1. Volume 16, Issue 3, Pages 466-473 (March 2008)
Freeze-dried Tendon Allografts as Tissue-engineering Scaffolds for Gdf5 Gene Delivery 
Patrick Basile, Tulin Dadali, Justin Jacobson, Sys Hasslund, Michael Ulrich-Vinther, Kjeld Søballe, Yasuhiko Nishio, M Hicham Drissi, Howard N Langstein, David J Mitten, Regis J O'Keefe, Edward M Schwarz, Hani A Awad 
Molecular Therapy 
Volume 16, Issue 3, Pages (March 2008)
DOI: /sj.mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Transduction efficacy of freeze-dried tendon grafts in vitro and in vivo. (a) 3-mm Freeze-dried mouse flexor digitorum longus (FDL) tendon allografts were loaded with 5 × 109 transducing units of rAAV-lacZ, and incubated on a confluent monolayer of human embryonic kidney 293 cells for 48 hours (arrow). Representative micrographs of X-gal stained cultures show (b) large numbers of LacZ+ cells proximal to the graft, and (c) sparse staining in peripheral fields away from the graft. rAAV-lacZ loaded FDL allografts were also transplanted into FDL tendon defects of mice (n = 4). Representative micrographs of one end of the rAAV-lacZ loaded FDL allografts stained with antibodies against β-galactosidase at (d) 7 days after transplantation and (e) 14 days after transplantation. It is important to note the lack of viable cells and absence of any staining in the freeze-dried allografts (asterisks) that are surrounded by hypercellular and intensely stained fibrotic tissue. (c) The specificity of the staining was verified by the absence of non-specific staining in negative controls (f, secondary antibody only). S indicates remnants of the repair suture. rAAV, recombinant adeno-associated virus.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Kinetics and biodistribution of recombinant adeno-associated virus (rAAV)-mediated transduction through the use of processed tendon allografts. (a) Temporal bioluminescence images (BLIs) of a representative mouse grafted with a freeze-dried flexor digitorum longus tendon allograft loaded with rAAV-Luc, recorded over 21 days, show the localized biodistribution of rAAV-Luc transduction (heat map-yellow arrows) at the site of allograft implantation in the hind foot. (b) Kinetics of in vivo rAAV transduction, based on average BLI signal intensity, computed from measurements of total integrated light signal (photons emitted/cm2/s) emitted from a standardized region of interest in a standard 3-minute time interval (mean value ± SEM; n = 4).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Functional verification of the rAAV-Gdf5 vector. Human embryonic kidney 293 (HEK293) cells were grown in 6-well plates and transfected with: (1) pUC19, (2) pSPORT6-Gdf5, or (3) pAAV-Gdf5, and 48 hours later total RNA was harvested from the cells. The messenger RNA was reverse transcribed and used as the template for polymerase chain reaction (PCR) with Gdf5-specific primers. (4) The pSPORT-Gdf5 plasmid was used as template in the positive control. (a, top) The ethidium bromide–stained agarose gel shows the predicted 485-base pair PCR product. HEK293 cells were grown in 6-well plates and infected with the indicated amount of rAAV-lacZ or rAAV-Gdf5 (5.0 × 107 particles/ml). After 48 hours in culture, the supernatants were collected and 30 μl was used for Western blotting with GDF-5-specific antibodies. Ten nanograms of recombinant murine GDF-5 was used as a positive control. (a, bottom) Autoradiography of the Western blot reveals the predicted 13.7-kd GDF-5 protein. Microwound monolayer assay: (b) 80% confluent 3T3 cells were growth-arrested for 24 hours, and then microwounded by passing a pipette tip across the culture well and treated with 0.5% bovine calf serum (BCS) and 5.0 × 107 particles/ml of either rAAV-lacZ or rAAV-Gdf5. (c) The average width of the defect was digitally measured over time and the wound width normalized to the time zero width [w (t)/w (0)] versus time was plotted. (d) Healing time constants (τ) for the different treatments were computed and plotted as mean values ± SEM. Note that higher τ values indicate slower wound healing rates. (e) In a separate experiment, 3T3 cells grown to 80% confluence were microwounded and treated with 0.5% BCS and incremental doses of rmGDF-5. The data presented are mean values ± SEM for the healing time constant (τ) for the different doses of the GDF-5 protein treatments. Asterisks indicate significant differences (P < 0.01; n = 6 per treatment) compared to untreated controls. GDF-5, growth and differentiation factor 5.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
rAAV-Gdf5 loading of freeze-dried allografts improves the metatarsophalangeal (MTP) flexion range of motion and the gliding function of reconstructed flexor digitorum longus (FDL) tendons while maintaining their biomechanical properties. Mice had their FDL tendons reconstructed with freeze-dried allografts loaded with rAAV-Gdf5 (treated) or rAAV-lacZ (controls) and killed at 14 and 28 days after surgery (n = 9 per treatment per time point). The operated hind feet were removed and subjected to the MTP flexion test to determine (a) the MTP joint flexion range, and (b) the gliding coefficient. The tendons were then isolated and tested biomechanically to determine (c) their breaking (maximum) tensile force, and (d) their linear tensile stiffness. The data presented are mean values ± SEM. Asterisks indicate significant differences compared to time-matched controls (P < 0.05). GDF-5, growth and differentiation factor 5; rAAV, recombinant adeno-associated virus.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
rAAV-Gdf5 loading of freeze-dried allografts mediates de novo GDF-5 protein synthesis by the host cells at the periphery of the implanted allograft. Representative immunohistochemical sections of (a) the rAAV-lacZ-loaded and (b) the rAAV-Gdf5-loaded flexor digitorum longus tendon allografts at 14 days after grafting, stained with anti-mouse GDF-5 antibody. It is important to note the matrix-bound GDF-5 (positive staining indicated by arrows), presumably synthesized by the transduced host cells surrounding the rAAV-Gdf5-treated allografts (asterisk), that is absent in the rAAV-lacZ-treated graft. GDF-5, growth and differentiation factor 5; rAAV, recombinant adeno-associated virus.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
rAAV-Gdf5 loading of freeze-dried allografts mediates cellular repopulation of the graft and remodeling of the fibrotic scar tissue. Representative histological sections of (a,c,e) the rAAV-lacZ-loaded and (b,d,f) rAAV-Gdf5-loaded flexor digitorum longus (FDL) tendon allografts at 14 days after grafting, stained with Alcian Blue and Orange G. (a,b) Micrographs at ×4 show the implanted grafts with their anatomical relationships to the surrounding tissue. Boxed regions shown in the magnified micrographs (×20) depict (c,d) the distal ends of both grafts, and (e,f) the middle segment of the grafts. The tissues represented by numbers are: 1) talus, 2) tarsal bones, 3) metatarsal bone, 4) FDL tendon allograft, and 5) fibrotic/inflammatory tissue. S indicates remnants of suture. (f) Arrows indicate a remodeled tissue that appears to align and integrate with the rAAV-Gdf5-loaded allograft, and that also seems to have been repopulated with host cells compared to e the mostly acellular rAAV-lacZ-loaded allograft. rAAV, recombinant adeno-associated virus.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions