title: Characterization of platelet lysate cultured mesenchymal stromal cells and their potential use in tissue-engineered osteogenic devices for the treatment of bone defects.
abstract: Mesenchymal stromal cells (MSCs), seeded onto a scaffold and associated with platelet-gel, may represent an innovative treatment to improve bone repair. The preparation of MSCs for clinical use requires the fulfillment of Good Manufacturing Practice indications. The aim of this study was to validate a Good Manufacturing Practice-grade protocol of tissue engineering for bone regeneration, seeding platelet lysate (PL)-cultured MSCs onto an hydroxyapatite clinical-grade scaffold. Six large-scale experiments were performed. MSC expansions were performed comparing fetal bovine serum 10% and PL 5%. We demonstrated that PL lots contain high levels of growth factors possibly responsible of accelerated growth rate, since the number of colony-forming unit-fibroblast and population doublings were always significantly higher in PL cultures. MSCs were characterized for their phenotype and multilineage differentiation capacity, demonstrating appropriate features for both conditions. Gene expression analysis revealed higher expression of typical osteogenic genes of PL-cultured MSCs, when compared to fetal bovine serum MSCs. Cell transformation was excluded by analysis of karyotype, absence of growth without anchorage, and p53/c-myc gene expression. Scaffolds were precoated with retronectin before MSC seeding. MSC adhesion, distribution, and proliferation were demonstrated through the whole surface of the scaffold by scanning electron microscopy analysis or by immunofluorescence and MSC osteogenic differentiation through quantitative reverse transcriptase-polymerase chain reaction of typical osteogenic genes. The present report offers a model of an MSC-based bioengineered device, using an hydroxyapatite clinical-grade scaffold, and supports its potential use in tissue engineering to repair bone defects.
authors.affiliation: Laboratory of Cell Therapy Stefano Verri, Pediatric Department, Matilde Tettamanti Research Center, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy.
authors.forename: Agnese
authors.initials: A
authors.lastname: Salvadè
authors.forename: Pamela
authors.initials: P
authors.lastname: Della Mina
authors.forename: Diego
authors.initials: D
authors.lastname: Gaddi
authors.forename: Francesca
authors.initials: F
authors.lastname: Gatto
authors.forename: Antonello
authors.initials: A
authors.lastname: Villa
authors.forename: Marco
authors.initials: M
authors.lastname: Bigoni
authors.forename: Paolo
authors.initials: P
authors.lastname: Perseghin
authors.forename: Marta
authors.initials: M
authors.lastname: Serafini
authors.forename: Giovanni
authors.initials: G
authors.lastname: Zatti
authors.forename: Andrea
authors.initials: A
authors.lastname: Biondi
authors.forename: Ettore
authors.initials: E
authors.lastname: Biagi
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000737:chemistry*; D001847:Bone Diseases / Q000628:therapy; D001861:Bone Regeneration / Q000502:physiology; D018786:Bone Substitutes / Q000138:chemical synthesis* / Q000627:therapeutic use; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002457:Cell Extracts / Q000494:pharmacology*; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects; D009983:Orthopedic Equipment; D010012:Osteogenesis / Q000502:physiology; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects*; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D018786:Bone Substitutes; D002457:Cell Extracts; D003470:Culture Media