The aim of this study was to investigate a potential novel biological transport disc that avoids secondary injury to the body and facilitates bone healing. Twenty-seven dogs were divided randomly into three groups: group A were treated with human bone morphogenetic protein 2 (BMP-2) modified bone mesenchymal stem cell (BMSC) sheets combined with freeze-dried bone allograft as biological transport disc; group B were treated with BMSC sheets combined with freeze-dried bone allograft as transport disc (control); and group C were treated with direct extension only (blank). There were nine dogs in each group. Non-vascular transport distraction osteogenesis was performed in groups A and B to repair the mandibular bone defects, and in group C only mandibular truncation surgery was performed. The regeneration of bone was evaluated through X-ray, haematoxylin and eosin assay, and immunohistochemistry. After 2, 4, and 8 weeks of distraction, new bone density values in group A were 49.00 ± 1.16, 66.63 ± 2.62, and 72.78 ± 2.67, respectively, and these were significantly different to values in groups B ( P = 0.0005, P = 0.0004, P = 0.0012) and C ( P < 0.0005, P = 0.0001, P = 0.0003). The average grey value for BMP-2 expression in group A after 4 weeks of distraction was 195.63 ± 4.45, which was significantly different when compared to groups B ( P = 0.0022) and C ( P = 0.0006). This novel biological transport disc represents an effective non-secondary injury method to enhance new bone formation in non-vascular transport distraction osteogenesis.
A variety of methods are currently used for the treatment of bone defects, discrepancies, and deformities, including external fixators and the Ilizarov technique. Skeletal reconstruction of large bone defects resulting from trauma, infection, tumour resection, and skeletal abnormalities is often required in orthopaedic and oral and maxillofacial surgery. These large defects, of a size beyond the normal potential for self-healing, are often refractory to treatment and represent a challenge for surgeons.
Mandibular distraction osteogenesis has emerged as an effective surgical technique for the treatment of congenital retrognathia, micrognathia, and mandibular hypoplasia. Increasing surgeon experience and innovations in surgical devices have led to the expansion of the use of this attractive technique throughout the craniofacial skeleton, allowing for the correction of maxillary hypoplasia, midface hypoplasia, and craniosynostosis.
Transport distraction osteogenesis is mainly applied to the repair of relatively large bone defect segments. This technique requires the bone to be cut on one side, which then forms a transport disc, with the soft tissue and periosteum kept attached. However, for patients with serious bone defects and bone deformities, it is not always possible to create an appropriate transport disc in the bone defect area. Furthermore, the failure of autologous bone transport disc surgery in these patients is a significant limitation to the widespread use of the traditional transport distraction osteogenesis procedure.
By chance, the present authors discovered that free autologous bone blocks that did not have periosteum and the surrounding soft tissue attached were still able to promote distraction osteogenesis. Hence, the concept of ‘non-vascular transport distraction osteogenesis’, also referred to as ‘free transport distraction osteogenesis’, which is different from the traditional transport distraction osteogenesis, was proposed. Following this, a non-vascular transport distraction osteogenesis animal model was successfully constructed and the mechanism studied. This new method appears to provide an excellent solution to the problem of repairing large maxillofacial bone defects, for which it is not possible to use traditional transport distraction osteogenesis.
On the basis of this prior work, the present study was performed to investigate a potential new type of non-free autologous bone transport disc that is both safe and effective and does not result in secondary injury to the body. This new non-free autologous bone transport disc may also facilitate new bone formation. To broaden the indications for distraction osteogenesis and extend its application, this experimental animal research employed a biological transport disc to repair mandibular defects. These transport discs were made from a freeze-dried bone scaffold, with the application of bone morphogenetic protein 2 (BMP-2) as a growth factor and bone marrow mesenchymal stem cells (BMSCs) as seed cells. This represents a new distraction model for maxillofacial bone defect repair.
Materials and methods
This study was approved by the necessary ethics committee. Twenty-seven healthy male Chinese rural dogs, aged approximately 2 years and weighing between 13 and 15 kg, were used in this study. The dogs were divided randomly into three groups, with nine dogs in each group: group A (experimental group), group B (control group), and group C (blank group). The protocol design and procedures were approved by the animal research centre of the study institution.
