title: The effects of tetracycline-loaded silk fibroin membrane on proliferation and osteogenic potential of mesenchymal stem cells.
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.
  
  
  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.
authors.affiliation: Department of Periodontics, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
authors.forename: Seong-Ho
authors.initials: SH
authors.lastname: Jin
authors.affiliation: Department of Agricultural Biology, National Academy of Agricultural Science, Rural Development Administration, Suwon, Republic of Korea.
authors.forename: HaeYong
authors.initials: H
authors.lastname: Kweon
authors.affiliation: Department of Periodontics, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea. Electronic address: jbassoonis@yahoo.co.kr.
authors.forename: Jun-Beom
authors.initials: JB
authors.lastname: Park
authors.affiliation: Department of Oral and Maxillofacial Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea. Electronic address: omfskim1@nate.com.
authors.forename: Chang-Hyen
authors.initials: CH
authors.lastname: Kim
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005349:Fibroins / Q000494:pharmacology*; D005881:Gingiva / Q000166:cytology; D006801:Humans; D008422:Materials Testing; D008567:Membranes, Artificial; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects*; D010012:Osteogenesis / Q000187:drug effects*; D047011:Silk / Q000494:pharmacology*; D013752:Tetracycline / Q000494:pharmacology*; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D008567:Membranes, Artificial; D047011:Silk; D005349:Fibroins; D013752:Tetracycline
keywords: Fibroins; Gingiva; Mesenchymal stromal cells; Multipotent stem cells; Silk; Tetracycline; Tissue engineering