| Objective:Periodontal tissue defects are common in periodontal diseases and periodontal trauma. The most ideal healing mode for periodontal tissue defects is the functional regeneration of alveolar bone, cementum and periodontal ligament, with new periodontal attachment formation. Treatment technology, such as periodontal flap surgery, bone operation, guided tissue regeneration(GTR), get regeneration to some extent. But the regeneration of the connection between periodontal soft and hard tissues is still unable. Periodontal new attachment, anatomical structures reconstruction and support function of natural periodontal tissue, are becoming research hotspots. The establishment of tissue engineering theory and the development of tissue engineering technology have brought new idea for periodontal treatment, and becoming a researching hotspot of periodontal regeneration.The construction of seed cells-scaffold complex is the research core of periodontal tissue engineer. Previous studies have showed that periodontal ligament cells(PDLCs), as the basis of periodontal tissue regeneration, the sources are limited, and most researches have focused on the utilizing of scaffold materials with or without any surface modifications. These make it hard to attain efficient tissue regeneration and functional reconstruction. Therefore, suitable source of seed cells, appropriate scaffold materials, the construction of seed cells-scaffold complex have becoming problems to be resolved urgently in periodontal tissue engineering technology.To investigate the feasibility of construction and application of tissue engineered periodontal complex, and provide experimental data and theoretical basis for clinical research. Beagle gingival fibroblasts(GFs) were used as seed cells, and Bio-Gide collagen membrane and small intestinal submucosa(SIS) were used as scaffold material to construct tissue engineered periodontal complex in vitro. Repair periodontal defects with the tissue engineered periodontal complex in vivo, and observe the tissue regeneration and establishment of new attachment.Methods: 1 Selection of scaffold material and degradation test 1.1 Preparation of SISSwine jejunum(50 cm) obtained from the closed breeding swine, and prepared by Abraham method. 1.2 Bio-Gide collagen membrane 1.3 Implantation of Bio-Gide collagen membrane and SISBio-Gide collagen membrane and SIS were implanted in both sides of the subcutaneous, sutured the wound and marked the location. 1.4 Observation of the degradation proceed by HE staining 2 Primary cultures of canine fibroblast and mineralized induction in vitro 2.1 Cell culture and identification of canine GFsCanine GFs were cultured by explants method, identified by immunohistochemical staining for vimentin and Scanning Electron Microscope. 2.2 Mineralized induction and identification of canine GFsBeagle dog GFs 2-3 generation of logarithmic growth phase cells were used, replace cell culture medium with mineralized induced medium. On the 11 th day, the mineralization nodules formed and identified with 2% alizarin red staining and 1% silver nitrate staining. 3 The construction of tissue engineered periodontal complex 3.1 The construction of tissue engineered periodontal ligamentBeagle canine GFs(2×106 / ml cells) were seeding on double side of the Bio-Gide membrane.3.2 The construction of tissue engineered mineralized membraneBeagle canine GFs(2×106 / ml cells) were seeding on the SIS, stationary culture for 3 days, and induced cultured with mineralized induced medium for 8 days. 3.3 The construction of tissue engineered periodontal complex(mineralized membrane + periodontal membrane + mineralized membrane complex) 4 Engineered periodontal complex reconstruction in vivo 4.1 Establishment of periodontal tissue defects animal modelNormal group: without trauma, keep the normal periodontal structure.Trauma group: gingival flap restoration and tight suture were done after the establishment of periodontal defects.Tissue-engineered periodontal membrane: tissue-engineered periodontal membrane was implanted into the periodontal defects after the establishment of periodontal defects.Tissue-engineered periodontal complex group: tissue engineered periodontal complex was implanted into the periodontal defects after the establishment of periodontal defects. 4.2 Observation of tissue engineered periodontal complex in vivoThree Beagle dogs were executed on 10 th, 20 th and 30 th day respectively, both mandible and maxillary were obtained and fixed in 10% formalin, followed by decalcifying and HE staining.Results: 1 Scaffold material selection and degradation test 1.1 Gross observationAll experimental animals were healthy survived, no inflammation such as wound swelling, oozing or bleeding. All incisions were primary healing with no infection or graft detachment. Animals ate and lived normally. No abnormal manifestations such as necrosis, blackening, suppuration, or effusion at any time. And no wrapping formed. 