title: Attachment, Viability and Adipodifferentiation of Pre-adipose Cells on Silk Scaffolds with and Without Co-expressed FGF-2 and VEGF.
abstract: OBJECTIVE
  For application of stem cells and progenitor cells in regenerative medicine, scaffolds for carrying the cells play a key role. One promising biomaterial for scaffold generation is silk because of its mechanical strength, good cytocompatibility and low immunogenicity. Furthermore, bioengineering of silk proteins enable co-expression of various growth, differentiation and angiogenic factors on silk fibers, which may promote cell growth, differentiation and angiogenesis. This study aimed to test cytocompatibility and growth/differentiation of pre-adipose cells on scaffolds with and without expressed growth factors fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF).
  
  
  METHODS
  Disk-form scaffolds of 15×3 mm (diameter × thickness) were manufactured in two different densities using silk with and without expressed growth factors FGF-2 or VEGF. Pre-adipose cells were prepared from fatty tissues of patients undergoing operation. Cells (1.6×10(6)) were seeded onto each of the silk-scaffold disks, that were placed into wells of 12-well culturing plates. Adipose-differentiation was induced using differentiation medium containing DMEM/F-12, insulin, pantothenate, biotin, triiodothyronine (T3), transferrin, dexamethasone, isobuthylmethylxanthine and rosiglitazone. Cells on the scaffolds were visualized using a confocal microscope. Viability and adiponectin were measured on days 0, 7 and 14. Expression of adipose-differentiation markers was assessed by means of real-time polymerase chain reaction (RT-PCR).
  
  
  RESULTS
  Pre-adipose cells attached well onto the silk fibers. The highest initial viability was measured on the low-density scaffolds with expressed VEGF. Adipose-differentiation was evident in visible oil droplets and significantly increased adiponectin protein levels were seen in ELISA. Furthermore, increased expression of adipose-differentiation genes were measured in RT-PCR. Adipose-differentiation was more profound in cells on high-density scaffolds. In concordance, viability of cells on high-density scaffolds did not increase, while that of cells on low-density scaffolds doubled over the 14-day experimental period. Slightly enhanced adipose-differentiation was observed in cells on scaffolds with expressed FGF-2 or VEGF.
  
  
  CONCLUSIONS
  Silk scaffolds exhibit excellent cytocompatibility for human pre-adipose cells and have application potential in tissue engineering and regenerative medicine. VEGF and FGF-2 expressed on silk fibers could have a potential positive effect on pre-adipose cells, while the effect of VEGF should be further addressed in vivo.
authors: Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany h.hanken@uke.de.
authors: Henning
authors: H
authors: Hanken
authors: Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Friedemann
authors: F
authors: Göhler
authors: Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Ralf
authors: R
authors: Smeets
authors: Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Max
authors: M
authors: Heiland
authors: Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Alexander
authors: A
authors: Gröbe
authors: Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Reinhard E
authors: RE
authors: Friedrich
authors: General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Philipp
authors: P
authors: Busch
authors: Department of Plastic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Marco
authors: M
authors: Blessmann
authors: Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Lan
authors: L
authors: Kluwe
authors: Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Philip
authors: P
authors: Hartjen
mesh_terms: D000273:Adipose Tissue / Q000187:drug effects*; D001672:Biocompatible Materials / Q000737:chemistry / Q000627:therapeutic use; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D044968:Regenerative Medicine; D047011:Silk / Q000737:chemistry / Q000627:therapeutic use*; D023822:Tissue Engineering; D054457:Tissue Scaffolds / Q000009:adverse effects; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001672:Biocompatible Materials; D047011:Silk; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2
keywords: Adipose tissue engineering; adipocyte differentiation; incorporated growth factors; pre-adipose cells; silk fibroin scaffolds