Seong-Ho Jin1, HaeYong Kweon2, Jun-Beom Park3, Chang-Hyen Kim4. 1. Department of Periodontics, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea. 2. Department of Agricultural Biology, National Academy of Agricultural Science, Rural Development Administration, Suwon, Republic of Korea. 3. Department of Periodontics, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea. Electronic address: jbassoonis@yahoo.co.kr. 4. Department of Oral and Maxillofacial Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea. Electronic address: omfskim1@nate.com.
Abstract
BACKGROUND: The main objective of this study was to investigate the effect of tetracycline-loaded silk fibroin membranes (TC-SFMs) on the proliferation and the osteogenic differentiation of human mesenchymal stem cells. MATERIALS AND METHODS: Four groups (0, 1, 5, and 10% concentration) of TC-SFMs were prepared for the experiments. We investigated cumulative tetracycline (TC) release profile for 7 d. Human gingiva-derived mesenchymal stem cells (GMSCs) were isolated from our previous study and seeded to the TC-SFMs. WST-8 assay (Cell Counting Kit-8; SigmaeAldrich Co, St. Louis, MO), staining of Phalloidin-FITC, and scanning electron microscope analyzed the cellular attachment and viability. Staining of Alizarin Red S (Sigma-Aldrich Co.) and osteogenic marker (osteocalcin) analyzed osteogenic differentiation. Additionally, quantitative polymerase chain reaction measured the expression of osteogenic lineage genes, including bone gamma-carboxyglutamic acid protein, bone sialoprotein, runt-related transcription factor 2, and collagen type I α1 according to TC concentration (0.05, 0.1, 0.25, and 0.5 mg/mL). RESULTS: The release of TC from TC-SFMs plateaued and neared completion in 24 h. Significantly higher viability was noted achieved in 1% and 5% TC-SFMs. The morphology of GMSCs on TC-SFMs at 0% and 1% concentration showed spindle shapes, but cells in 10% TC-SFMs appeared spheroid. During Alizarin Red S staining at 21 d of osteogenic differentiation, calcium and osteocalcin formation were significantly lower in the 10% TC-SFM group than in the 0, 1, and 5 groups. Compared with the control group, bone gamma-carboxyglutamic acid protein showed significantly low expression rate at TC concentration ≥0.05 mg/mL. Bone sialoprotein was low at TC concentration ≥0.1 mg/mL. Likewise, runt-related transcription factor 2 and collagen type I α1 were low at TC concentration of 0.5 mg/mL. CONCLUSIONS: Within the limits of this study, 1% and 5% TC-SFMs showed higher proliferation and osteogenic potential of GMSCs than 10% TC-SFM. Therefore, the use of 1% to 5% range of TC may be more suitable to silk fibroin membrane for stem cell tissue engineering.
BACKGROUND: The main objective of this study was to investigate the effect of tetracycline-loaded silk fibroin membranes (TC-SFMs) on the proliferation and the osteogenic differentiation of human mesenchymal stem cells. MATERIALS AND METHODS: Four groups (0, 1, 5, and 10% concentration) of TC-SFMs were prepared for the experiments. We investigated cumulative tetracycline (TC) release profile for 7 d. Human gingiva-derived mesenchymal stem cells (GMSCs) were isolated from our previous study and seeded to the TC-SFMs. WST-8 assay (Cell Counting Kit-8; SigmaeAldrich Co, St. Louis, MO), staining of Phalloidin-FITC, and scanning electron microscope analyzed the cellular attachment and viability. Staining of Alizarin Red S (Sigma-Aldrich Co.) and osteogenic marker (osteocalcin) analyzed osteogenic differentiation. Additionally, quantitative polymerase chain reaction measured the expression of osteogenic lineage genes, including bone gamma-carboxyglutamic acid protein, bone sialoprotein, runt-related transcription factor 2, and collagen type I α1 according to TC concentration (0.05, 0.1, 0.25, and 0.5 mg/mL). RESULTS: The release of TC from TC-SFMs plateaued and neared completion in 24 h. Significantly higher viability was noted achieved in 1% and 5% TC-SFMs. The morphology of GMSCs on TC-SFMs at 0% and 1% concentration showed spindle shapes, but cells in 10% TC-SFMs appeared spheroid. During Alizarin Red S staining at 21 d of osteogenic differentiation, calcium and osteocalcin formation were significantly lower in the 10% TC-SFM group than in the 0, 1, and 5 groups. Compared with the control group, bone gamma-carboxyglutamic acid protein showed significantly low expression rate at TC concentration ≥0.05 mg/mL. Bone sialoprotein was low at TC concentration ≥0.1 mg/mL. Likewise, runt-related transcription factor 2 and collagen type I α1 were low at TC concentration of 0.5 mg/mL. CONCLUSIONS: Within the limits of this study, 1% and 5% TC-SFMs showed higher proliferation and osteogenic potential of GMSCs than 10% TC-SFM. Therefore, the use of 1% to 5% range of TC may be more suitable to silk fibroin membrane for stem cell tissue engineering.