title: Human cartilage fragments in a composite scaffold for single-stage cartilage repair: an in vitro study of the chondrocyte migration and the influence of TGF-β1 and G-CSF.
abstract: OBJECTIVE
  Minced chondral fragments are becoming popular as a source of cells for cartilage repair, as a growing interest is developing towards one-stage procedures to treat cartilage lesions. The purpose of this study is to (A) compare cell outgrowth from cartilage fragments of adult and young donors using two different types of scaffolds and (B) evaluate the influence of transforming-growth-factor-β1 (TGF-β1) and granulocyte colony-stimulating factor (G-CSF) on chondrocyte behaviour.
  
  
  METHODS
  In part (A) cartilage fragments from adult and young donors were either loaded onto an HA-derivative injectable paste scaffold or onto an HA-derivative membrane scaffold. Construct sections were then examined for cell counting after 1, 2 and 3 months. In part (B) only membrane scaffolds were prepared using cartilage fragments from young donors. Constructs were cultured either in standard growth medium or in the presence of specific growth factors, such as TGF-β1 or G-CSF or TGF-β1 + G-CSF. After 1 month, construct sections were examined for cell counting. Expression of chondrocyte markers (SOX9, CD151, CD49c) and proliferative markers (β-catenin, PCNA) was assessed using immunofluorescence techniques, both in unstimulated construct sections and in cells from unstimulated and stimulated construct cultures.
  
  
  RESULTS
  Part (A): histological analysis showed age-dependent and time-dependent chondrocyte migration. A significant difference (p < 0.05) was observed between young and older donors at the same time point. No difference was detected between the two types of scaffolds within the same group at the same time point. Part (B): after 1 month, the number of migrating cells/area significantly increased due to exposure to TGF-β1 and/or G-CSF (p < 0.05). Immunofluorescence revealed that outgrowing cells from unstimulated scaffold sections were positive for SOX9, CD151, CD49c and G-CSF receptor. Immunofluorescence of cells from construct cultures showed an increase in β-catenin in all stimulated groups and an increased PCNA expression in G-CSF-exposed cultures (p < 0.05).
  
  
  CONCLUSIONS
  Outgrowing cells may represent a subset of chondrocytes undergoing a phenotypic shift towards a proliferative state. TGF-β1, and to a greater extent G-CSF, may accelerate this outgrowth. The clinical relevance of this study may involve a potential future clinical application of scaffolds preloaded with growth factors as an additional coating for chondral fragments. Indeed, a controlled delivery of G-CSF, widely employed in various clinical settings, might improve the repair process driven by minced human cartilage fragments during one-stage cartilage repair.
authors.affiliation: Department of Orthopaedics and Traumatology, University of Torino, Turin, Italy. antonio.marmotti@inwind.it
authors.forename: A
authors.initials: A
authors.lastname: Marmotti
authors.forename: D E
authors.initials: DE
authors.lastname: Bonasia
authors.forename: M
authors.initials: M
authors.lastname: Bruzzone
authors.forename: R
authors.initials: R
authors.lastname: Rossi
authors.forename: F
authors.initials: F
authors.lastname: Castoldi
authors.forename: G
authors.initials: G
authors.lastname: Collo
authors.forename: C
authors.initials: C
authors.lastname: Realmuto
authors.forename: C
authors.initials: C
authors.lastname: Tarella
authors.forename: G M
authors.initials: GM
authors.lastname: Peretti
mesh_terms: D000328:Adult; D000367:Age Factors; D002356:Cartilage / Q000166:cytology; D018929:Cell Culture Techniques; D002465:Cell Movement; D019902:Chondrocytes / Q000166:cytology* / Q000637:transplantation*; D005269:Femur / Q000601:surgery; D005455:Fluorescent Antibody Technique; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D006820:Hyaluronic Acid; D039422:Integrin alpha3 / Q000378:metabolism; D008875:Middle Aged; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000378:metabolism; D060185:Tetraspanin 24 / Q000378:metabolism; D013997:Time Factors; D054457:Tissue Scaffolds; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D055675:Viscosupplements; D051176:beta Catenin / Q000378:metabolism
chemical_list: C554715:CD151 protein, human; D039422:Integrin alpha3; D018809:Proliferating Cell Nuclear Antigen; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D060185:Tetraspanin 24; D053773:Transforming Growth Factor beta1; D055675:Viscosupplements; D051176:beta Catenin; D016179:Granulocyte Colony-Stimulating Factor; D006820:Hyaluronic Acid