rhBMP-2 graft was demonstrated to have the best effect. In the group of the AXCB/rhBMP-2 graft, the area percentage of bone regeneration increased,

Conflicts of interest: All authors declare no conflicts of interest.

* Corresponding author. Medical Centre of Stomatology, First Affiliated Hospital of Jinan University, 613 Huangpu Road West, Guangzhou 510630, China.

E-mail address:tlrf@jnu.edu.cn(R.-f. Lai). http://dx.doi.org/10.1016/j.kjms.2014.10.008

1607-551X/Copyrightª2014, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved. Available online atwww.sciencedirect.com

ScienceDirect

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and the implanted materials were gradually degraded and replaced by autogenous bone regen-eration over time. We conclude that the AXCB graft soaked with rhBMP-2 showed good osteo-genic effect in the repair of bone defects and good biocompatibility. AXCB serves as a good carrier of rhBMP-2, which promotes bone formation.

Copyrightª2014, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.

Introduction

Large bone defects in the oral and maxillofacial region are frequently seen in human patients, and the proper repair of such defects is challenging due to the anatomical complexity of this region and the associated cosmetic is-sues. The primary method to repair the bone defect is bone transplantation, which includes autologous bone graft, bone allograft, and xenograft. Autologous bone graft pro-vides not only a scaffold but also a certain number of os-teoblasts, and it has the best osteogenic effect. Therefore, it is considered the gold standard for bone defect repair

[1e3]. The autologous cancellous bone is usually taken from the iliac cancellous bone, the distal femur, the greater trochanter, or the proximal tibia [4]. However, autologous bone graft has limitated bone sources, and it requires a second operation area, which increases trauma to patients and the duration of operation[5]. Bone allograft is another way of providing a scaffold for bone regenera-tion, but it may carry a high risk of disease transmission. In addition, ethical issues may also limit the clinical applica-tion of allogeneic bone graft [6,7]. Xenograft is a good source of scaffold for bone regeneration, and the most frequently used, but it is associated with a potential risk of disease transmission (e.g., bovine spongiform encephalop-athy from bovine bone xenograft). Nevertheless, with the development of the advanced specific-antigen-extraction technology, the risk of disease transmission from xeno-graft is no longer a health concern[8]. Repairing bone de-fects with tissue engineering has been the focus of many studies. One of the significant findings is that the addition of appropriate growth factors may promote bone defect repair by accelerating the process of bone induction or formation. It is widely accepted that bone morphogenetic protein-2 (BMP-2) induces osteogenesis by promoting the differentiation of both mesenchymal cells and the bone marrow precursor cells inside the bone marrow into chon-drocytes and osteoblasts, and it may also accelerate bone tissue regeneration and repair [9,10]. However, the po-tential synergistic effect of BMP-2 and xenogeneic bone in osteogenesis has not been well explored. Recombinant human BMP-2 (rhBMP-2) acts as an essential factor in bone regeneration for mandibular defect repair. rhBMP-2 has been demonstrated to have bone-induction activity, which can stimulate bone growth naturally in the human body. Using a suitable scaffold for carrying rhBMP-2 seems to play a key role in the procedure. However, thus far, there is no recognized scaffold that is accepted for clinical use. In this study, we investigated the biocompatibility of antigen-extracted xenogeneic cancellous bone (AXCB) as a scaf-fold and its osteogenic efficiency, in combination with

rhBMP-2 (AXCB/rhBMP-2), in repairing defects of the mandibular bone in rabbits. We aim to identify a better new approach for bone defect repair in the oral and maxillofa-cial region using the allogeneic bone as a scaffold.

Materials and methods

The experiments were performed in 48 adult male New Zealand white rabbits weighing 3.0e3.5 kg. All experi-mental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. The study protocol was approved by the Institutional Animal Care and Use Committee at Jinan University Health Science Center, Guangzhou, China.

