ABSTRACT
The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.
Subject(s)
Adenoviridae/genetics , Alginates/chemistry , Cartilage, Articular/cytology , Chondrocytes/cytology , Extracellular Matrix Proteins/biosynthesis , Aged , Aged, 80 and over , Cell Culture Techniques/methods , Cells, Cultured , Chondrocytes/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Female , Femur Head/cytology , Femur Head/metabolism , Gene Expression Regulation , Genetic Markers , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Humans , Male , Middle Aged , Polymerase Chain Reaction , Statistics, Nonparametric , Transduction, Genetic , Transfection/methodsABSTRACT
INTRODUCTION: Factors which impair fracture healing after intramedullary (IM) nailing of long bone fractures range from surgical and biological factors to mechanical parameters. Mechanical parameters known to prolong bony consolidation are share forces at the site of the fracture. Fracture near press-on interlocking reduces share forces directly at the fracture site and is hypothesised to enhance callus mineralisation. A sheep model of midshaft tibia osteotomies evaluates the technique. MATERIALS AND METHODS: Fracture near interlocking was achieved by surfacing a custom made nail with special hutches that enable firm screw seating on top of the nail ("golf ball" structure). Virtual (fine element analysis (FEA)) and biomechanical pilot tests were completed before in vivo application in 12 adult female German black sheep. Midshaft tibia osteotomy was performed creating a subcritical 7 mm gap for delay in union. One group (n=6) was treated with reamed IM nailing employing the custom made nail and in addition to proximal and distal standard interlocking a fracture near press on interlocking was employed. A second group of six sheep without additional press on interlocking served as control. 10 weeks after operation the quality of fracture healing was determined by micro-CT. RESULTS: The FEA showed that axial loading up to 4000N did not lead to implant fatigue. Fracture near press on interlocking led to significantly more callus mineralisation compared to the conventional interlocking procedure (0.567 g/cm(3) ± 0.106 g/cm(3) versus 0.434 g/cm(3) ± 0.0836 g/cm(3), p=0.043). CONCLUSIONS: Fracture near press on interlocking increases callus mineralisation in a subcritical osteotomy model in sheep. The results indicate that the reduction of share forces at the fracture site after nailing procedures may be effective in reducing the time until bony consolidation.