Gene mediated insulin-like growth factor-I delivery to the synovium.

The feasibility of articular gene therapy using insulin-like growth factor- I transgene expression in synovial tissues was assessed in vitro by transfe ction of synovial explant and monolayer cultures. Synovial membrane was har vested from horses and distributed for explant culture in multiwell plates or digested for monolayer culture in multiwell plates and chamber slides. S ynovial monolayers were cultured for 48 h after infection with 0, 100, 200, or 500 moi adenovirus-IGF-I (AdeIGF-I) to establish an optimum dose. Expla nts were then either infected with AdeIGF-I or adenoviral LacZ and cultured for 8 days, treated with 100 ng/ml recombinant IGF-I as a positive control , or remained as uninfected untreated culture controls. Expression of IGF-I in explants and monolayers was assessed by in situ hybridization and quant itative polymerase chain reaction (PCR), and translation confirmed by IGF-I radioimmunoassay (RIA) and tissue immunoreaction. Effects of IGF-I on syno vial function was assessed by proteoglycan and hyaluronan assay, and northe rn blot assessment of decorin and collagen type I expression. Significant t ransgene expression in synovial cells was present for all AdeIGF-I concentr ations. Similarly, medium IGF-I concentrations were significantly elevated in AdeIGF-I infected synovial monolayer and explant cultures at all time po ints. Peak IGF-I concentration of 246 +/- 43 ng/ml developed in explant cul tures on day 4; IGF-I levels in control explant groups were unchanged over baseline values. In situ hybridization and immunolocalization for IGF-I ind icated focal IGF-I expression in intimal and subintimal layers of infected explants, with diffuse immunoreaction throughout infected subintimal and fi brous layers. For monolayer cultures, intracellular immunoreaction to IGF-I was markedly higher in infected cells, and was most prominent at 100 moi. Effects of IGF-I on synoviocyte cultures were evident on northern blots, wh ich showed decreased decorin expression and elevated type I collagen produc tion in AdeIGF-I infected monolayers. Proteoglycan concentration in the med ium from explant cultures rose over the initial 4 days but was similar betw een treatment groups. The concentration of hyaluronan in medium from explan t cultures did not differ significantly within or between treated and contr ol groups during the 8-day study period. These data indicate that IGF-I can be successfully introduced to synovial structures by adenoviral vectors an d results in effective IGF-I ligand synthesis without untoward synovial mor phologic, effects. (C) 2001 Orthopaedic Research Society. Published by Else vier Science Ltd. All rights reserved.