Preparation of AXCB

The iliac bones were separated from healthy pigs, and cut into 15 mm6 mm4 mm pieces for the preparation of AXCB. The bone pieces were soaked in acetone for 48 hours to remove the fatty composition, demineralized in 0.6M hydrochloric acid, completely washed with water, treated with epoxide as a crosslinking fixative material for collagen crosslinking fixation, washed with water again (Fig. 1A), and then freeze-dried to prepare them for rhBMP-2 incor-poration. The rhBMP-2 was produced by recombinant expression inEscherichia coliat the Institute of Biomedical Engineering, Jinan University (Guangzhou, China), and pu-rified to>98%. It was then dissolved in gelatin solution with 0.1% acetic acid (10 mg/mL). The biological activity of the rhBMP-2 was tested before use. Each piece of AXCB was soaked in 1 mL of gelatin solution containing rhBMP-2 (2.0 mg/mL) for 24 hours, sterilized by gamma-ray irradi-ation with a radiirradi-ation dosage of 25 kGy, then freeze-dried and stored frozen until use (Fig. 1B).

Animal experiment

Forty-eight adult New Zealand white rabbits weighing 3.0e3.5 kg (obtained from the Experimental Animal Center of Guangdong Province) were randomly divided into four groups (AXCB graft with rhBMP-2, AXCB graft only, autologous bone graft, and nongraft control), with three subgroups (4, 8, and 12 weeks) for each group (4 animals for each condition). In the autologous bone graft group, bilateral abdominal incisions parallel to the iliac crest were performed. A 15 mm6 mm4 mm bone piece was excised from the iliac bone on each side, and then placed in saline solution until use. Preparation of the AXCB and soaking with rhBMP-2 were

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performed as described earlier. A bone defect of 15 mm6 mm4 mm was then created on both mandibles for all animals in the four groups (Fig. 1C). In the graft groups, the bone defect created was implanted with the prepared AXCB soaked with rhBMP-2, AXCB only, or with autologous bone (Fig. 1D), whereas in the control (nongraft) group, the skin incision was directly closed with sutures after the crea-tion of the bone defect. All animals were then housed under the same conditions and monitored for postoperative activ-ities, emotional response, and wound healing. The animals were killed by excessive carbon dioxide asphyxiation at 4 weeks, 8 weeks, or 12 weeks after the surgery, and the whole mandible was harvested from each side for investigation.

X-ray examination

X-ray examination of the harvested mandibles was per-formed (DR3000; Kodak, Rochester, NY, USA), and the im-aging data were scored based on the LaneeSandhu scoring method by three technicians who were blinded to the data

[11]. The scoring criteria are presented in Table 1. The Leica Image Analysis System (Leica, Inc., Solms, Germany) was used for the assessment of bone regeneration following the mandibular defect.

Bone mineral density measurement

The obtained bone samples were fixed with formalin in phosphate buffer, and bone mineral density was measured for the repaired area using the bone density meter platform Lunar Prodigy (GE, Fairfield, CT, USA). The bone mineral content is presented as mm/cm3.

Figure 1. Implantation of AXCB incorporated with rhBMP-2 in a mandibular defect in rabbits. (A) The prepared AXCB. (B) AXCB scaffold incorporated with rhBMP-2. (C) Creation of a 15 mm6 mm6 mm mandibular defect; (D) Implantation of AXCB/rhBMP-2 into the created mandibular defect. AXCBZantigen-extracted xenogeneic cancellous bone; rhBMP-2Zrecombinant human bone morphogenetic protein-2.

Table 1 Scoring criteria based on the LaneeSandhu scoring method.

Item Classification Score

Bone formation

No new bone formation 0 New bone occupied 25% of the defect 1 New bone occupied 50% of the defect 2 New bone occupied 75% of the defect 3 Bone

union

Clear fracture line 0 Fuzzy fracture line 2 Disappeared fracture line 4 Bone

modeling

No bone modeling 0

Marrow cavity formed 2 Cortical bone modeling 4

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Preparation of bone samples for pathological staining

The obtained bone samples were decalcified, embedded in paraffin, sectioned into 3-mm slices, and mounted onto slides for hematoxylin and eosin (H and E) staining. The stained slides were observed under optical microscope at 100 magnification for evaluation of new bone formation and calcification, new blood vessel and fibrous tissue gen-eration, inflammatory cell infiltration, and implanted scaffold degradation.

