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

11th ISABS CONFERENCE 201 Croat Med J. 2019;60:201-11 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 Rod1, Igor Matić2, Maja Antunović2, Vesna Vetma2, Ivan Pavičić3, Damir Hudetz1, Inga Marijanović2, Dragan Primorac1,4,5,6,7,8,9,10, Alan Ivković11,12,13 St. Catherine Specialty Hospital, Zabok/Zagreb, Croatia 1 Department of Molecular Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia 2 Institute for Medical Research and Occupational Health, Croatia 3 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. Eberly College of Science, The Pennsylvania State University, University Park, PA, USA 4 School of Medicine, University of Split, Split, Croatia 5 School of Medicine, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia 6 Faculty of Dental Medicine and Health Osijek, Josip Juraj Strossmayer University of Osijek, Osijek, Croatia 7 Faculty of Medicine, University of Rijeka, Rijeka, Croatia 8 Henry C. Lee College of Criminal Justice and Forensic Sciences, University of New Haven, West Haven, CT, USA 9 Children’s Hospital Srebrnjak, Zagreb, Croatia 10 Department for Orthopedic Surgery, University Hospital “Sveti Duh,” Zagreb, Croatia 11 Department of Biotechnology, University of Rijeka, Rijeka, Croatia 12 Department of Histology and Embryology, University of Zagreb School of Medicine, Zagreb, Croatia 13 Received: June 11, 2018 Accepted: March 13, 2019 Correspondence to: Eduard Rod St Catherine Specialty Hospital Bracak 8 49210 Zabok, Croatia www.cmj.hr 202 11th ISABS CONFERENCE Regenerative medicine looks for the ways to stimulate the inherent ability of our body to regenerate damaged tissue by activating or promoting natural healing processes, thus lowering the number of needed surgical or other medical interventions. It focuses on the interaction of cells, biological signals, and the environment (1). One of the most important topics in orthopedics is bone regeneration. Essential components needed for bone regeneration are osteogenic cells, osteoinductive growth factors, and an osteoconductive structure in the local environment (2,3). Various human cells have the potential to differentiate toward osteolineage cells. For example, bone-marrow derived mesenchymal stem cells (4) and stem cells derived from anterior cruciate ligament (ACL) remnant have been used to promote bone healing (5). Moreover, the insights into the creation of heterotopic bone (6) and the disease fibrodysplasia ossificans progressiva (7) have revealed a significant ability of human muscles to create bone. Muscle tissue contains various populations of progenitor cells: satellite, non-satellite, and perivascular cells. Non-satellite mesenchymal progenitors and perivascular cells play the main role in heterotopic bone formation (8). Although human muscle-derived cells can enhance bone regeneration (9), they often lack sufficient biological stimuli by growth factors, the most potent of which are bone morphogenetic proteins (BMP) -2, -4, and -7 (10-12). Successful osteogenesis depends on appropriate spatial and temporal expression of BMPs. BMP-2 has been used to promote bone regeneration (4,5,9), but it is soluble and disappears soon after implantation, so that many researchers have sought the delivery system to retain BMP-2 at the target site. This search led to the BMP-2-expressing viral vectors, as a concept representing gene therapy. The goal of gene therapy is to implement genetic material into cells, thus providing sufficient concentration of the missing substrate and alleviating the disease symptoms (13). This new genetic material is transferred to cells using virus vectors carrying genes needed for treatment. In recent years, many of these novel techniques have been developed for the treatment of musculoskeletal diseases, both for healing of bone and soft tissues (1,14-16). However, these methods have their limitations and need to be further improved. An example of this kind of treatment is the conventional ex vivo gene therapy, which was the initial modality of gene therapy and the first one used in clinical trials on humans (17). It consists of several steps: harvesting of cells from the body, in vitro cul- www.cmj.hr Croat Med J. 2019;60:201-11 tivation, infection of cells with virus vectors, and replantation of genetically modified cells back to the donor (13). Such a strategy has been proven successful, although not ideal for application during orthopedic procedures. Tissue transportation and cultivation in a laboratory requires two-steps surgical procedures, making the whole process expensive, long lasting, and inconvenient for the patient. To avoid such a complicated procedure, the expedited ex vivo strategies have been developed. The idea behind them is to perform a successful gene therapy inside the operating theater during a single procedure and without the need for extensive tissue cultivation. Several studies of gene therapy used for bone and cartilage healing proved this to be possible, but the whole process lasted minimally two hours (4,18). Although this is a significant step forward compared with the conventional ex vivo gene therapy, the average duration of orthopedic procedures is often shorter than two hours. Consequently, (...truncated)