Izvorni znanstveni članak
https://doi.org/10.3325/cmj.2019.60.201
Optimization of an ex vivo gene transfer to the hamstrings tendons muscle remnants: potential for genetic enhancement of bone healing
Eduard Rod
; St. Catherine Specialty Hospital Zabok/Zagreb, Croatia
Igor Matić
; Department of Molecular Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia
Maja Antunović
; Department of Molecular Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia
Vesna Vetma
; Department of Molecular Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia
Ivan Pavičić
; Institute for Medical Research and Occupational Health, Croatia
Damir Hudetz
; St. Catherine Specialty Hospital Zabok/Zagreb, Croatia
Inga Marijanović
; Department of Molecular Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia
Dragan Primorac
; St. Catherine Specialty Hospital Zabok/Zagreb, Croatia
Alan Ivković
; Department for Orthopedic Surgery, University Hospital “Sveti Duh,” Zagreb Croatia,
Sažetak
Aim To 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.
Methods This 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). Results The 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.
Conclusion This study confirms that Ad.BMP-2 can transduce
human muscle tissue and direct it toward osteogenic
differentiation within 30 minutes.
Ključne riječi
Hrčak ID:
240029
URI
Datum izdavanja:
15.6.2019.
Posjeta: 555 *