Quantitative analysis of the new bone

The bone samples were fixed in 10% neutral buffered formalin, immersed in Technovit 7200VLC (Heraeus-Kulzer, Germany) after dehydration, and sectioned into five thin slices of approximately 40e80mm and mounted onto slides after solidification for 24 hours. The slides were H and E stained, and histomorphometry was performed using the Leica Image Analysis System (Leica, Inc., Solms, Germany).

Statistical analysis

All results are presented as the meanstandard deviation. The data were compared and intergroup differences were

analyzed using one-way analysis of variance withpost hoc

Bonferroni test, and the variance ratio (F) was calculated. Statistical analyses were performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Apvalue < 0.05 was taken to be statistically significant.

Results

Although all rabbits became awake on the day the surgery was performed, they had reduced activity and limited food intake. However, their activity and food intake returned to normal 1e2 days after the surgery. All surgical wounds healed without swelling and oozing, and the sutures were taken out 7 days after the surgery. The rabbits in each group then showed slowly increasing body weight, activity, and food intake. Neither signs of loss, displacement, and discharge of the implants nor fracture or wound infection was observed during the observation period.

Radiographic evaluation

We used lateral and vertical radiography to evaluate bone regeneration and healing of the mandible defect during follow up. As shown in Fig. 2, a clear boundary was observed around the margin of the AXCB graft and the AXCB/rhBMP-2 graft 4 weeks after the surgery; in addition,

Figure 2. X-ray images of the rabbit mandible defect. (A) A representative result at 4 weeks after the surgery in the AXCB group. (B) A representative result at 4 weeks after the surgery in the AXCB/rhBMP-2 group. A clear boundary was observed around the margin of the AXCB graft and the AXCB/rhBMP-2 graft; in addition, we also observed limited bony fusion between the surrounding autologous bone and the implanted AXCB/rhBMP-2 graft. (C) A representative result at 8 weeks after the surgery in the AXCB group. (D) A representative result at 8 weeks after the surgery in the AXCB/ rhBMP-2 group. A fuzzy boundary was observed around the margin of the AXCB graft and the AXCB/rhBMP-2 graft, and the bony fusion between the surrounding autologous bone and the implanted AXCB/rhBMP-2 graft was observed to be obvious, and it was much clearer than the AXCB graft group. (E) A representative result at 12 weeks after the surgery in the AXCB group. (F) A representative result at 12 weeks after the surgery in the AXCB/ rhBMP-2 group. A much fuzzier boundary was observed around the margin of the AXCB graft and the AXCB/rhBMP-2 graft. The bony fusion between the surrounding autologous bone and the implanted AXCB/rhBMP-2 graft was clearly observed. The bony fusion was much clearer than that in the AXCB graft group. (G) A representative result at 12 weeks after the surgery in the blank group. A low-density shadow with a size of approximately 10 mm5 mm was clearly observed. (H) A representative result at 12 weeks after the surgery in the autograft group. A fuzzy boundary was observed. AXCBZantigen-extracted xenogeneic cancellous bone; rhBMP-2Zrecombinant human bone morphogenetic protein-2.

