Bone Formation in a Rat Tibial Defect Model Using Carboxymethyl Cellulose/BioC/Bone Morphogenic Protein-2 Hybrid Materials

The objective of this study was to assess whether carboxymethyl cellulose- (CMC-) based hydrogel containing BioC (biphasic calcium phosphate (BCP); tricalcium phosphate (TCP) : hydroxyapatite (Hap) = 70 : 30) and bone morphogenic protein-2 (BMP-2) led to greater bone formation than CMC-based hydrogel containing BioC without BMP-2. In order to demonstrate bone formation at 4 and 8 weeks, plain radiographs, microcomputed tomography (micro-CT) evaluation, and histological studies were performed after implantation of all hybrid materials on an 8 mm defect of the right tibia in rats. The plain radiographs and micro-CT analyses revealed that CMC/BioC/BMP-2 (0.5 mg) led to much greater mineralization at 4 and 8 weeks than did CMC/BioC or CMC/Bio/BMP-2 (0.1 mg). Likewise, bone formation and bone remodeling studies revealed that CMC/BioC/BMP-2 (0.5 mg) led to a significantly greater amount of bone formation and bone remodeling at 4 and 8 weeks than did CMC/BioC or CMC/BioC/BMP-2 (0.1 mg). Histological studies revealed that mineralized bone tissue was present around the whole circumference of the defect site with CMC/BioC/BMP-2 (0.5 mg) but not with CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) at 4 and 8 weeks. These results suggest that CMC/BioC/BMP-2 hybrid materials induced greater bone formation than CMC/BioC hybrid materials. Thus, CMC/BioC/BMP-2 hybrid materials may be used as an injectable substrate to regenerate bone defects.


Introduction
More than a million people in the United States and other countries require bone graft to regenerate bone defects caused by fracture, trauma, or tumor resection [1,2]. Autograft has been used widely as bone graft due to its excellent osteoinductive and osteoconductive properties [2]. Allograft has also been used as a bone substitute because it prevents donor site morbidity [2]. However, both autograft and allograft have disadvantages, such as limited availability, donor site morbidity, transmission of infectious diseases, and immunerejection reactions [2,3]. To address these shortcomings, a new approach to bone graft has been developed.
Hydroxyapatite (HAp) and calcium phosphate (CaP) have been successfully used as bone graft materials for the regeneration of bone defects due to their physical and chemical properties and their structural similarity to natural bone. CaP-based materials have been approved by the Food and Drug Administration (FDA) for clinical applications in the fields of dental and orthopedic surgery. CaP-based materials possess bioactive, biocompatible, and osteoconductive properties [4]. Although CaP-based materials have been widely used in a clinical setting, they have no osteogenic or osteoinductive properties; however, such properties are needed for the repair and regeneration of bone defects. The osteoinductive properties of natural bone are the result of bone morphogenic proteins (BMPs) and osteogenic proteins that exist in the extracellular matrix (ECM) [5][6][7][8][9]. Thus, in order to produce more effective bone regeneration, CaP-based materials combined with osteoinductive materials are required.
Carboxymethyl cellulose (CMC) has natural biodegradable and biocompatible properties [22,23] and has been used as a biomedical membrane [24,25]. In an in vitro study, Leone et al. demonstrated that amidated carboxymethyl cellulose (CMCA) hydrogel is a potential filler for cartilage defects [26]. Moreover, a recent report demonstrated that a CMC/HAp hybrid hydrogel induced more osteoblast-like cell proliferation, osteogenic markers including Runx2, ALP, and collagen type I, and mineralization than did a CMC-based hydrogel without HAp [27].
On the basis of these results, we hypothesized that a CMC-based hydrogel containing BioC (biphasic calcium phosphate (BCP); tricalcium phosphate (TCP): hydroxyapatite (Hap) = 70 : 30) and BMP-2 would promote greater bone formation in a rat tibial defect model than would a CMCbased hydrogel containing BioC without BMP-2.

Rat Treatments.
Eight-week-old Sprague-Dawley rats (Orient Bio Co., Seongnam City, Korea) were used for the in vivo evaluation of CMC/BioC, CMC/BioC/BMP-2 (0.1 mg), and CMC/BioC/BMP-2 (0.5 mg) groups. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Korea University Medical Center (KUIACUC-2012-128). Experimental animals were divided into three groups: group I ( = 4) was implanted with CMC/BioC, group II ( = 4) was implanted with CMC/BioC/BMP-2 (0.1 mg), and group III ( = 4) was implanted with CMC/BioC/BMP-2 (0.5 mg). The rats were anesthetized with tiletamine/zolazepam (50 mg/kg; Zoletil) and xylazine (10 mg/kg; Rompun). After shaving the right tibia, the periosteum and soft tissue were carefully retracted and two 0.9 mm K-wires (Zimmer, Warsaw, IN) were fixed to the right tibia. K-wires were clamped bilaterally with the author's own-designed external fixator (U&I, Gyeonggi-do, Korea). An 8 mm defect of the right tibia was created with a cutting burr, and 150 L of CMC/BioC, CMC/BioC/BMP-2 (0.1 mg), or CMC/BioC/BMP-2 (0.5 mg) was injected into the right tibia defect, as appropriate, respectively. The subcutaneous tissue and skin were sutured with absorbable 4-0 vicryl (Ethicon, Somerville, NJ, USA) ( Figure 1). The rats were allowed free movement in cages after recovery from anesthesia. One rat in group I had been dead for two days after treatment, so another rat has been treated with the same manners with others in group I.

