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700. Localized Lentivirus Delivery Using a Modified Collagen Gel
TISSUE ENGINEERING &OTHER CELL THERAPIES I Tissue Engineering & Other Cell Therapies I 699. Smad8/BMP-2 Engineered Mesenchymal Stem Cells Induce Rapid Achilles Tendon Full Defect Regeneration
Ayelet Ben-Arav,1 Jess G. Snedeker,2 Yoram Zilberman,1 Zulma Gazit,1,3 Ralph Müller,4 Gadi Pelled,1,3 Dan Gazit.1,3 1 Skeletal Biotech Lab, Hebrew University of Jerusalem, Jerusalem, Israel; 2University of Zurich, Balgrist, Zurich, Switzerland; 3 Department of Surgery, Cedars Sinai Medical Center, Los Angeles, CA; 4ETH, Zürich, Switzerland. Tendon tissue regeneration continues to be an important goal in the field of orthopedic medicine. We have previously shown that overexpressing Smad8 and BMP-2 genes in C3H10T1/2 MSC line induced tenogenic differentiation. Furthermore, Smad8/BMP-2 engineered MSCs were able to regenerate a partial defect in a rat Achilles tendon (AT). In the present study we investigated the ability of these cells to regain the mechanical function of a fully resected AT. We hypothesized that the implantation of Smad8/BMP-2 modified MSCs in a full defect of AT would induce tissue regeneration leading to improved biomechanical properties. AT were separated from the plantaris and soleus tendons. A full 2mm defect was created in the distal tendon region. AT was either implanted with genetically engineered (GE) MSCs, non-engineered (NE) MSCs, fibrin gel with no cells (FG), or sutured together (natural healing). The cells were labeled with CM-DiI in order to allow cell detection in histological analysis and were suspended in fibrin gel prior to implantation. Three weeks post implantation the tendons were excised and processed for further histological or biomechanical analyses. Mechanical tests were performed with a universal testing machine. The tensile test was displacement controlled at 10 mm/min. Tendons were preconditioned with 10-cycle preloading to 10% nominal strain before testing to failure. H&E staining was performed on histology sections.
Figure 1: Biomechanical analysis. Stiffness (A) and Elastic modulus (B).
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Figure 2. Histological analysis using H&E staining (A-D, F). A. Intact tendon. B. Natural healing. C. FG only. D. GE MSCs. E. Fluorescent microscopy imaging of CM-DiI-labeled GE MSCs. F. NM MSCs. Biomechanical analysis of healing AT showed that tendons implanted with GM MSCs showed the highest effective stiffness (70% greater than natural healing, p<0.02, Fig. 1A) and elastic modulus (Fig. 1B). Other mechanical parameters such as force, stress and maximum load showed no significant differences between the groups. Histological analysis revealed tendon-like morphology with elongated cells in the GE MSCs group (Fig. 2). Fluorescence microscopy showed that CM-DiI-labeled cells were present at the repair site at the time of sacrifice (Fig. 2E). This is the first study showing improved functional recovery of Achilles tendon regenerated by gene-modified MSCs.
700. Localized Lentivirus Delivery Using a Modified Collagen Gel
Seungjin Shin,1 Lonnie D. Shea.1 Chemical and Biological Engineering, Northwestern University, Evanston, IL. 1
Gene therapy with recombinant lentiviral vectors has been widely applied. However, the localization of viral vector delivery to the target sites remains a challenge. We have investigated the use of hydrogels to localize delivery of lentiviral vectors, and modifying the hydrogel with binding sites that retain the vector locally and maintain its activity. Collagen hydrogels loaded with lentiviral vectors released the vectors locally, with the release controlled by the properties of the gel. The presence of the gel retained the activity of the virus relative to the absence of a gel, and increasing the solids content of the hydrogel served to limit release. Cells seeded onto the lentiluciferase encapsulating hydrogels were readily infected; however, the extent of luciferase production was affected by the cell migration rate and the transduction efficiency was increased as the cells migrate into the hydrogels. We subsequently investigated the incorporation of binding sites within the hydrogel that can function to limit release of lentivirus. Cells were seeded onto lentivirus loaded hydrogels into which hydoxylapatite(HA) particles or Phosphatidylserine(PS) containing microspheres were incorporated. HA particles and PS microspheres decreased the number of infected cells outside of the hydrogel. Association of the lentivirus with HA and PS microspheres increased the activity of the complexes, with HA increasing the virus activity for 72 hours. In vivo studies with collagen and HA-loaded collagen demonstrated sustained and localized transgene expression for at least 4 weeks with the maximum expression level at two weeks after implantation. Interestingly, expression significantly increased with the HA-loaded collagen relative to the collagen alone. Taken together, these studies demonstrate the ability to efficiently deliver lentiviral vectors from collagen hydrogels to yield both in vitro and in vivo transgene expression, which would have broad applicability to numerous applications in regenerative medicine and in model systems that investigate tissue formation. S267
