Despite numerous advances in orthopaedic and plastic surgery, the repair of bone defects remains challenging. The most desirable material for bone repair is autologous bone graft, due to its excellent osteoconduction, osteoinduction, and osteogenesis properties.[
Recent progress in the fields of biotechnology and tissue engineering has offered new options for repair of traumatic and non-traumatic bone defects. Mesenchymal stem cells (MSCs), which are multipotent adult stem cells of mesodermal origin, have been shown to play a critical role in tissue engineering. MSCs are an excellent potential source of cells for bone tissue engineering due to their excellent renewal ability and osteogenic differentiation capabilities.[
The use of MSCs with an appropriate scaffold has been demonstrated to be promising in guiding bone tissue neoformation after implantation in the host. Cell repopulation can be achieved either by direct cell loading or indirect cell induction with osteogenic factors.[
Bone marrow mesenchymal stem cells (BM-MSCs) were isolated from rabbits and cultured as reported previously.[
Human demineralized cancellous bone (HDCB), which is a type of DBM and has been proven to be usable as scaffold material, was used in this experiment. HDCB was supplied from the Bone Bank at the National Institute of Burns. Fresh bones were aseptically harvested within the first 12 h after being shown to be free of any infectious disease. Bones were treated with H2O2, a mixture of methanol/chloroform, hydrochloric acid, and phosphate buffer pH 7.4. Subsequently the bones were dehydrated for 24 h until the water content remaining in the bones was less than 5%. The bones were cut into blocks with dimensions of 1.5 cm × 0.3 cm × 0.5 cm. A medullary hole was made in the bone blocks with a diameter of 1.5 mm. The block was packaged and sterilized by gamma irradiation at a dose of 25 kGy. The sterilized bones were then preserved at 4℃.
Culture-expanded BM-MSCs were seeded evenly onto the HDCB scaffold. DHCB/BM-MSCs were cultured in T flasks (Thermo Scientific Nunc A/S, Denmark) filled with 5mL DMEM containing 10% FBS and antibiotics. The grafts were placed in a vacuum desiccator and treated at a pressure of 100 Torr for 100 seconds, after which they were incubated at 37℃, 5% CO2 for 2 weeks. The medium was replaced every 3 days.[
Twenty-eight males 8-week-old New Zealand white rabbits with a body weight of approximately 1.5 kg from the Experimental Laboratory of the Medical Training and Research Center, Hue Central Hospital, were used for the study. The protocol for this experimental study was approved by the Committee of the Medical Training and Research Center. The rabbit bone defect model was established as described previously.[
The procedure for the transplantation of cancellous bone graft into the segmental radial defect
Following sacrifice, both reconstructed radiuses were harvested and completely cleared from the soft tissues. The status of callus growth, degradation, bone healing, and NB formation at the bone graft in the radius were observed.
Radius bone specimens in each group were X-rayed for evaluation of bone formation and remodeling (Titan 2000, COMED Medical Systems CO. Ltd., Korea). Assessment of NB formation and remodeling was based on the modified Lane and Sandhu radiological scoring system.[1] Three experts blindly assessed radiological scores, which were the sum of the scores of bone formation and remodeling. The score for NB formation was assigned as 0 (no NB formation), 1 (< 25% NB formation), 2 (25-50% NB formation), 3 (50-75% NB formation), or 4 (> 75% NB formation). The score assigned to the assessment of union was 0 (nonunion), 1 (possible union), or 2 (radiographic union). The proximal and distal unions of the bone graft were separately evaluated. The remodeling score assigned was 0 (no evidence of remodeling), 2 (intramedullary remodeling), or 4 (cortical remodeling). The maximum number of points, which could be achieved, was 10 for each reconstructed bone.
Fifty-two specimens from the bone graft sites of the radius were successfully fixed with 10% paraformaldehyde, decalcified with sodium formate and embedded in paraffin. Four specimens in group 1 experienced technical failures. Three sagittal sections were cut with a slow speed saw from each site at the distal, proximal and middle lines of the bone graft. Sections were then prepared and stained with hematoxylin and eosin. The micrographic images from the light microscope were quantified. Images from each section were taken to evaluate the bone formation ratio by a qualified pathologist blinded to the study. The NB formation ratio was calculated by the percentage area of bone tissue within the defect site, and a mean value was determined for each section.
The specimens of the radius of each group were loaded onto a multifunctional mechanical tester (Instron 5582 Universal Tester, USA) for performance of a uniaxial compression test. The specimen was placed between compression plates. Force was applied to the specimens at a constant speed of 1 mm/min until fracture occurred. Compressive stress and strain were calculated and plotted. Stress value at the point of yield (load-to-failure) was determined.
The data were presented as mean and standard deviation. The Student's
The wounds healed completely after one week and the rabbits were noted to regain full movement within two weeks. All rabbits survived with normal behavior. No complications such as infection or necrosis were recorded prior to sacrifice.