Cell isolation and culture
Canine bone marrow was harvested from the tibias of small Chinese rural dogs. The marrow was subjected to the whole bone marrow adherent culture method for the isolation of BMSCs. BMSCs were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 15% foetal bovine serum (Gibco, Invitrogen, Grand Island, NY, USA) and 1% antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) in a humidified environment (37.8 °C, 5% CO 2 ). Cellular outgrowth of canine BMSCs was observed after 7 days of culture and reached 80% confluence after 10 days. The cells were then scraped using cell scrapers and expanded. Cells between the second and fourth passages were used for all experiments.
Fabrication of cell sheets
Third passage BMSCs were transfected with the human BMP-2 gene using lentivirus as a vector. Positive and stable transfected canine BMSCs were inoculated into a 6-cm diameter cell culture dish at a density of 1 × 10 6 . The cells were stimulated with 50 μg/ml ascorbic acid to form an extracellular matrix. After 10 days of culture, the cell sheets were gently harvested using the plastic cap of a penicillin bottle. During this process, the cell sheet was not allowed to dry completely.
Fabrication of the biological transport disc
Freeze-dried bone blocks, produced by deep-freezing, degreasing, freeze-drying, and sterilization, were obtained from fresh allogeneic dog mandibles. These were rehydrated before use by flushing with sterile saline (>10 times) and then immersing them in sterile saline for more than 8 h; attention was paid to maintaining sterile conditions so as to avoid contaminating the freeze-dried bone. The biological transport discs were fabricated by covering the rehydrated freeze-dried bone blocks in intact cell sheets, producing a composite; these were stored prior to application.
Surgical protocol and animal model
The surgical procedures were performed under anaesthesia and in sterile conditions. Anaesthesia was induced by intraperitoneal injection of 1 ml/kg sodium pentobarbital (Shanghai West Tang Biotechnology Company, China). The mandibular surgical site was prepared preoperatively by hair removal, sterilization, and the application of local anaesthetic. The exposed bone surface was cut and a mandibular segmental bone defect of 2.5 cm in length by 1 cm in height was prepared; the distractor was then preset and fixed ( Fig. 1 ). Finally, the wound was sutured and flushed with gentamicin. General nursing care, a regular diet, and antibiotics to prevent infection were provided after the operation. The postoperative recovery period for the dogs was 5 days. Distraction was started on the sixth day after surgery. The distraction device was activated at a rate of 1 mm once a day for 10 days. In group A, a biological transport disc comprising human BMP-2 modified BMSC sheets combined with the freeze-dried bone allograft was applied (experimental group). In group B, a transport disc comprising BMSC sheets combined with freeze-dried bone allograft was applied (control group). Animals in group C (blank group) underwent only mandibular truncation surgery in the same location with direct pulling to 10 mm and fixation.
The morphology of the cell sheets was examined by haematoxylin and eosin (H&E) staining. Calcium nodule formation by BMSCs was examined by alizarin red staining. The dogs were sacrificed at 2, 4, or 8 weeks following the completion of distraction, and tissue was collected from the distraction gap. The regenerated bone mandibles were assessed by X-ray, H&E staining, and immunohistochemistry of BMP-2.
SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. Experimental data were recorded as the mean ± standard deviation (SD); data were compared by one-way analysis of variance (ANOVA). A P -value of <0.05 was deemed to indicate a significant difference.
Cell isolation and cell sheet delivery
Most cells displayed a typical elongated spindle or vortex shape. Cells were cultured in osteoblast-inducing conditional medium for approximately 21 days and were then stained with alizarin red. Calcium nodule formation was dyed orange and observed under an inverted phase contrast microscope ( Fig. 2 ).
After cell growth for 5 days, cell sheets began to form; after 7–10 days, the cells formed an intact cell sheet and the marginal cells began to form folds. At this point, the intact cell sheet was peeled off carefully using the plastic cap of a penicillin bottle. The white membrane, which has a certain thickness and elasticity, was harvested. H&E staining showed the BMSC sheets to consist of several layers of cells with tight connections among the cells ( Fig. 3 ).
