Paolo Arrigoni1,2,3, Jacob C Ruprecht1,4, Dawn A D Chasse1, Katherine A Glass4, Benjamin Andress5, Farshid Guilak6,7, J Brice Weinberg8,9, Amy L McNulty1,5. 1. Department of Orthopaedic Surgery, Duke University School of Medicine, Durham, NC, USA. 2. Orthopedics and Traumatology Clinic, IRCCS Policlinico San Matteo Foundation, Locomotor System Diseases Unit, University of Pavia, Pavia, Italy. 3. Department of Clinical Surgical, Diagnostic and Pediatric Sciences, Locomotor System Diseases Unit, University of Pavia, Pavia, Italy. 4. Department of Biomedical Engineering, Duke University, Durham, NC, USA. 5. Department of Pathology, Duke University School of Medicine, Durham, NC, USA. 6. Department of Orthopaedic Surgery, Washington University, St. Louis, MO, USA. 7. Shriners Hospital for Children, St. Louis, MO, USA. 8. Department of Medicine, Duke University School of Medicine, Durham, NC, USA. 9. VA Medical Center, Durham, NC, USA.
Abstract
OBJECTIVES: The utilization of viral vectors to deliver genes of interest directly to meniscus cells and promote long-term modulation of gene expression may prove useful to enhance meniscus repair and regeneration. The objective of this study was to optimize and compare the potential of lentivirus (LV) and adeno-associated virus (AAV) to deliver transgenes to meniscus cells in both intact meniscus tissue and isolated primary cells in monolayer. DESIGN: Porcine meniscus tissue explants and primary meniscus cells in monolayer were transduced with LV or self-complementary AAV2 (scAAV2) encoding green fluorescent protein (GFP). Following transduction, explants were enzymatically digested to isolate meniscus cells, and monolayer cells were trypsinized. Isolated cells were analyzed by flow cytometry to determine percent transduction. RESULTS: LV and scAAV2 showed a high transduction efficiency in monolayer meniscus cells. scAAV2 was most effective at transducing cells within intact meniscus tissue but the efficiency was less than 20%. Outer zone meniscus cells were more readily transduced by both LV and scAAV2 than the inner zone cells. Higher virus titers and higher cell density resulted in improved transduction efficiency. Polybrene was necessary for the highest transduction efficiency with LV, but it reduced scAAV2 transduction. CONCLUSIONS: Both LV and scAAV2 efficiently transduce primary meniscus cells but only scAAV2 can modestly transduce cells embedded in meniscus tissue. This work lays the foundation for viral gene transfer to be utilized to deliver bioactive transgenes or gene editing machinery, which can induce long-term and tunable expression of therapeutic proteins from tissue-engineered constructs for meniscus repair and regeneration.
OBJECTIVES: The utilization of viral vectors to deliver genes of interest directly to meniscus cells and promote long-term modulation of gene expression may prove useful to enhance meniscus repair and regeneration. The objective of this study was to optimize and compare the potential of lentivirus (LV) and adeno-associated virus (AAV) to deliver transgenes to meniscus cells in both intact meniscus tissue and isolated primary cells in monolayer. DESIGN: Porcine meniscus tissue explants and primary meniscus cells in monolayer were transduced with LV or self-complementary AAV2 (scAAV2) encoding green fluorescent protein (GFP). Following transduction, explants were enzymatically digested to isolate meniscus cells, and monolayer cells were trypsinized. Isolated cells were analyzed by flow cytometry to determine percent transduction. RESULTS: LV and scAAV2 showed a high transduction efficiency in monolayer meniscus cells. scAAV2 was most effective at transducing cells within intact meniscus tissue but the efficiency was less than 20%. Outer zone meniscus cells were more readily transduced by both LV and scAAV2 than the inner zone cells. Higher virus titers and higher cell density resulted in improved transduction efficiency. Polybrene was necessary for the highest transduction efficiency with LV, but it reduced scAAV2 transduction. CONCLUSIONS: Both LV and scAAV2 efficiently transduce primary meniscus cells but only scAAV2 can modestly transduce cells embedded in meniscus tissue. This work lays the foundation for viral gene transfer to be utilized to deliver bioactive transgenes or gene editing machinery, which can induce long-term and tunable expression of therapeutic proteins from tissue-engineered constructs for meniscus repair and regeneration.
Authors: Chisa Hidaka; Clemente Ibarra; Jo A Hannafin; Peter A Torzilli; Mannix Quitoriano; Shih-Shi Jen; Russell F Warren; Ronald G Crystal Journal: Tissue Eng Date: 2002-02
Authors: Andre F Steinert; Glyn D Palmer; Ramille Capito; Jochen G Hofstaetter; Carmencita Pilapil; Steven C Ghivizzani; Myron Spector; Christopher H Evans Journal: Tissue Eng Date: 2007-09