To make the tissue under concept of tissue engineering, it is necessary to cultivate cells in proper scaffolds. Silk fibers are useful materials for range of biomedical applications as well as scaffolds for tissue engineering. The final goal of our project is to use natural Thai silk fibers as a novel scaffold for tissue replacement and/or regeneration. Objectives: The aims of this study are to fabricate electrospun Thai silk fibroin nanofibers and test for their cytotoxic effect on human gingival fibroblast cells. Methods: Protein fibroin from Thai Bombyx mori cocoons was extracted by Na2CO3, CaCl2/C2H5OH/H2O solution and was then dialyzed in deionized water at 4oC for 72 hrs. Subsequently, the dialyzed product was lyophilized to yield a sponge. After dissolved fibroin sponge in formic acid, the fibroin nanofibers were obtained by electrospinning under 15 KVp, 17 mA at loading rate 0.5 ml/hr. The electrospun fibroin nanofibers were studied for their ultrastructures as well as cytotoxicity by microscopy and cell culture system. Results: Based on this protocol, the fibroin nanofibers demonstrated grayish-white highly order fibers. The results from the co-culture of fibroin nanofibers with human gingival fibroblasts demonstrated that cells could proliferate and attach to the nanofibers suggested non-toxic property of the fibers. In conclusion, we successfully fabricated the Thai silk fibroin nanofibers which were not toxic to human gingival fibroblasts in vitro. After optimum physical property adjustment and extensive examination in vivo, the Thai silk fibroin nanofibers might be able to use as natural, non-toxic and promising scaffolds for tissue engineering.
Periodontal diseases are chronic inflammatory diseases caused by pathogenic bacteria in dental plaque, characterized by complex interactions among fibroblasts and immunocompetent cells, such as T cells. In T cells, Ig superfamily members activate tyrosine kinases to activate integrins. The role of chemokine stimulation has not been clarified. Therefore, we used T cell-like Jurkat cells expressing the receptor for stromal cell-derived factor-1α (CXCL12, a CXC chemokine) and investigated adhesion to fibronectin, an extracellular matrix protein and a β1-integrin ligand. The results confirmed that CXCL12 stimulation enhanced adhesion of Jurkat cells to fibronectin with anti -α4, -α5 and β1-integrin antibodies inhibited adhesion to fibronectin. Although CXCL12 stimulation also facilitated actin polym-erization, no effects were seen with anti -α4, -α5 or β1-integrin antibodies. The above findings suggest that CXCL12 -induced adhesion of Jurakat cells to fibronectin is dependent on integrin activation, and increased adhesion accelerates immune reactions in inflamed tissue.
Collagen XII, a fibril-associated collagen (FACIT), is characterized by a short triple-helical domain with three extended noncollagenous NC3 domains, and may contribute to the stability of collagen I fibrils. Differential splicing within the NC3 domain results in large and small variants of collagen XII. Little is known about the role of this FACIT collagen in bone metabolism. In this study, we demonstrated the gene expression of collagen XII splice variants in human osteoblast-like cells, and their responses to IL-1β as a bone resorptive agent. Western blot analysis also showed the protein expression of both long- and short-form splice variants, α1(XIIA) and α1(XIIB) respectively, in osteoblast-like cells. When cells were cultured for 48 h with IL-1β, mRNA expression levels of collagen I, osteocalcin and whole collagen XII decreased compared with controls (p<0.01). Interestingly, after IL-1β stimulation, the level of α1(XII) chain decreased with time, although α1(XIIA) increased significantly by 2 h, and then decreased to a lower level compared with non-stimulated controls. Our results clarify that the collagen XII gene is transcribed in human osteoblast-like cells, and indicate that the transcription of splice variants may be influenced by the bone resorptive agent.