Bone Formation Analyses
Plain Radiographs. At 4 and 8 weeks after injection, samples were fixed in 3.7% paraformaldehyde solution. Radiographs of the specimens were obtained with a plain radiograph apparatus (In Vivo DXS 4000 Pro System, Carestream Health, Rochester, NY, USA) at 43 KVP, 2 mA, and 44 cm filmradiation beam distance for a 1.5 s exposure time.
Microcomputed Tomography (Micro-CT) Evaluation. At predetermined time intervals of 4 and 8 weeks, bone volume was obtained with a micro-CT system (Albira II Imaging System, Carestream Health). The CT system was operated at a voltage of 40 kV, and a current of 250 A was used with a nominal resolution of 9 m/pixel. Image analysis was performed with a bone analyzer (Molecular Imaging Analysis software; Carestream Health Inc., Woodbridge, VA, USA).
Histological Study. The specimens were retrieved at 4 and 8 weeks. After decalcification, the samples were embedded in paraffin. The tissues were cross-sectioned at an 8 m thickness in the longitudinal parallel direction and stained with hematoxylin and eosin (H&E) and Masson's trichrome staining. The cytoplasm of osteoblasts and bone formation were assessed with H&E staining. Mineralized bone matrix and osteoid were evaluated by Masson's trichrome staining.

Statistical Analysis.
Data are presented as mean ± standard deviation. Statistical comparisons were carried out via one-way analysis of variance using Systat software (Chicago, IL, USA). Differences were considered statistically significant at * < 0.05 and * * < 0.001.

Discussion
Osteoconductive materials, such as CaP-based materials, allow a framework for vascular invasion and cellular infiltration but do not induce mesenchymal cells to differentiate into mature bone cells. Osteoinductive materials, including growth factors such as BMPs, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), induce new bone formation but they do not provide a framework for vascular invasion and cellular infiltration. Thus, osteoconductive materials combined with osteoinductive materials may be ideal for bone regeneration. The objective of this study was to assess whether a CMCbased hydrogel containing BioC and BMP-2 induced a greater amount of new bone formation than did a CMC-based hydrogel containing BioC without BMP-2. The results from the plain radiographs and micro-CT demonstrated that bone formation was significantly greater in the CMC/BioC/BMP-2

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(0.5 mg) group than in the CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) group at 4 and 8 weeks. Thus, these findings demonstrate that CMC/BioC/BMP-2 (0.5 mg) induces rapid bone formation at an earlier stage than does CMC/BioC or CMC/BioC/BMP-2 (0.1 mg). As expected from the plain radiographs and micro-CT data, the histological study data revealed that the CMC/BioC/BMP-2 (0.5 mg) group had more mineralized bone tissue at the defect site than did the CMC/BioC and CMC/BioC/BMP-2 (0.1 mg) groups at 4 weeks. In all groups, a greater amount of mineralized bone tissue was observed at the defect site at 8 weeks than at 4 weeks. The entire defect area was covered with mineralized bone tissue at 8 weeks in the CMC/BioC/BMP-2 (0.5 mg) group but not in the CMC/BioC or CMC/BioC/BMP-2 (0.1 mg) group. These results are consistent with those of previous studies. Lin et al. [28] reported that BMP-2-immobilized heparinbound demineralized bone matrix (HC-DBM) showed higher alkaline phosphatase (ALP) activity (2 weeks), more calcium deposition (4 and 8 weeks), and more bone formation than that of controls after subcutaneous implantation in rats. Zhao et al. [29] demonstrated that a BMP-2absorbed monoclonal antibodies conjugated DBM (MAbs-DBM) group experienced greater osteogenic differentiation in an in vitro study and greater ectopic bone formation in an in vivo study than the control group. In a previous study, we found that woven bone covered the whole circumference more often in a BMP-2-coated tricalcium phosphate/hydroxyapatite group than in a tricalcium phosphate/hydroxyapatite group in a rat model of femoral distraction osteogenesis [30,31]. Kim et al. [32] and Park et al. [33] reported that BMP-2-coated biphasic calcium phosphate (BCP) granules or blocks supported significantly greater bone formation than BCP granules or blocks in a rat model of calvarial defects. Moreover, Choi et al. [34] showed that implantation of BMP-2/BCP granules onto 6 mm diameter defects of the maxillary sinus of rabbits led to enhanced bone formation compared with the control group. Finally, a recent report demonstrated that new bone formation at a bone defect in the middle ear after mastoid surgery was greater in the presence of BMP-2/BCP scaffolds [35].
Our results suggest that CMC/BioC/BMP-2 hybrid materials induce greater bone formation at an earlier stage through release of BMP-2 than CMC/BioC hybrid materials. However, our in vivo study has some limitations. For example, longterm evaluation greater than 8 weeks is needed to compare the quality and architecture of new bone formation between control and implanted groups. More comprehensive analyses that include the histology and angiogenesis of new bone and biomechanical testing are needed. Finally, the effects of longterm release of BMP-2 require further investigation.
In conclusion, CMC/BioC/BMP-2 (0.5 mg) hybrid materials implanted in a rat tibial defect model led to greater bone formation than did CMC/BioC and CMC/BioC/BMP-2 (0.1 mg) hybrid materials. Thus, CMC/BioC/BMP-2 hybrid materials may be useful in an injectable substrate for the clinical application of the regeneration of bone defects in the orthopedic field.