title: Determination of effective rAAV-mediated gene transfer conditions to support chondrogenic differentiation processes in human primary bone marrow aspirates.
abstract: The genetic modification of freshly aspirated bone marrow may provide convenient tools to enhance the regenerative capacities of cartilage defects compared with the complex manipulation of isolated progenitor cells. In the present study, we examined the ability and safety of recombinant adeno-associated virus (rAAV) serotype 2 vectors to deliver various reporter gene sequences in primary human bone marrow aspirates over time without altering the chondrogenic processes in the samples. The results demonstrate that successful rAAV-mediated gene transfer and expression of the lacZ and red fluorescent protein marker genes were achieved in transduced aspirates at very high efficiencies (90-94%) and over extended periods of time (up to 125 days) upon treatment with hirudin, an alternative anticoagulant that does not prevent the adsorption of the rAAV-2 particles at the surface of their targets compared with heparin. Application of rAAV was safe, displaying neither cytotoxic nor detrimental effects on the cellular and proliferative activities or on the chondrogenic processes in the aspirates especially using an optimal dose of 0.5 mg ml(-1) hirudin, and application of the potent SOX9 transcription factor even enhanced these processes while counteracting hypertrophic differentiation. The current findings demonstrate the clinical value of this class of vector to durably and safely modify bone marrow aspirates as a means to further develop convenient therapeutic approaches to improve the healing of cartilage defects.
authors.affiliation: Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Rey-Rico
authors.affiliation: Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Frisch
authors.affiliation: Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany.
authors.forename: J K
authors.initials: JK
authors.lastname: Venkatesan
authors.affiliation: Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Schmitt
authors.affiliation: 1] Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany [2] Department of Orthopaedic Surgery, Saarland University Medical Center, Homburg/Saar, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Madry
authors.affiliation: Center of Experimental Orthopaedics, Saarland University Medical Center, Homburg/Saar, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Cucchiarini
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000502:physiology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D020219:Chondrogenesis; D000229:Dependovirus / Q000235:genetics*; D017930:Genes, Reporter; D006493:Heparin / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D061251:Primary Cell Culture; D055758:SOX9 Transcription Factor / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes; D000097573:Red Fluorescent Protein
chemical_list: D000925:Anticoagulants; D006493:Heparin; D006629:Hirudins; D008164:Luminescent Proteins; D055758:SOX9 Transcription Factor; D000097573:Red Fluorescent Protein; C527932:SOX9 protein, human