1.2 Histological observation 1.2.1 Histological observation at 2nd week Two weeks after the implantation of Bio-Gide collagen membrane, collagen fibers in Bio-Gide collagen membrane ruptured, a small amount of fibroblasts grew among collagen fibers, with infiltration of a small amount of lymphocytes and plasma cells.Two weeks after the implantation of SIS scaffolds, structure of SIS scaffolds was still legible, fibroblasts and a large number of new capillaries were visible, with infiltration of lymphocytes, plasma cells. 1.2.2 Histological observation at 4th weekFour weeks after the implantation of Bio-Gide collagen membrane, collagen fibers in Bio-Gide collagen membrane swelled and ruptured, fibroblasts grew among collagen fibers.Four weeks after the implantation of SIS scaffolds, SIS scaffolds degraded obviously, loose fiber structure and fiber fracture had appeared, inflammatory cells like lymphocytes, plasma cells decreased. 1.2.3 Histological observation at 6th weekSix weeks after implantation of Bio-Gide collagen membrane, collagen fibers in Bio-Gide collagen membrane swelled and ruptured obviously, large amount of fibroblasts appeared.Six weeks after implantation of SIS scaffolds, SIS scaffolds continued to degrade, but still a little amount of scaffold structure was visible with locally infiltration of lymphocytes and plasma cells. Large amount of fibroblasts appeared. 1.2.4 Histological observation at 8th weekEight weeks after implantation of Bio-Gide collagen membrane, collagen fibers ruptured or disappeared. Large amount of fibroblasts appeared.Eight weeks after implantation of SIS scaffolds, most of SIS scaffolds degraded, with locally infiltration of lymphocytes and plasma cells. Large amount of fibroblasts appeared. 1.2.5 Histological observation at 10 th weekTen weeks after the implantation of Bio-Gide collagen membrane, the structure disappeared. Bio-Gide collagen membrane degraded completely, and replaced by fibrous connective tissue.Ten weeks after the implantation of SIS scaffolds, almost all SIS scaffold degraded, a large amount of fibroblasts appeared. 1.2.6 Histological observation at 12 th weekTwelve weeks after implantation of SIS scaffolds, SIS completely degraded and replaced by large amount of fibroblasts, adipose tissue, new capillaries and collagen. 2 Primary culture of canine GFs and mineralized in vitro 2.1 Primary culture of canine GFs and identificationOn the 3rd day of primary cultured, spindle cells crawled out to tissue blocks around, most were fusiform, with plump soma, uniform cytoplasm, round or oval large nuclei in the middle, cytoplasm extending outward 2-3 neurites ranging from length, small part were flat polygon cells. On 6th-7th day, Beagle canine GFs grew tissue-centered radially and swirly observed under inverted microscope.The second generation Beagle canine GFs against showed positive for vimentin, cytoplasm was colored pale brown uniformly.Observation under electron microscopic, visible Beagle canine GFs were larger volume, contours were clear, most were flat structure raised of fusiform or stellate, with regular oval nuclei and clear nucleolus. 2.2 Identification of mineralized Beagle canine GFsOn the 11 th day of Beagle canine GFs mineralized induction, mineralized nodules formed with different sizes, deep color or black, round or oval.Alizarin red staining results showed orange calcified nodules of different sizes formed, as a complex of alizarin red and calcium salt.Silver nitrate(1%) staining results showed black and dark brown calcium nodules with different size formed, which indicated that there was plenty of calcium salt deposition, mineralized nodule formation.3 The construction of tissue engineered periodontal complex 3.1 Construction of tissue engineered periodontal ligamentScanning electron microscope results showed that Bio-Gide collagen membrane had a good porous network structure, which provide canine GFs strong attachment, vigorous growth and full stretch. Cells with many cell neurites were often long fusiform, and grew around or across the pore. Cells adhered to scaffolds, secreted a large number of matrix, formed fibrous structure. Compared with cells not composed, fiber reticular tissue and basically had fibrous structure of periodontal ligament formed in complex, which meant successful construction of tissue engineered periodontal ligament in vitro. 3.2 Construction of tissue engineered mineralized membraneInoculated GFs on one side of SIS scaffolds tissue, and induced by mineralized culture medium for 8 days. Observation under inverted microscope showed the formation of mineralized nodules.Scanning electron microscope results showed that SIS had a good porous network structure, which gave canine GFs strong attachment, vigorous growth. Cells with many cell neurites were often long fusiform, mineralized nodules formed as well. 3.3 Construction of tissue engineered periodontal complexOne layer of tissue engineered periodontal membrane between two layers of tissue engineered mineralized membrane were constructed, as sandwich-like tissue engineered periodontal complex, to simulation of alveolar bone, cementum and periodontal ligament. The results showed cavernous structure, with certain mechanical properties and plasticity. 