Genetic manipulation of human mesenchymal progenitors to promote chondrogenesis using "bead-in-bead" polysaccharide capsules.
Articular cartilage defects arising from trauma or degenerative diseases fail to repair spontaneously. We have adopted a non-viral gene delivery and tissue engineering strategy, in which Sox-9 transfected human mesenchymal progenitors have been encapsulated within alginate/chitosan polysaccharide capsules to promote chondrogenesis. Human bone marrow stromal cells and articular chondrocytes were transfected with flag-tagged Sox-9 plasmid and after 7 days in static culture, large regions of cell-generated matrix containing cartilage proteoglycans were observed as confirmed by positive Alcian blue staining and Sox-9 immunohistochemistry. Further, after 28 days, in vitro and in vivo, samples encapsulated with Sox-9 transfected cells demonstrated large regions of cartilaginous matrix as confirmed by positive Alcian blue staining, Sox-9 and type-II collagen immunohistochemistry, absent in samples encapsulated with untransfected cells. Extracted protein from in vivo constructs was further assessed by western blot analysis and positive expression of Sox-9 and type-II collagen was observed in Sox-9 transfected constructs which was absent in untransfected cells. Regions of cartilage-like matrix were significantly increased in Sox-9 constructs in comparison with untransfected constructs, confirming Sox-9 gene delivery enhances chondrogenesis in targeted cell populations, outlining the potential to promote cartilaginous construct formation with therapeutic implications for regeneration of human articular cartilage tissue defects.
Babister
Jodie C
JC
Bone and Joint Research Group, Developmental Origins of Health and Disease, Institute of Developmental Sciences, University of Southampton, General Hospital, Southampton, United Kingdom.
Tare
Rahul S
RS
Green
David W
DW
Inglis
Stefanie
S
Mann
Stephen
S
Oreffo
Richard O C
RO
D000464:Alginates / Q000737:chemistry; D001853:Bone Marrow / Q000378:metabolism; D002214:Capsules; D002454:Cell Differentiation; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000235:genetics*; D005109:Extracellular Matrix / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D015202:Protein Engineering; D055758:SOX9 Transcription Factor; D017154:Stromal Cells / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000464:Alginates; D002214:Capsules; D006603:Hexuronic Acids; D006609:High Mobility Group Proteins; D011134:Polysaccharides; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors; D020723:Glucuronic Acid