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we also observed limited bony fusion between the sur-rounding autologous bone and the implanted AXCB/rhBMP-2 graft (Figs. 2A and 2B). Eight weeks after the surgery, a fuzzy boundary was observed around the margin of the AXCB graft and the AXCB/rhBMP-2 graft, and the bony fusion between the surrounding autologous bone and the implanted AXCB/rhBMP-2 graft was observed very clearly. This bony fusion was much clearer than that observed in the AXCB graft group (Figs. 2C and 2D). Twelve weeks after the surgery, a much fuzzier boundary was observed around the margin of the AXCB graft and the AXCB/rhBMP-2 graft. The bony fusion between the surrounding autologous bone and the implanted AXCB/rhBMP-2 graft was observed very well, and was much clearer than that in the AXCB graft group (Figs. 2E and 2F). A low-density shadow of approximately 10 mm5 mm was clearly observed in the blank group 12 weeks after the surgery (Fig. 2G). With regard to the autograft group, a fuzzy boundary was observed 12 weeks after the surgery (Fig. 2H). The bone density of the AXCB/ rhBMP-2 graft group increased with time according to the observation of the radiography results. New bone formation was assessed by the LaneeSandhu scoring method. The average scores were: 1.00, 7.50, and 11.00 in the autograft group; 1.00, 5.25, and 7.50 in the AXCB/rhBMP-2 group; and 0.20, 2.75, and 3.75 in the AXCB-alone group at 4 weeks, 8 weeks, and 12 weeks after the surgery, respectively, which indicated that the scaffold graft alone had a limited effect on bone regeneration and that the addition of rhBMP-2 greatly enhanced bone regeneration, comparable to the autogenous bone graft (Table 2). New bone generation increased over time.

Bone mineral density

A bone mineral density test revealed significant differences in bone mineral density across the groups 12 weeks after the surgery (autogenous bone group> xenogeneic AXCB/ rhBMP-2 group>xenogeneic AXCB-only group;p< 0.05). Moreover, the bone mineral density significantly increased over time (at 4, 8, and 12 weeks) within the AXCB/rhBMP-2 graft group and the autograft group (p<0.05;Table 3).

Histological observation

In the group with implantation of AXCB, at 4 weeks after the surgery, some fibrous tissue, capillary proliferation, trace of trabecular bone degradation, a small number of osteoblasts, and a small amount of new bone formation were found around the edge of the implant (Fig. 3A). At 8

weeks after the surgery, there was partial degradation of trabecular bone; in addition, a large number of osteoblasts around the edge of the implant were observed (Fig. 3B). At 12 weeks after the surgery, there was a small amount of matured trabecular bone tissue (Fig. 3C). In the group with implantation of AXCB soaked with rhBMP-2, at 4 weeks after the surgery, the trabecular bone of the implant was partially degraded, extensive new bone formation was observed, and around the new bone, there were a large number of osteoblasts and mesenchymal cells, capillary ingrowth, and osteoid formation (Fig. 3D). At 8 weeks after the surgery, there was a small area of unabsorbed implant, a large amount of trabecular bone tissue and new bone formation, and capillary ingrowth, with many surrounding osteoblasts and mesenchymal cells (Fig. 3E). At 12 weeks after the surgery, there was almost complete degradation of the implant, as well as a large amount of new bone formation with mature trabecular bone and some bone marrow (Fig. 3F). Histological examination showed rigorous bone regeneration around the implant. In the nongraft control group, the mandible showed only a small amount of new bone formation, and the created bone defect was mainly occupied by fibrous tissue at 12 weeks after the surgery (Fig. 3G). In the autograft group, a large amount of new mature trabecular bone was seen, and the mandibular defect was mostly occupied by the newly formed bone (Fig. 3H).

Quantitative assessment of bone regeneration

The percentage of area with new bone formation was calculated under microscopic view of the H and E-stained slides. Statistical analysis suggested that the area of bone regeneration in the mandibular defect was significantly greater in the AXCB/rhBMP-2 graft group than in the AXCB graft group (p < 0.05), and there was no significant dif-ference in the area of new bone formation between the autograft group and the AXCB/rhBMP-2 graft group (p > 0.05). There was a significant increase of bone regeneration over time (at 4, 8, and 12 weeks after the surgery) within the AXCB/rhBMP-2 graft group and the autograft group (p<0.05;Table 4).

Discussion

Tumors, especially malignant tumors, severe trauma, and congenital malformations in the oral and maxillofacial re-gion often lead to a large area of bone defect. Because of

Table 2 Average scores of bone regeneration in each group based on the LaneeSandhu scoring method.

