Optimization of an exvivo gene transfer to thehamstrings tendons muscleremnants: potential for geneticenhancement of bone healing

摘要

Aim To assess whether an adenoviral vector carrying thebone morphogenetic protein genes (Ad.BMP-2) can transducehuman muscle tissue and direct it toward osteogenicdifferentiation within one hour.Methods This in vitro study, performed at the Departmentof Molecular Biology, Faculty of Science, Zagreb from 2012to 2017, used human muscle tissue samples collected duringanterior cruciate ligament reconstructions performedin St Catherine Hospital, Zabok. Samples from 28 patientswere transduced with adenoviral vector carrying firefly luciferasecDNA (Ad.luc) by using different doses and timesof transduction, and with addition of positive ions fortransduction enhancement. The optimized protocol wasfurther tested on muscle samples from three new patients,which were transduced with Ad.BMP-2. Released bonemorphogenetic protein 2 (BMP-2) levels in osteogenic mediumwere measured every three days during a period of21 days. Expression of osteogenic markers was measuredat day 14 and 21. After 21 days of cultivation, muscle tissuewas immunohistochemically stained for collagen typeI detection (COL-I). Results The new transduction protocol was established using108 plaque-forming units (P 0.001) as an optimal doseof adenoviral vector and 30 minutes (P 0.001) as an optimalcontact time. Positive ions did not enhance transduction.Samples transduced with Ad.BMP-2 according to theoptimized protocol showed enhanced expression of osteogenicmarkers (P 0.050), BMP-2 (P 0.001), and COL I.Conclusion This study confirms that Ad.BMP-2 can transducehuman muscle tissue and direct it toward osteogenicdifferentiation within 30 minutes.