1. Volume 53, Issue 2, Pages 369-381 (April 2013)
Cellular and molecular mechanisms of accelerated fracture healing by COX2 gene therapy 
K.-H. William Lau, Vishal Kothari, Amitava Das, Xiao-Bing Zhang, David J. Baylink 
Bone 
Volume 53, Issue 2, Pages (April 2013)
DOI: /j.bone
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2. Fig. 1
Generation of multiple (three) tibial fractures in mice and local administration of viral vector expressing a modified human COX2 gene to the fracture site. Panel A is a representative radiographic image of three consecutive fractures (indicated by three parallel arrows) created at and around the mid-shaft of tibia by the three-point bending technique after insertion of the stabilizing pin inside the marrow cavity (indicated by the single arrow). Panel B is the X-ray picture showing the administration of a viral vector at or near the three fractures using a 30-G needle (indicated by the arrow). Panel C shows the relative expression levels of human COX2 mRNA in the fracture callus of lenti-COX2- or lenti-gfp-treated mice at 21days post-fracture. Results are shown as relative fold of COX2 mRNA in contralateral tibia and reported as mean±SEM, n=6 mice per treatment group (each qRT-PCR assay was done in duplicate). Statistical significance was determined by two-tailed Student's t-test.
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3. Fig. 2
COX2 gene therapy promotes bony bridging of fracture gaps and reduces callus size. Panel A shows representative radiographs of fractured tibia at day 7, 14, 17, or 21 post-fracture of lenti-COX2-treated mice (bottom panels) and control lenti-gfp-treated mice (top panels). Arrows indicate fracture gaps. At day 21 post fracture, 9 out of 10 lenti-gfp-treated control mice showed no radiographic evidence for bony bridging of the fracture gaps. In contrast, all 10 lenti-COX2-treated mice showed radiographic evidence for bony bridging. Panel B shows the mean±SEM (n=10 per treatment group) of the fracture callus size (measured by pQCT) at day 17 and 21 post fracture in fractures injected with lenti-COX2 or lenti-gfp vector. *P<0.05 by two-tailed Student's t-test. Two-way ANOVA: treatment effect, P<0.0001; time effect, P>0.05; and treatment×time interaction, P>0.05.
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4. Fig. 3
The smaller callus size in COX2-treated fractures was in a large part due to reduction in cartilage area within the callus. Top: Alcian blue staining of cartilaginous callus in healing fractures at 14, 17, and 21days post-fracture. Cartilage was stained in blue (indicated by the arrows). Bars=200μm. Bottom: Quantification of the cartilage area (reported as % of total callus area, mean±SEM, n=3 to 4 per group). *P<0.05 by two-tailed Student's t-test. Two-way ANOVA: treatment, P<0.0001; time, P>0.05; and interaction, P>0.05.
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5. Fig. 4
COX2 gene therapy markedly increases the number of osteocalcin-expressing osteoblasts (A), alkaline phosphatase (Alpl) mRNA expression (B), and osteopontin (Opn) mRNA expression (C) at day 14 to day 21 post-fracture. Panel A shows representative IHC staining of osteocalcin-expressing cells on thin sections of the healing fracture calluses of mice 14, 17, and 21days, respectively, after receiving the lenti-gfp or lenti-COX2 vectors. Bars=50μm. Bottom summarizes the number of osteocalcin positive cells along the woven bone surface counted in four randomly selected fields. Results are shown as the average number per 4 fields (mean±SEM, n=3 per treatment group). *P<0.05; **P<0.01; and ***P<0.001 by two-tailed Student's t-test. Two-way ANOVA: treatment, P<0.0001; time, P<0.0001; and treatment×time interaction, P< Panels B and C show the relative Alpl (B) or Opn (C) mRNA levels, respectively, each of which was determined by qRT-PCR and was normalized against the housekeeping gene, β-actin (Actb). Results are shown as fold of respective mRNA level in the contralateral intact tibia and reported as mean±SEM, n=3–6 per treatment group for both B and C. *P<0.05 by two-tailed Student's t-test. In panel B, two-way ANOVA of AlPl mRNA: treatment, P<0.001; time, P>0.05; treatment×time interaction, P>0.05. In panel C, two-way ANOVA of Opn mRNA: treatment, P<0.0001; time, P<0.0001; and treatment×time interaction, P<0.007.