Groups 4 wks after surgery 8 wks after surgery 12 wks after surgery

AXCB graft group 0.200.00 2.750.50 3.750.50

AXCB/rhBMP-2 graft group 1.001.16a,* 8.250.50a,* 10.501.00a,b,* Autograft group 1.000.43 9.500.76 11.001.23b,* *p<0.05.

AXCBZantigen-extracted xenogeneic cancellous bone; rhBMP-2Zrecombinant human bone morphogenetic protein-2. a Versus the AXCB graft group at 4 weeks, 8 weeks, and 12 weeks after the surgery.

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Figure 3. Histological images of the rabbit mandible defect (100). (A) A representative result at 4 weeks after the surgery in the AXCB group. Some fibrous tissue, capillary proliferation, a trace of trabecular bone degradation, a small number of osteoblasts, and a small amount of new bone formation were found around the edge of the implant. (B) A representative result at 4 weeks after the surgery in the AXCB/ rhBMP-2 group. The trabecular bone of the implant was partially degraded, extensive new bone formation was observed, and around the new bone there were a large number of osteoblasts and mesenchymal cells, capillary ingrowth, and osteoid formation. (C) A representative result at 8 weeks after the surgery in the AXCB group. There was partial degradation of trabecular bone, and also a large number of osteoblasts around the edge of the implant. (D) A representative result at 8 weeks after the surgery in the AXCB/rhBMP-2 group. There was a small area of unabsorbed implant, a large amount of trabecular bone tissue and new bone formation, and capillary ingrowth, with many surrounding osteoblasts and mesenchymal cells. (E) A repre-sentative result at 12 weeks after the surgery in the AXCB group. There was a small amount of matured trabecular bone tissue. (F) A representative result at 12 weeks after the surgery in the AXCB/ rhBMP-2 group. There was almost complete degradation of the implant, as well as a large amount of new bone formation with mature trabecular bone and some bone marrow. (G) A represen-tative result at 12 weeks after the surgery in the blank group. The mandible showed only a small amount of new bone formation, and the created bone defect was mainly occupied by fibrous tissue at 12 weeks after the surgery. (H) A representative result at 12 weeks after the surgery in the autograft group. A large amount of new mature trabecular bone was seen, and the mandibular defect was mostly occupied by the newly formed bone. AXCB Z antigen-extracted xenogeneic cancellous bone; rhBMP-2Zrecombinant human bone morphogenetic protein-2.

Table 3 Bone mineral density of the animals in each group at 4 weeks, 8 weeks, 12 weeks after the surgery.

Groups 4 wks after surgery 8 wks after surgery 12 wks after surgery AXCB graft group 195.0312.36 223.3128.17 231.4321.96 AXCB/rhBMP-2 graft group 223.1320.45 250.6214.87 326.8533.70a,b,*

Autograft group 241.1913.57 277.6526.70 351.103785b,*

Data are presented as milligram/cubic centimeter. *p<0.05.

AXCBZantigen-extracted xenogeneic cancellous bone; rhBMP-2Zrecombinant human bone morphogenetic protein-2. aVersus the AXCB graft group and the autograft group at 12 weeks after the surgery.

b Versus 4 weeks and 8 weeks after the surgery in the AXCB/rhBMP-2 graft group and the autograft group.

Table 4 Percentage of area with new bone formation in each group at 4 weeks, 8 weeks, 12 weeks after the surgery. Groups 4 wks after surgery 8 wks after surgery 12 wks after surgery AXCB graft group 14.035.02 28.4911.35 55.8710.20 AXCB/rhBMP-2 graft group 27.724.68a,* 53.9021.92a,* 77.359.83a,b,* Autograft group 30.191.46 49.732.68 68.183.92b,* *p<0.05.

AXCBZantigen-extracted xenogeneic cancellous bone; rhBMP-2Zrecombinant human bone morphogenetic protein-2. aVersus the AXCB graft group at 4 weeks, 8 weeks, 12 weeks after the surgery.