Ayelet Ben-Arav,1 Jess G. Snedeker,2 Yoram Zilberman,1 Zulma Gazit,1,3 Ralph Müller,4 Gadi Pelled,1,3 Dan Gazit.1,3 1 Skeletal Biotech Lab, Hebrew University of Jerusalem, Jerusalem, Israel; 2University of Zurich, Balgrist, Zurich, Switzerland; 3 Department of Surgery, Cedars Sinai Medical Center, Los Angeles, CA; 4ETH, Zürich, Switzerland. Tendon tissue regeneration continues to be an important goal in the field of orthopedic medicine. We have previously shown that overexpressing Smad8 and BMP-2 genes in C3H10T1/2 MSC line induced tenogenic differentiation. Furthermore, Smad8/BMP-2 engineered MSCs were able to regenerate a partial defect in a rat Achilles tendon (AT). In the present study we investigated the ability of these cells to regain the mechanical function of a fully resected AT. We hypothesized that the implantation of Smad8/BMP-2 modified MSCs in a full defect of AT would induce tissue regeneration leading to improved biomechanical properties. AT were separated from the plantaris and soleus tendons. A full 2mm defect was created in the distal tendon region. AT was either implanted with genetically engineered (GE) MSCs, non-engineered (NE) MSCs, fibrin gel with no cells (FG), or sutured together (natural healing). The cells were labeled with CM-DiI in order to allow cell detection in histological analysis and were suspended in fibrin gel prior to implantation. Three weeks post implantation the tendons were excised and processed for further histological or biomechanical analyses. Mechanical tests were performed with a universal testing machine. The tensile test was displacement controlled at 10 mm/min. Tendons were preconditioned with 10-cycle preloading to 10% nominal strain before testing to failure. H&E staining was performed on histology sections.
Figure 1: Biomechanical analysis. Stiffness (A) and Elastic modulus (B).
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Figure 2. Histological analysis using H&E staining (A-D, F). A. Intact tendon. B. Natural healing. C. FG only. D. GE MSCs. E. Fluorescent microscopy imaging of CM-DiI-labeled GE MSCs. F. NM MSCs. Biomechanical analysis of healing AT showed that tendons implanted with GM MSCs showed the highest effective stiffness (70% greater than natural healing, p<0.02, Fig. 1A) and elastic modulus (Fig. 1B). Other mechanical parameters such as force, stress and maximum load showed no significant differences between the groups. Histological analysis revealed tendon-like morphology with elongated cells in the GE MSCs group (Fig. 2). Fluorescence microscopy showed that CM-DiI-labeled cells were present at the repair site at the time of sacrifice (Fig. 2E). This is the first study showing improved functional recovery of Achilles tendon regenerated by gene-modified MSCs.
700. Localized Lentivirus Delivery Using a Modified Collagen Gel
Seungjin Shin,1 Lonnie D. Shea.1 Chemical and Biological Engineering, Northwestern University, Evanston, IL. 1
Gene therapy with recombinant lentiviral vectors has been widely applied. However, the localization of viral vector delivery to the target sites remains a challenge. We have investigated the use of hydrogels to localize delivery of lentiviral vectors, and modifying the hydrogel with binding sites that retain the vector locally and maintain its activity. Collagen hydrogels loaded with lentiviral vectors released the vectors locally, with the release controlled by the properties of the gel. The presence of the gel retained the activity of the virus relative to the absence of a gel, and increasing the solids content of the hydrogel served to limit release. Cells seeded onto the lentiluciferase encapsulating hydrogels were readily infected; however, the extent of luciferase production was affected by the cell migration rate and the transduction efficiency was increased as the cells migrate into the hydrogels. We subsequently investigated the incorporation of binding sites within the hydrogel that can function to limit release of lentivirus. Cells were seeded onto lentivirus loaded hydrogels into which hydoxylapatite(HA) particles or Phosphatidylserine(PS) containing microspheres were incorporated. HA particles and PS microspheres decreased the number of infected cells outside of the hydrogel. Association of the lentivirus with HA and PS microspheres increased the activity of the complexes, with HA increasing the virus activity for 72 hours. In vivo studies with collagen and HA-loaded collagen demonstrated sustained and localized transgene expression for at least 4 weeks with the maximum expression level at two weeks after implantation. Interestingly, expression significantly increased with the HA-loaded collagen relative to the collagen alone. Taken together, these studies demonstrate the ability to efficiently deliver lentiviral vectors from collagen hydrogels to yield both in vitro and in vivo transgene expression, which would have broad applicability to numerous applications in regenerative medicine and in model systems that investigate tissue formation. S267