Effect Of Low Intensity Pulsed Ultrasound On Repairing Bone Defect By Cell-scaffold Complex Composed By Rabbit Bone Marrow Stromal Cells And β-tricalcium Phosphate

Bone defect causes a dilemma in a variety of clinical situations, including trauma,tumor resection, and reconstruction. Although bone tissue usually has the ability to repair itself, but a bone defect of enough size needs to be bridged, the repair attempt fails in most cases, resulting in the formation of a pseudarthrosis, non-union of the fracture, and loss of function. Autologous bone transplantation is the most effective method for restoration, because it provides three essential elements: osteoconduction, osteoinduction, and osteogenic cells. But autologous bone grafts are available in limited shapes and sizes, and significant donor site morbidity is another major disadvantage to this approach. Allografts are believed to be osteoconductive, but confer the risk of disease transmission and immune rejection. Bone cement is readily available and dose not cause either rejection or disease transmission issues. However, it dose not allow bone formation and growth, as it is not biodegradable and may lower the threshold for injection at the surgical site. These treatments have a number of limitations, which could not meet the clinical requirement.Tissue engineering bone is a new alternative that has a potential to overcome many of the drawbacks mentioned above. Tissue engineering involves the use of cell-scaffold complex composed with living cells and scaffold to repair bone defect. Despite bone tissue engineering has been used for decades, little progress has been made with respect to clinical application. One of main problems is the lack of understanding of how the technique shoud work more efficiency.Low intensity pulsed ultrasound (LIPUS) is acoustic at frequencies above the limit of human hearing. It is a form of mechanical energy that can be transmitted into the body as high-frequency acoustical pressure waves. The micromechanical strains produced by these pressure waves in body tissue can result in biochemical events at the cellular level and may promote bone formation in a manner comparable with bone responses to mechanical stress postulated by Wolff's law. Studies of animal models have demonstrated a bone response to LIPUS, including the promotion of cartilage-related gene expression and increased rates of endochondral ossification. In vitro studies have suggested that LIPUS produces significant multifunctional effects that are directly relevant to bone formation and resorption. Clinical investigations involving LIPUS have shown successful healing of pseudoarthroses, delayed unions, and nonunions.Since LIPUS is currently in clinical use for stimulation of bone healing and there is considerable interest in the clinical application of tissue engineering bone, it is possible that combination of the two therapies may provide better results than with either treatment alone. The purpose of this study was to offer a experimental evidence in the application of LIPUS in bone tissue engineering by repairing bone defect with cell-scaffold complex stimulated by LIPUS, which composed with bone marrow stromal cells (BMSCs)andβ-tricalcium phosphate (β-TCP).PartⅠ: Effect of low intensity pulsed ultrasound on proliferative ability of rabbit bone marrow stromal cells in vitroObjective To explore the effect of low intensity pulsed ultrasound on the proliferation of rabbit bone marrow stromal cells (BMSCs) in vitro.Methods Some bone marrow of a rabbit were drown out and cultured by differentiation culture medium to harvest BMSCs and the different generation BMSCs were randomly assigned to the experimental groups and the control groups. The experimental groups, which stimulated by low intensity pulsed ultrasound (LIPUS) 20min/day, and the control groups were all observed with lightmicroscope. Doubling time, divisional index, seeding efficiency were calculated and growth curves were drawn.Results Proliferative ability of BMSCs stimulated by LIPUS increased significantly compared with the control group(P<0.05).Conclusion LIPUS can enhance the proliferative ability of rabbit BMSCs in vitro cultured with differentiation culture medium.PartⅡ: Effect of low intensity pulsed ultrasound on osteogenic potential of rabbit bone marrow stromal cells in vitroObjective To explore the effect of low intensity pulsed ultrasound on the osteogenesis of rabbit bone marrow stromal cells (BMSCs) in vitro.Methods Rabbit bone marrow was drown out and cultured by conditioned culture medium to harvest BMSCs and the third generation BMSCs were randomly assigned to the experimental groups (n=4) and the control groups (n=4). The experimental groups, which stimulated by low intensity pulsed ultrasound (LIPUS) 20min/day for one week,two weeks or three weeks, and the control groups were subjected to Von Kossa staining and alkaline phosphatase (ALP) activity test and observed with invert microscope.Results BMSCs stimulated with LIPUS became larger, with increased nuclei and decreased nucles to cytoplasm; those without stimulation became flat, and have lower refractive index. ALP activity of BMSCs stimulated by LIPUS for one week,two weeks or three weeks increased significantly compared with the control groups (P<0.05)and calcium pixel values of the experimental group is also higher than those of the control group.Conclusion LIPUS can accelerate the osteogenesis of rabbit BMSCs in vitro cultured with conditioned culture medium.PartⅢ: Effect of low intensity pulsed ultrasound on biocompatibility betweenβ-tricalcium phosphate and rabbit bone marrow stromal cellsObjective To study the effect of low intensity pulsed ultrasound (LIPUS) on the biocompatibility betweenβ-tricalcium phosphate (TCP) and rabbit bone marrow stromal cells (BMSCs).Methods Some bone marrow of a rabbit were drown out and cultured by differentiation culture medium to harvest BMSCs and the third generation BMSCs were randomly assigned to the experimental groups and the control groups. The morphology of BMSCs in the experimental groups, which stimulated by LIPUS 20min/day for one week, two weeks, or three weeks, and the control groups were all observed with phase-contrast microscope and scanning electron microscope.Results BMSCs could fully attach to and extend to TCP, and excreted extracellular matrix. The number of BMSCs on the experimental group increased significantly compared with the control groups at the first week and second week(P<0.05).But the number of BMSCs on the experimental group increased significantly compared with the control groups at the third week. Conclusion LIPUS stimulation upregulated the biocompatibility between TCP and BMSCs compared to the control group at early stage and have not same obvious effect at late stage.PartⅣ: Repair of rabbit radius defect by tissue-engineering bone formd byβ-tricalcium phosphate and rabbit bone marrow stromal cells with the addition of LIPUS stimulationObjective To explore the effect of low intensity pulsed ultrasound (LIPUS) on the ability of repairing rabbit radius defect by cell-scaffold complex composed with bone marrow stromal cells (BMSCs)andβ-tricalcium phosphate (TCP).Methods Bone marrow of a rabbit were drown out and cultured by differentiation culture medium to harvest BMSCs and the third generation BMSCs were cultivated on TCP for one week in vitro. Bilateral distal radial defects were made using a 1-mm saw in 20 mature New Zealang white rabbits. The cell-scaffold complexs stimulated by LIPUS were implated in rabbit radial defects (cell-scaffold complexs without stimulation as control). All the rabbits were killed at 4 or 8 weeks after operation. The defect healing state were observed with naked eyes, X-ray and histological technic. The callus area (CA) was measured with slide gaud and the gray density of CA was analyzed by computer image analysis system.Results It was found that the callus on the experimental group was growing more quickly and apparently with naked eyes than that on the control groups. POD 4 weeks, there was significant statistical difference (P<0.05) between CA of the experimental group and that of the control group with image analysis system. But POD 6 weeks, the bone density between experimental group and control group were not statistically significant ( P > 0.05 ) . Histological slices in the experimental group showed that the absorption, organization of hematoma, the formation of trabeculae and matrix were earlier than those in control group and there were more fibroblast, osteoblasts and callus in the experimental group.Conclusion Cell-scaffold complex composed with BMSCs andβ-TCP stimulated by LIPUS can repair bone defect defect more effectively than that without LIPUS stimulation at early stage and have not same obvious effect at late stage.