| Purpose:In this study, we seeded the BMSCs into PLGA/β-TCP scaffold to construct tissue engineering artificial bone in vivo, to explore the potential value on repairing the mandibular segmental defect in canine.Methods:1. PLGA was polymerized with LA and GA, according to LA/GA=70/30, mole ratio. Then, infrared spectral analysis, nuclear magnetic resonance spectrum analysis, gel permeation chromatography. Mixed the PLGA, β-TCP powder as certain proportion [PLGA/β-TCP=6/4, quality ratio]. Using NaCl as porogen material, massive porous PLGA/β-TCP Scaffolds was fabricated by a method of solution casting/porogen leaching. Then soaked scaffolds in deionized water to dissolve NaCl, which resulted the porosity of the PLGA/β-TCP scaffolds. Specific gravity method was used to measure the porosity of the scaffold. Bone marrow stromal cells adhesion on PLGA/β-TCP was observed by SEM scanning.2. Bone marrow was extracted from dogs thighbone after general anesthesia. Bone marrow stromal cells were isolated by density gradient centrifugation, cultured and proliferated in vitro. Then indentified the cell type of osteoblasts after differentiated and seeded them into PLGA/β-TCP scaffolds. Six Beagle adult dogs were assigned randomly into experimental and control group averagely. A3cm unilateral mandible segmental defect was made in each animal. Two different kinds of bone grafts were implanted respectively. Experiment group:BMSCs/PLGA/β-TCP compound; Control group: PLGA/β-TCP. The dog in each group was sacrificed at the4th,12th,24th week post-operation respectively. The defects were evaluated by radiography, gross observation, histology and image analysis.Results:1.The porosity of the PLGA/β-TCP scaffolds was approximately85%, the pore size of it was mainly distributed in the range of150μm～300μm. BMSCs cells could attach to it with a spindle shape or a polygonal shape and extend much pseudopods.2. It was verified that BMSCs can be effectively obtained with method of density gradient centrifugation. BMSCs were induced into osteoblasts in vitro, Alizarin red staining showed red nodes in extracellular matrix, Alkaline phosphatase and BGP immunocytochemistry and immunofluorescence showed positive expression.3. A marked osteogenesis screenage by CT scanning, while low-density image in the control group at4weeks postoperatively; HE staining showed that the scaffold have a different biodegradation in experimental and control group, there were many osteoblasts, osteoclasts and ehondral cells, and immunohistochemistry of osteopontin was positive expressed in the experimental group, while a mount of foreign body giant cells and inflammatory cells appeared in the control goup.Until12th week, dense bone screenage was showed in the experimental group. The control group, however, have a lower-density image than it is at4th week. Histology observation showed that bone islands was formed, hyperplasia of osteoblasts grew aroud it, scaffold materials obvious degradation than control group, in which abundant fibrous connective tissue formed. The experimental group was strong positiveness of osteopontin.At24th week, CT scanning of the experimental group showed that cancellous bone have formed in the central area of the defect,cortical bone formation aroud it, but the control showed group soft tissue screenage. HE staining showed that room in the defects was filled with neonate osseous tissue completely and the bone medullary cavity had completely recanalized. The scaffolds degradation was completely, while fibrous healing occured in non-induced group.Conclusion:1. PLGA/β-TCP scaffolds showed good cellular biocompatibility and could serve as effective scaffold for bone tissue engineering.2. BMSCs can be used as a seed cell for bone tissue engineering.3. Canine segmental mandibular defects can be repaired with the tissue-engineered bone generated by BMSCs and PLGA/β-TCP scaffold. |
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