Optimization of an ex vivo gene transfer to the hamstrings tendons muscle remnants: potential for genetic enhancement of bone healing

Abstract

AimTo assess whether an adenoviral vector carrying the bone morphogenetic protein genes (Ad.BMP-2) can transduce human muscle tissue and direct it toward osteogenic differentiation within one hour.MethodsThis in vitro study, performed at the Department of Molecular Biology, Faculty of Science, Zagreb from 2012 to 2017, used human muscle tissue samples collected during anterior cruciate ligament reconstructions performed in St Catherine Hospital, Zabok. Samples from 28 patients were transduced with adenoviral vector carrying firefly luciferase cDNA (Ad.luc) by using different doses and times of transduction, and with addition of positive ions for transduction enhancement. The optimized protocol was further tested on muscle samples from three new patients, which were transduced with Ad.BMP-2. Released bone morphogenetic protein 2 (BMP-2) levels in osteogenic medium were measured every three days during a period of 21 days. Expression of osteogenic markers was measured at day 14 and 21. After 21 days of cultivation, muscle tissue was immunohistochemically stained for collagen type I detection (COL-I).ResultsThe new transduction protocol was established using 108 plaque-forming units (P < 0.001) as an optimal dose of adenoviral vector and 30 minutes (P < 0.001) as an optimal contact time. Positive ions did not enhance transduction. Samples transduced with Ad.BMP-2 according to the optimized protocol showed enhanced expression of osteogenic markers (P < 0.050), BMP-2 (P < 0.001), and COL I.ConclusionThis study confirms that Ad.BMP-2 can transduce human muscle tissue and direct it toward osteogenic differentiation within 30 minutes.