At 12 weeks after surgery, radii implanted in group 1 showed a small amount of callus and fibrous-like tissue in the interspaces between the defects and grafts. Partial degradation of the HDCB grafts was found. There was a significant amount of callus and bony union filled more than half of the defects in groups 2 and 3. The HDCB grafts in these groups were almost degraded. In group 4, good bony union was observed. Bone defects were almost completely remodeled with NB tissue and the HDCB grafts were completely degraded in this group
Gross observations of the reconstruction of radius at 3 months after surgery. (a) Small amount of callus and fibrous‑like tissue in the interspaces between defect and human demineralized cancellous bone graft in Group 1; (b) callus formed in the defect repair by periosteum‑wrapped human demineralized cancellous bone graft in Group 2; (c) significant amount of callus and bony union filled in the defect repair with the human demineralized cancellous bone graft seeded with mesenchymal stem cells in Group 3; (d) complete bone healing in the defect repair by periosteum‑wrapped human demineralized cancellous bone graft seeded with bone marrow mesenchymal stem cells in Group 4
At 3 months postoperatively, there were a small amount of callus formation at the defect gaps in group 1. NB formation was found to account for over half of the material at the reconstructed bone in groups 2 and 3. Bone regeneration in the radius in group 4 was observed to be the best, where callus formation was greatest in comparison to the other groups
Modified Lane and Sandhu radiological scores, mean new bone formation in Histology (%), and mean compressive strength (MPa) of the rabbit's radius in each group at 3 months after surgery
Group | Scaffold implantation | Mean radiological scores | Mean compressive strength (MPa) | Mean new bone in Histology (%) |
---|---|---|---|---|
1 | HDCB only | 2.95 ± 0.58 | 31.14 ± 6.72 | 29.60 ± 8.33 |
2 | Periosteum-wrapped HDCB | 5.57 ± 0.51 | 73.00 ± 7.20 | 49.79 ± 11.69 |
3 | HDCB/BM-MSCs | 6.41 ± 1.03 | 80.57 ± 8.50 | 64.12 ± 11.31 |
4 | Periosteum-wrapped HDCB/BM-MSCs | 8.58 ± 0.64 | 129.31 ± 5.99 | 80.50 ± 4.96 |
HDCB: Human demineralized cancellous bone, BM-MSCs: Bone marrow mesenchymal stem cells
Results of X‑ray at the 3 months postoperation. (a) A few calluses at the defect gap in Group 1; (b) significant new bone information at the reconstructed bone in Group 2; (c) more new bone formation between graft and bone tissue in Group 3; (d) almost remodeling of new formed bone along the entire gap of the bone defect in Group 4, and the cortical bone bridged to the adjacent native bone
Inflammation was not observed in the grafted bone segment. Poor NB formation and capillary network were found at the interface between the graft and radius in group 1. Both ends of the original radius were united with newly regenerated bone in groups 2 and 3, while the HDCB scaffold was mostly degraded and cortical bone was only observed at the center of the defects. A larger amount of NB was generated along the entire scaffold structure and more capillaries were formed in the area of NB in group 4. Group 4 showed superior bone union, cancellous bone, cortical bone, marrow formation, and capillary formation in comparison to the other groups. Cortical bone was also found along the entire gap of the bone defect bridging adjacent native bone
HE stained histological sections from the grafted bone of four groups at 3 months after implantation (original magnification, ×40). NB: New bone, VC: Vascular cavity, BM: Bone marrow, P: Periosteal membrane
Radii of rabbits with partial or complete union were subjected to biomechanical testing. Results of the biomechanical tests are summarized in Table 1. Group 4 showed the highest compressive strength (
This study demonstrates the presence of NB formation and bone healing, as shown both radiologically and histologically, on demineralized cancellous bone graft seeded with BM-MSCs. Results were improved when BM-MSCs were associated with periosteum.
MSCs, periosteal cells and osteoblasts have all been successfully used for bone tissue engineering.[
The anatomy of the periosteum, its nutrient transport, and its osteoinductive and osteoconductive capacities have been well described.[
Based on clinical observation, radiologic examination, histological analyses, and biomechanical measurements, the current study supports the essential role of periosteum in the process of bone repair. In addition, the regenerative effect of combining BM-MSCs with periosteum showed better outcomes in both the quantity and quality as compared to BM-MSCs alone. Furthermore, the MB-MSCs used in the current study are derived from an allogenic source, which is more convenient for isolation and expansion when compared with periosteum-derived cells. To further enhance current bone tissue engineering strategies, a successful cellular replacement for periosteum or tissue-engineered periosteum should be investigated. Zhang
Xenogeneic demineralized cancellous bone grafts, which have the advantages of favorable cellular compatibility and histocompatibility as a scaffold, have widely been used for the repair of short bony defects showing the induction of NB formation and good mechanical properties. Osteoinductive structures in demineralized bone graft include a series of low-molecular-weight glycoproteins with bone morphogenetic proteins. These proteins promote chondroblastic differentiation of mesenchymal cells and create NB formation via endochondral osteogenesis.[
In the group repaired by periosteum-wrapped HDCB graft seeded with BM-MSCs, bone healing and union were significantly accelerated as compared to the other 3 groups. Increased density at the graft site and early fusion of cortical bone were observed. In addition to NB formation demonstrated histologically, a significant amount of regenerated capillary vasculature between the NB was also being observed in a high proportion of grafted bone pores. Zhang
For improved biochemical analysis for bone regeneration, a three-point bending test should be performed to evaluate the degree of scaffold integration with the host bone.
In conclusion, this study demonstrates that repair of bone defect in a rabbit model can be achieved through bone grafting using BM-MSCs implanted on a xenogeneic demineralized cancellous bone scaffold. NB formation was optimized with preservation of the periosteum at the site of injury. The combination of biocompatible material, the ability for self-renewal, differentiation of mesenchymal stem cells with the augmenting effects of periosteum may prove to be an extremely promising approach in the fields of orthopaedic and plastic surgery.
I acknowledge my colleagues at the Department of Hematology, Hue Central Hospital. I would also like to specially thank Dr. Bui Duc Phu, Dr. Nguyen Duy Thang, Dr. Phan Thi Thuy Hoa, Dr. Phan Hoang Duy, Dr. Dang Cong Thuan and Dr. Fréderic Schuind, for their excellent help and support.
Nil.
There are no conflicts of interest.