4 Reconstruction of tissue engineered periodontal complex in vivo 4.1 Establishment of animal model of periodontal tissue defectsPeriodontal condition of experimental animal was good before operation, without gingiva swelling or periodontal pockets, and no alveolar bone defect during flap surgery.Experiment is divided into four groups, normal control group, periodontal trauma group, tissue-engineered periodontal membrane and tissue-engineered periodontal complex group corresponding left upper jaw, upper right, left mandibular and right mandible respectively. The 2nd, 3rd and 4th premolar in each quadrant of each Beagle dog involved in this study, Self-control was used in this experiment to ensure the same experimental conditions among three groups.Surgical methods cause Beagle canines tissues of acute injury, 2 mm periodontal defect, from the tooth neck direct a third root, formed on mesial and distal side of mesial root of each experimental teeth, periodontal membrane and cementum were removed by subgingival curette. 4.2 Gross observationAll experimental animals postoperative moved and ate normally and survived until the end of the research. After the implantation of tissue engineered material, all incisions were primary healing with no infection or complications. No graft detachment, no local or systemic obvious adverse reaction appeared. 4.3 Histologically observation 4.3.1 Histologically observation at the 10 th postoperative dayNormal control group: Beagle canine premolar root furcation area including cementum, periodontal ligament, alveolar bone, and cancellous bone. Cancellous bone was composed of bone trabeculae and bone marrow. Bone trabeculae were thick and dense, different size of bone marrow gap distributed within fat cells.Periodontal trauma group: On the 10 th postoperative day, no new cementum and periodontal ligament repaired, only a small amount of new alveolar bone.Tissue engineered periodontal ligament group: On the 10 th postoperative day, no periodontal membrane formed.Tissue engineered periodontal complex group: On the 10 th postoperative day, cementum reconstructed, new periodontal ligament fibers were visible, new alveolar bone formation, and cementum construction started. 4.3.2 Histological observation at the 20 th postoperative dayNormal control group: Beagle canine premolar root furcation area including cementum, periodontal ligament, alveolar bone, and cancellous bone. Among them, cancellous bone was composed of bone trabeculae and bone marrow. Bone trabeculae were thick and dense, different size of bone marrow gap distributed within fat cells.Periodontal trauma group: On the 20 th postoperative day, no new cementum, periodontal ligament were not repaired, a small amount of new alveolar bone.Tissue engineered periodontal ligament group: On the 20 th postoperative day, cementum was in reconstructing, new alveolar bone and periodontal fibers were visible.Tissue engineered periodontal complex group: On the 20 th postoperative day, cementum and alveolar bone reconstruction completed, periodontal fiber structure was clear. 4.3.3 Histological observation at the 30 th dayNormal control group: Beagle canine premolar root furcation area including cementum, periodontal ligament, alveolar bone, and cancellous bone. Among them, cancellous bone was composed of bone trabeculae and bone marrow. Bone trabeculae were thick and dense, different size of bone marrow gap distributed within fat cells.Periodontal trauma group: On the 30 th postoperative day, no new cementum and periodontal ligament were repaired.Tissue engineered periodontal ligament group: On the 30 th postoperative day, cementum was in reconstruction, osteoblasts and new periodontal fibers were visible.Tissue engineered periodontal complex group: On the 30 th postoperative day, cementum repair completed, with mature periodontal ligament and clear perforating fiber.Conclusions:1 Gingival fibroblasts have strong potential ability of differentiation and induced mineralization, which form the mineralized nodules. Provide seed cells for the construction of tissue engineered periodontal mineralization membrane.2 Successful construction of tissue engineered periodontal membrane with gingival fibroblasts seeded on the Bio-Gide collagen membrane.3 Successful construction of tissue engineered mineralization membrane with gingival fibroblasts inducing mineralization seeded on the small intestinal submucosa.4 Double tissue engineered mineralization membrane, with tissue engineered periodontal membrane between them, constructing the "sandwich" structure of tissue-engineered periodontal complex.5 Tissue engineered periodontal membrane in animals can repair periodontal tissue defect with periodontal tissue regeneration, but the alveolar bone and cementum formation is slower.6 The "sandwich" structure of tissue engineered periodontal complex(mineralized membrane + periodontal membrane + mineralized membrane complex) could repair periodontal defects and obtain the ideal structure of periodontal tissue in vivo. |
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