Mouse segmental femoral graft model.
All animal studies were conducted in accordance with principles and procedures approved by the University of Rochester Committee for Animal Resources. We used 8-week-old C57BL/6 mice for femoral grafting as we have previously described13
. We cleaned allografts from ICR mice with 70% ethanol, rinsed them three times in saline to remove residual ethanol, and then froze them at −80 °C for at least 24 h before use. This procedure is based on the methods used by the Musculoskeletal Transplant Foundation to prepare clinical allografts. Graft healing was followed radiographically using a Faxitron X-ray system as described previously15
In vivo treatments.
For loss-of-function studies with biologics, we administered antibodies against VEGF (R&D Systems, Inc.), RANK:Fc fusion protein (a gift from Immunex, Inc.), or goat IgG (Sigma) in phosphate-buffered saline (PBS) by intraperitoneal injection every 3 d until mice were killed, as described previously31
. Ex vivo
gene transfer to live autografts were performed by harvesting the specimen, incubating it in 20 μl of sterile PBS containing 5 × 107
transducing units of rAAV for 10 min at room temperature and rapidly placing it back in the original donor. We performed in vivo
gene transfers to processed allografts by pipetting 5 × 107
particles of rAAV in 50 μl of a 1% sorbitol-PBS solution onto the cortical surface of the grafts. The allografts were then frozen at −80 °C, lyophilized and stored at −80 °C until they were transplanted. We used at least six mice in each treatment group.
Histological and histomorphometric analysis.
Following killing of mice, the grafted femurs were processed and stained with hematoxylin, eosin, Orange G and alcian blue (H&E), or for tartrate-resistant acid phosphatase (TRAP) activity and counterstained with hematoxylin as we have described previously31,32
. We performed X-gal (Sigma) staining on sections counterstained with eosin as we have described previously32
. Histomorphometric analysis was carried out using Osteometrics software as we have described previously13
Real-time quantitative RT-PCR assays and microarrays.
We harvested autografts and allografts from killed mice, immediately froze them in liquid nitrogen, minced them using a 6750 Freezer/Mill (SPEX CertiPrep, Inc.), and extracted total RNA using TRIsol (Invitrogen Corp.). We made single-stranded cDNA using a reverse transcription kit (Invitrogen) and used it as template for real-time PCR with SYBR Green PCR Master Mix (Applied Biosystems) and gene-specific primers in a Rotor-Gene 2000 (Corbett Research) as previously described15
. The mean cycle threshold (Ct) values from quadruplicate measurements were used to calculate the gene expression, with normalization to β-actin as an internal control. The primer sequences for Tnfsf11
are: forward, 5′-TCTCATAACCTGATGAAAGG-3′; reverse 5′-GCATCTTGATCCGGATCCAG-3′. The primer sequences for Vegfa
are: forward, 5′-GATGTGAATGCAGACCAAAG-3′; reverse, 5′-CACATCTGCAAGTACGTTCG-3′. The Functional Genomics Core Facility performed the microarray experiments, under the direction of A. Brooks. The experiments were performed by pooling the RNA extracted from six independent samples per group (autografts or allografts) in duplicate. Total RNA from day 10 samples were biotinylated and amplified using the T7 linear amplification approach previously described33
. Affymetrix m430_2.0 arrays, which represent approximately 45,000 mouse probe sets, were run following the manufacturer's protocol. Signal values were calculated using a probe level analysis normalization tool (Robust Multichip Analysis, RMA) before making pair-wise comparisons between allograft and autograft samples.
Preparation of rAAV vectors.
vectors have been described previously. Plasmids containing cDNA for Vegfa36
) and Flt1
) were used for subcloning into the pAAV-BGHA transfer vector using oligonucleotide primers containing restriction sites for Not
I and Eco
RI at the 5′ and 3′ end, respectively. After ligation and transformation, positive clones grown in E. coli
were confirmed by restriction digests and DNA sequencing. The resulting plasmids were used to produce the rAAVs through a helper-virus-free method, which were titered by dot blot39
. The function of each rAAV vector was verified by assessing protein production in vitro by enzyme-linked immunosorbent assay (ELISA; R&S systems) as described35
. The functional activities of the rAAV-RANKL and rAAV-VEGF vectors were also confirmed by in vitro
assays, respectively. In vivo
expression of VEGF and RANKL was assessed by serum ELISA as we have described previously35
. The transduction efficiency of rAAV-β-gal was determined in vitro by X-gal staining and by assaying for β-galactosidase activity using the Galacto-Light system (Tropix Inc.) as described previously34
Statistical analysis. An observer blinded to the treatment performed the histomorphometry. Data were calculated as the mean ± s.d., and the groups were compared using two-tailed analysis of variance (ANOVA). Statistical significance was set at P < 0.05.
Accession numbers. The GEO accession numbers for the primary data files are GSM37204 and GSM37205.