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anatomical particularity and three-dimensional structural complexity, the restoration of bone defects in the oral and maxillofacial region remains a challenge for surgeons. The restoration of the original shape of the facial skull is a prerequisite for the restitution of facial appearance. Sur-geons are trying to develop new approaches for the enhancement of bone regeneration to replace autogenous bone grafts. Recent progress in regenerative medicine and bone tissue engineering raises the hope of repairing bone defects with a combination of biomaterials and growth factors. Application of the large cancellous bone (ilium) as a morphogen carrier for rhBMP-2 in skeletal repair has been extensively studied during the past decade [12,13]. It is widely accepted that BMP-2 induces osteogenesis by pro-moting the differentiation of both mesenchymal cells and the bone marrow precursor cells inside the bone marrow into chondrocytes and osteoblasts, and it may also accel-erate bone tissue regeneration and repair. BMPs have been successfully applied in the reconstruction of long bones, spine, and the facial skeleton in preclinical studies[14].

At present, a large number of animal experiments and

in vitroexperiments confirmed that rhBMP-2 does not cause systemic toxicity, cytotoxicity, or cell mutagenicity. The earliest application of rhBMP-2 in the clinical setting was in a lumbar interbody fusion device. This randomized study included 14 patients, each of whom was implanted with two lumbar interbody fusion devices with rhBMP-2 at a dose of 1.5 mg/mL by the retroperitoneal and transperitoneal approach. Significant differences of Oswestry scores were found between the experimental group and the control group before and after the surgery at 3 months, 6 months, 12 months, and 24 months, respectively, using X-ray and computed tomography examinations [15]. Villavicencio et al[16]reported a clinical observation on 74 patients who underwent a lumbar interbody fusion operation using rhBMP-2/gelatin sponge or allograft. The radiographic spi-nal fusion rate in their study was 100% at 12 months and 24 months after the surgery, respectively, and no rhBMP-2-related complications, such as bone overgrowth, ectopic bone, nerve damage, and other complications were observed. In addition, rhBMP-2 has not been found to mote the growth of tumor cells. These studies have pro-vided a theoretical basis that the rhBMP-2 can be safely and effectively used for clinical treatment. However, rhBMP-2 is an exogenous cell growth factor, and therefore its clinical application in supraphysiological doses has the potential risks of soft-tissue edema, skin rash, inflammation reaction, heterotopic ossification, and adverse immune response consequences. Therefore, the safe use of rhBMP-2 for human patients still needs long-term preclinical and clinical studies. In addition, there are also some problems that should be resolved to achieve the ideal effect of bone regeneration enhanced by rhBMP-2 in clinical and scientific research. On the one hand, pure rhBMP-2 in vivo can be rapidly decomposed and diluted by protease inhibitors. Various studies have shown that rhBMP-2 in vivo can decompose within a few hours after implantation, and therefore, it has only a very limited time to maintain the effective concentrationin vivo [17]. It was widely recog-nized that the local concentration of rhBMP-2 was of more clinical value than the total dose. The present research has not determined the therapeutic use of high rhBMP-2

concentrations in the nonhuman primate animal body. A concentration in the range of 0.4e1.5 mg/mL has been applied in long bone trauma patients. On the other hand, pure rhBMP-2 itself does not have any filling, supporting, or other mechanical properties, and therefore, it cannot directly fill into the bone defect, which seriously restricts the bone repair effect. At present, many vehicles have been used as rhBMP-2 carriers, including polymer ceramic, bone cement, polymer, collagen, chitosan, hyaluronic acid, allograft, and xenograft.