Results of biological transport disc fabrication
Several sterilized freeze-dried bone blocks were prepared. The intact cell sheet was peeled off and used to cover the freeze-dried bone, forming a composite; the biological transport disc was then ready for use ( Fig. 4 ).
All operations were completed successfully and all of the experimental animals survived to the scheduled time in good condition. There was no opening or contact between the surgical site and the oral cavity, and there were no wound infections.
On examination of the X-rays obtained for groups A and B, both ends of the mandibular bone incision line were clearly identifiable at 2 weeks after distraction. A low density shadow was observed in the distraction gap, and the transport disc was clearly visible and distinguished from the surrounding host tissue. At 4 weeks after distraction, the density shadow in the distraction gap was greater than it had been at 2 weeks for both group A and group B. In particular, group A demonstrated a higher density in the upper part of the distraction gap and near the host bone; the distraction gap was filled with a triangular shadow. The density shadows in groups B and C were less evident than that in group A. At 8 weeks after distraction, the density shadow was greater than that observed at 2 and 4 weeks after distraction. The density shadow in group A was greater than that in group B. The original shape of the transport disc could not be distinguished: only a high density shadow could be seen at the bottom of the original transport disc, and the transport disc itself could not be differentiated clearly from the surrounding bone tissue boundaries ( Fig. 5 ).
In group C (blank group), there were no obvious changes on the X-ray image at 2 weeks after distraction. Both ends were clearly visible, and there was an obvious low density shadow in the distraction gap. At 4 weeks after distraction, the two bone ends appeared slightly ‘fuzzy’, and an obvious low density shadow was apparent in most of the distraction gap. At 8 weeks after distraction, the distraction gap had narrowed significantly, but there was still a low density shadow in the middle of the gap ( Fig. 5 ). The bone density values in the distraction gap for the three study groups are given in Table 1 .
|Group||A||B||C||P -value A vs. B||P -value A vs. C|
|2 weeks||49.00 ± 1.16||38.79 ± 1.24||26.63 ± 1.20||0.0005||0.0000|
|4 weeks||66.63 ± 2.62||48.62 ± 1.22||36.10 ± 2.12||0.0004||0.0001|
|8 weeks||72.78 ± 2.67||58.72 ± 1.26||50.10 ± 2.18||0.0012||0.0003|
a Group A: human BMP-2 modified BMSC sheets combined with freeze-dried bone allograft as biological transport disc; group B: BMSC sheets combined with freeze-dried bone allograft as transport disc; group C: direct extension group. Group A was compared to the other two groups for each fixation period; significant at P < 0.01.
At 2 weeks after distraction, a large amount of granulation tissue was observed on H&E staining in groups A and B. This tissue consisted of a large number of new thin-walled capillaries and fibroblasts, and an infiltration of scattered inflammatory cells. Compared with group B, the granulation tissue in group A was relatively mature, with increased fibrous tissue and thicker capillary walls. In group C, there was evidence of the early formation of trabecular bone near the two ends, which gradually thickened and coarsened, and there were a few scattered cartilage islands.
At 4 weeks after distraction, a large amount of new bone had formed in group A; the trabecular bone was funicular, interconnected, and formed reticular bone. The trabecular bone was disordered. A large number of osteoblasts, mesenchymal cells, and fibroblasts were observed around the new trabecular bone. In group B, there was a large amount of mature granulation tissue and fibrous tissue; funicular trabecular bone was visible within the fibrous tissue, surrounded by a large number of osteoblasts. In group C, there was a small amount of active proliferation of osteoblasts near the cartilage cells; some areas showed osteoid formation, with bone or cartilage cells buried within them. There was still a lot of fibrous connective tissue in the central region.
In group A, at 8 weeks after distraction, there was a considerable amount of lamellar bone arranged in concentric circles, in the centre of which were Haversian canals forming bone units. There were bone cells in every layer of the lamellar bone, and the bone tissue was close to maturity. In group B, the trabecular bone in the distraction gap was still reticulated and augmented. During the bone reconstruction period, a large number of osteoclasts were visible around the trabecular bone and were gradually forming a ring of lamellar bone. Only a small number of bone units were seen. In group C, a small amount of woven bone had formed at the two broken bone ends, but the central area was still filled with cartilage and connective tissue ( Fig. 6 ).