Microbubble-mediated delivery of human adenoviruses does not elicit innate and adaptive immunity response in an immunocompetent mouse model of prostate cancer

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Lack of standardization of clinically compliant culture protocols of mesenchymal stem cells for re-implantation in humans have hindered clinical progress in the field of tissue regeneration to repair maxillofacial and orthopedic defects. The goal of this study was to establish a clinically relevant osteogenic protocol for collection and expansion of autologous stem cells to be used at Marshall University for re-implantation and repair of maxillofacial and orthopedic conditions. Human bone marrow (hBM) samples were collected from patients undergoing intramedullary nail fixation for closed femoral fractures. hBM mesenchymal cells were expanded by growing them first in Petri dishes for two weeks, followed by a week of culture using Perfecta 3D Hanging Drop Plates®. Various scaffold materials were tested and analyzed for cellular integration, vitality, and differentiation capacity of harvested hBM-MSCs including: 60/40 blend of hydroxyapatite biomatrix; Acellular bone composite discs; Allowash®, cancellous bone cubes; PLGA (poly lactic-co-glycolic acid); and Woven chitin derived fiber. We found that the 3D spheroid culture allowed production of hBM mesenchymal cells that retained osteoblast differentiation capacity over a monolayer culture of hBM-MSCs without the need to use chemical or hormonal modulation. We also observed that hydroxyapatite and Allowash cancellous bone scaffolds allowed better cell integration and viability properties as compared to other materials tested in this study. In conclusion, the multimodal culture methodology we developed creates actively differentiating stem-cell spheroids that can then be readily utilized in clinical practices to improve the regeneration of tissues of the head and the body

Planning C2 pedicle screw placement with multiplanar reformatted cervical spine computed tomography

Object: Careful preoperative planning with thin-slice computed tomography (CT) scan is useful for hardware placement at C2. Prior studies have shown considerable variability in the proportion of C2 vertebrae considered safe for pedicle screw placement, depending on the imaging technique used. Our work sought to more carefully define that proportion using a refined imaging technique on a large number of submillimeter CT scans. Materials and Methods: We reviewed 150 submillimeter cervical spine studies randomly selected from CT scans performed at a Level 1 trauma center. OsiriX™ image analysis software was used to propagate a 5-mm cylinder through the plane of the pedicle on paracoronal reformatted CT scans. Hounsfield unit attenuation was used to determine whether the cylinder violated the pedicle. Binomial data were generated to determine the proportion of pedicles that would allow safe screw placement. Results: We analyzed 300 pedicles in 150 patients. Using a standard C2 pedicle starting point, 32% of pedicles were breached by the 5-mm diameter cylinder. When screw trajectory was adjusted by moving the cylinder to fit the pedicle isthmus, establishing an optimized starting point, only 14% of pedicles were breached. Average pedicle length was 27.3 mm for screws that would have crossed the isthmus versus 13.2 mm for screws that would have stopped short due to potential breach. Conclusions: Findings of the current work suggest that preoperative imaging analysis or navigation can be useful adjuncts when anatomical variants are present

Microbubble-mediated delivery of human adenoviruses does not elicit innate and adaptive immunity response in an immunocompetent mouse model of prostate cancer

Abstract Background Gene transfer to malignant sites using human adenoviruses (hAds) has been limited because of their immunogenic nature and host specificity. Murine cells often lack some of the receptors needed for hAds attachment, thus murine cells are generally non-permissive for human adenoviral infection and replication, which limits translational studies. Methods We have developed a gene transfer method that uses a combination of lipid-encapsulated perfluorocarbon microbubbles and ultrasound to protect and deliver hAds to a target tissue, bypassing the requirement of specific receptors. Results In an in vitro model, we showed that murine TRAMP-C2 and human DU145 prostate cancer cells display a comparable expression pattern of receptors involved in hAds adhesion and internalization. We also demonstrated that murine and human cells showed a dose-dependent increase in the percentage of cells transduced by hAd-GFP (green fluorescent protein) after 24 h and that GFP transgene was efficiently expressed at 48 and 72 h post-transduction. To assess if our image-guided delivery system could effectively protect the hAds from the immune system in vivo, we injected healthy immunocompetent mice (C57BL/6) or mice bearing a syngeneic prostate tumor (TRAMP-C2) with hAd-GFP/MB complexes. Notably, we did not observe activation of innate (TNF-α and IL-6 cytokines), or adaptive immune response (neutralizing antibodies, INF-γ+ CD8+ T cells). Conclusions This study brings us a step closer to demonstrating the feasibility of murine cancer models to investigate the clinical translation of image guided site-specific adenoviral gene therapy mediated by ultrasound-targeted microbubble destruction

Microbubble-mediated delivery of human adenoviruses does not elicit innate and adaptive immunity response in an immunocompetent mouse model of prostate cancer

Background Gene transfer to malignant sites using human adenoviruses (hAds) has been limited because of their immunogenic nature and host specificity. Murine cells often lack some of the receptors needed for hAds attachment, thus murine cells are generally non-permissive for human adenoviral infection and replication, which limits translational studies. Methods We have developed a gene transfer method that uses a combination of lipid-encapsulated perfluorocarbon microbubbles and ultrasound to protect and deliver hAds to a target tissue, bypassing the requirement of specific receptors. Results In an in vitro model, we showed that murine TRAMP-C2 and human DU145 prostate cancer cells display a comparable expression pattern of receptors involved in hAds adhesion and internalization. We also demonstrated that murine and human cells showed a dose-dependent increase in the percentage of cells transduced by hAd-GFP (green fluorescent protein) after 24 h and that GFP transgene was efficiently expressed at 48 and 72 h post-transduction. To assess if our image-guided delivery system could effectively protect the hAds from the immune system in vivo, we injected healthy immunocompetent mice (C57BL/6) or mice bearing a syngeneic prostate tumor (TRAMP-C2) with hAd-GFP/MB complexes. Notably, we did not observe activation of innate (TNF-α and IL-6 cytokines), or adaptive immune response (neutralizing antibodies, INF-γ+ CD8+ T cells). Conclusions This study brings us a step closer to demonstrating the feasibility of murine cancer models to investigate the clinical translation of image guided site-specific adenoviral gene therapy mediated by ultrasound-targeted microbubble destruction