Based on the theory of creeping substitution[18e20], an ideal bone graft used for bone defect repair should provide a platform for the three essential elements of bone regeneration (i.e., osteoinduction, osteoconduction, and osteogenesis). Osteoinduction is a process of inducing dif-ferentiation of mesenchymal stem cells into osteoblasts and chondrocytes, presumably by some morphogens. Osteoconduction is a property of the bone graft as a scaf-fold thatconductsthe ingrowth of the osteoblasts (differ-entiation and maturation) as well as that of the blood vessels, providing a platform for osteogenesis. The scaffold graft is gradually replaced by creeping substitution of the newly regenerated bone [21]. Some studies have shown that the creeping substitution occurs mainly in the facial layer and at the two ends of the implanted bone. Large bone defects may have very limited regeneration using heterologous bone graft. BMPs are good morphogens that induce both osteogenesis and angiogenesis[22,23], which may help to overcome the aforementioned limitation of large heterologous bone graft. rhBMP-2 is the growth factor most widely used for bone regeneration[24], but it diffuses quickly after application. Therefore, the development of favorable carriers with slow-releasing properties is critical. The bone morphology of the oral and maxillofacial re-gion in rabbits has similarities to that observed in humans. In this study, we soaked the rhBMP-2 into a piece of AXCB, which was then implanted into the bone defect of the same size created in the mandibles of New Zealand white rabbits, and subsequently assessed the bone regenerative effect

[25]. The results revealed that the AXCB/rhBMP-2 graft group had much better and more extensive bone regener-ation than the group grafted with AXCB only; in addition, the bone regeneration increased over time from 4 weeks to 12 weeks after the surgery, which indicated that rhBMP-2 had a significant bone regenerative effect over time with AXCB as a scaffold and that AXCB is probably a good carrier for rhBMP-2, allowing for the slow release of rhBMP-2 and thus improving its efficiency. By contrast, the AXCB was found to become gradually degraded over time, and at 12 weeks after the surgery, the implanted bone was almost completely replaced by newly regenerated bone tissue, which showed an apparent mature trabecular structure. There were no appreciable histological signs of inflamma-tion or immune rejecinflamma-tion of the graft, suggesting the good biocompatibility of AXCB.

Conclusion

In conclusion, the osteogenic effect of the AXCB graft soaked with rhBMP-2 proved to be much better than the AXCB graft alone (without rhBMP-2), which showed no

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significant difference with the autologous bone graft. The porous AXCB appears to be a good carrier for rhBMP-2, which allows for controlled release of rhBMP-2 to a certain extent. Antigen-extracted xenogeneic pig massive cancel-lous bone has shown good biocompatibility and it may potentially replace autologous bone graft in repairing large bone defects. This study has provided a new reference for bone regeneration in the oral and maxillofacial region.

Acknowledgments

This work was supported by a grant from the Guangdong Science and Technology Foundation (Grant No. 2012B061700091). We thank the Department of Nuclear Medicine, the Animal Center, and the Institute of Biomed-ical Engineering at Jinan University, and Guangdong Guan-Hao Science and Technology Development Co. Ltd for their generous support and kindly providing key materials used in the study.

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[19] Xu JC, Wu GH, Liu HL, Liu JT, Yan XJ, Chen JT. The effect of leptin on the osteoinductive activity of recombinant human bone morphogenetic protein-2 in nude mice. Saudi Med J 2010;31:615e21.

[20] Chuang JP, Chang CP, Shen HT, Kao J, Yan JL. Repair of the canine vertebral lamina with a combination of autologous micromorselized bone and poly-lactic acid gel after a total laminectomy. Kaohsiung J Med Sci 2010;26:357e65.

[21] Zhou M, Peng X, Mao C, Xu F, Hu M, Yu GY. Primate mandibular reconstruction with prefabricated, vascularized tissue-engineered bone flaps and recombinant human bone morphogenetic protein-2 implanted in situ. Biomaterials 2010;31:4935e43.

[22] Shi Q, Li Y, Sun J, Zhang H, Chen L, Chen B, et al. The osteogenesis of bacterial cellulose scaffold loaded with bone morphogenetic protein-2. Biomaterials 2012;33:6644e9. [23] Li HY, Chang J. Stimulation of proangiogenesis by calcium

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[25] Fu Y, Zhang Q, Sun Y, Liao W, Bai X, Zhang L, et al. Controlled-release of bone morphogenetic protein-2 from a microsphere coating applied to acid-etched Ti6AL4V implants increases biological bone growthin vivo. J Orthop Res 2014;32:744e51.

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