The use of FBS (Fetal Bovine Serum) is accompanied by a risk of disease transmission. The aim of this study was to evaluate autologous serum (AS) as an alternative culture medium for tissue engineering. Alkaline phosphatase (ALP) activity, DNA production (cell proliferation), and production of mineralized bone matrix were assessed when porcine mesenchymal stem cells (pMSCs) were cultured in autologous versus fetal bovine serum. Cells were cultured as follows: 1) DMEM (Dulbecco's modified high glucose eagle's medium), osteogenic supplements (OS: 100 nM dexamethasone, 50µg/ml ascorbic acid, and 10 mM beta-glycerophosphate), and 10% active autologous serum (AAS); 2) DMEM, OS, and 10% inactivated (heated) autologous serum (IAS); 3) DMEM, OS, and 10% FBS (control). At 3, 7, 14 and 21 days in culture, Alkaline phosphatase (ALP) activity, DNA production (cell proliferation), and production of mineralized bone matrix were measured. ALP activity and DNA production were greater at all time points in the FBS (control) group versus the experimental (AAS and IAS) groups. However, ALP activity relative to DNA production in the AAS and IAS groups was equal to the FBS group.
The purpose of this study was to evaluate gene expressions of human mesenchymal stem cells (hMSC) cultured in osteogenic differentiation medium (OM) which contained ascorbic acid, β-glycerophosphate and dexamethasone for 0 (control), 3, 5, 7, 14 and 21 days by 8.5k Genome Focus DNA microarray and real-time PCR. It was confirmed by the DNA microarray analysis that 327 genes of hMSC were significantly up-regulated by culture in OM especially at 14 and 21 days while 156 genes were down-regulated. Up-regulated genes included osteoblast-related genes such as secreted phosphoprotein 1 (osteopontin) gene and hypothetical protein expressed in osteoblast gene, along with angiogenesis-related genes and cell cycle arrestrelated genes, while down-regulated genes contained stroma-related genes and keratin-related genes. Expressions of several osteogenic differentiation marker genes such as (down-regulated) osteonectin gene and (up-regulated) BMP2 gene were also evaluated by real-time PCR. Gene expression database identified here might contribute to dental tissue engineering.
In the present study, we aimed to investigate the bone adaptability of commercially available biodegradable materials. Four natural polymer biodegradable materials, Avitene, Sponzel, Terdermis, Koken Tissue Guide and one synthetic polymer material, GC membrane, were immersed into Hanks' balanced salt solution (HBSS) without organic species with pH=7.4 for 14 days. HBSS was used as a simulated body fluid (SBF), and SBF immersion experiment was performed as preliminary experiment of in vivo animal experiment. Avitene and Sponzel have been dissolved in HBSS. Apatite formation was detected on Terdermis, Koken Tissue Guide and GC membrane. Micro X-ray diffraction measurement revealed the formation of hydroxyapatite on three biodegradable materials after HBSS immersion. We can make clear the biocompatibility of different biodegradable materials using simple HBSS immersion experiment.
In the present study, we attempted to mucosa reconstruction in an oral mucosa defect after removal of pleomorphic adenoma with the use of a cultured human oral epithelial cell sheet on human amniotic membrane (cell sheet), and obtained an excellent outcome. The patient was a 45-year-old female, in whom a hard movable tumor with a clear boundary, of which the size was the index finger tip, was detected immediately below the left mucosa of her upper lip. The lesion was diagnosed with a benign tumor in the minor salivary gland, and its removal was decided. To reconstruct the oral mucosa following tumor surgery, we planned treatment using the human cell sheet, and the patient gave consent after explanation of the surgical method and evidence safety. This surgical treatment was approved by the Human Studies Committee of Kyoto Prefectural University of Medicine. Punch biopsy was obtained for given oral epithelia, and produced a cell sheet by culturing the obtained oral epithelia on amniotic membrane for about 3 weeks. The cell sheet containing the amniotic membrane was sutured in the oral mucosa defect region, after removal of the tumor (10 x 7 mm), and fixed by the tie-over method. At present, about 3 months after surgery, neither contracture nor recurrence was detected, and the postoperative course was excellent.