title: Human dental pulp stem cells expressing transforming growth factor β3 transgene for cartilage-like tissue engineering.
abstract: OBJECTIVE
  The aim of this study was to engineer sizable three-dimensional cartilage-like constructs using stem cells isolated from human dental pulp stem cells (DPSCs).
  
  
  METHODS
  Human DPSCs were isolated from teeth extracted for orthodontic treatment and enriched further using immuno-magnetic bead selection for stem cell marker CD146. Chondrogenic lineage differentiation of DPSCs induced using recombinant transforming growth factor β3 (TGFβ3) was verified by pellet culture. Because the use of recombinant proteins is associated with rapid degradation and difficult in vivo administration, we constructed the recombinant adeno-associated viral vector encoding human TGFβ3 and determined the best multiplicity of infection for DPSCs. Transduced DPSCs were seeded on poly-l-lactic acid/polyethylene glycol (PLLA/PEG) electrospun fiber scaffolds demonstrating proper attachment, proliferation and viability as shown by scanning electron microscopy micrographs and CCK-8 cell counting kit. Scaffolds seeded with DPSCs were implanted in the back of nude mice.
  
  
  RESULTS
  Transduced DPSCs highly expressed human TGFβ3 for up to 48 days and expressed chondrogenic markers collagen IIa1, Sox9 and aggrecan, as verified by immunohistochemistry and messenger RNA (mRNA). Immunohistochemistry for TGFβ3/DPSC constructs (n = 5/group) showed cartilage-like matrix formation with glycosaminoglycans. In vivo constructs with TGFβ3/DPSCs showed higher collagen type II and Sox9 mRNA expression relative to non-transduced DPSC constructs (n = 5/group). Western blot analysis confirmed this expression pattern on the protein level (n = 3/group).
  
  
  CONCLUSIONS
  Immuno-selected DPSCs can be successfully differentiated toward chondrogenic lineage, while expressing the chondrogenic inducing factor. Seeded on PLLA/PEG electrospun scaffold, human DPSCs formed three-dimensional cartilage constructs that could prove useful in future treatment of cartilage defects.
authors.affiliation: Department of Orthodontics, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China. a.s.ortho@gmail.com
authors.forename: Ahmed
authors.initials: A
authors.lastname: Rizk
authors.forename: A Bakr M
authors.initials: AB
authors.lastname: Rabie
mesh_terms: D000818:Animals; D002356:Cartilage / Q000166:cytology / Q000254:growth & development*; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage; D020219:Chondrogenesis / Q000187:drug effects*; D003782:Dental Pulp / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects; D023822:Tissue Engineering; D053782:Transforming Growth Factor beta3 / Q000235:genetics / Q000494:pharmacology; D019076:Transgenes
chemical_list: D011994:Recombinant Proteins; D053782:Transforming Growth Factor beta3