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6. Fig. 5
Effects of fracture healing on Sdf1 mRNA expression (A), Cxcr4 mRNA (B) expression, and the relative size of SDF1-expressing cell population (C), and the COX2 gene therapy on expression levels of several MSC marker genes (D) during the early phase of the healing of tibial multiple fractures in the mouse. In panels A and B, bone and soft tissues of the healing callus of mice with multiple fractures in the tibia (and of corresponding area of the contralateral un-fractured tibia as internal control) of three to six mice per time-point were isolated. Total RNA was isolated and cDNA was synthesized. The mRNA levels of Sdf1 (A) and Cxcr4 (B) (normalized against Actb mRNA expression) were quantified in duplicate by qRT-PCR with primer sets specific for respective mouse gene. Results are shown as fold increase compared to corresponding unfractured control. Mean±SEM, n=4–6; *P<0.05 by two-tailed Student's t-test. In panel C, cells at the healing callus of four mice per treatment group were isolated by type I collagen digestion. The relative size of the SDF1-expressing cell population was analyzed by FACS at day 0, 4, and 7 post-fracture. Results are shown as percentage of the entire cell population (mean±SEM, n=4). *P<0.05 by two-tailed Student's t-test. In panel D, cells were isolated by type I collagen treatment from the 7-days healing calluses of fractured tibias of five to six mice each, treated with lenti-COX2 or lenti-gfp viral vector. Total RNA was isolated and cDNA was synthesized. The mRNA levels for three MSC marker genes (Nes, Poxdl, and Cd49f), normalized against Actb mRNA, were quantified by qPCR in duplicate with primer sets specific for respective mouse gene. Results are shown as fold increase compared to corresponding unfractured control. Mean±SEM, n=5–6; *P<0.05 by two-tailed Student's t-test.
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7. Fig. 6
The smaller callus size in COX2-treated fractures (A) was associated with reduced expression of Sox9 mRNA (B) and Col10α1 mRNA (C). In panel A, the callus size was measured with pQCT of fracture calluses of multiple fractures of lenti-gfp treated control mice and lenti-COX2-treated multiple fractures at day 14 post-fracture. Results are shown as mean±SEM, n=6 mice per group. In panel B, the Sox9 mRNA level in the fracture calluses of the same mice at day 14 post-fracture was measured by qRT-PCR. Results are shown as mean±SEM, n=3 mice per treatment group. In panel C, the Col10α1 mRNA at day 7, 14, 17, and 21 post-fracture was measured in healing calluses of lenti-gfp and lenti-COX2-treated fractures. Results are shown as relative fold of Col10α1 mRNA levels of contralateral intact tibia and reported as mean±SEM, n=4 per group. Two-way ANOVA: treatment, P<0.001, time, P<0.05; and treatment×time interaction, P<0.05. *P<0.05 by two-tailed Student's t-test.
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8. Fig. 7
COX2 gene therapy increased osteoclast/chondroclast number, resorption area, Acp5 mRNA expression, and Ctsk mRNA expression over controls in fracture calluses at late phases of fracture repair. Panel A shows representative histochemical staining of TRAP activity (stained reddish in color) on thin section of healing calluses of lenti-gfp-treated control fractures and lenti-COX2-treated fractures at day 14 and day 17 post-fracture, respectively. Bars=50μm. Panel B summarizes the number of TRAP positive, multinucleated osteoclast-like cells in four randomly selected fields within the calluses at day 14 or day 17 post-fractures of lenti-gfp- or lenti-COX2-treated mice, respectively. Results are shown as average number of osteoclast-like cells per 4 fields (mean±SEM, n=4–5 per group). Two-way ANOVA: treatment, P<0.001; time, P<0.001, and interaction, P>0.05. Panel C shows the relative TRAP positive area per total callus area. Results are shown as mean±SEM, n=4–5 per group. Two-way ANOVA: treatment, P<0.005, time, P<0.05, and interaction, P>0.05. Panels D and E are the relative expression levels of Acp5 mRNA and Ctsk mRNA, respectively. Results are shown as mean±SEM, n=4 per treatment group. Two-way ANOVA for Acp5 mRNA: treatment, P<0.01; time, P>0.05, and interaction, P>0.05. Two-way ANOVA for Ctsk mRNA: treatment, P<0.05; time, P<0.01; and interaction, P>0.05. *P<0.05 by two-tailed Student's t-test.
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9. Fig. 8
COX2 gene therapy promotes angiogenesis and vascularity (A) and Vegf and Vegfr mRNA expression (B) in healing fractures. In panel A, sections of fracture callus at day 17 post-fracture were immunohistochemically stained for CD31 (left), vWF (middle), and α-SMA (right) (brown in color, indicated by arrows in corresponding panel). Bars=200μm. In panel B, the mRNA expression levels of Vegfa and Vegfc and their receptors (Vegfr1 and Vegfr2) at the healing fractures at various time of healing (i.e., days 7, 14, 17, and 21 post-fracture, respectively) were measured by qRT-PCR and normalized against the housekeeping gene, Actb. Results are shown as relative folds of respective mRNA level in contralateral intact tibia, and are reported as mean±SEM, n=5 per time point per treatment group. *P<0.05 by two-tailed Student's t-test. Two-way ANOVA for Vegfa mRNA: treatment, P<0.05; time, P<0.05; and interaction, P>0.05; for Vegfc mRNA: treatment, P<0.005; time P<0.015; and interaction, P>0.05; for Vegfr1 mRNA: treatment, P<0.05; time, P>0.05; and interaction, P>0.05; and for Vegfr2 mRNA: treatment, P<0.05; time, P>0.05; and interaction, P<0.05.
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10. Fig. 9
Effect of endostatin on COX2-induced angiogenesis (A and B), callus size (C), TRAP-expressing osteoclasts within the remodeling callus (D), and osteocalcin-expressing osteoblasts area within the healing callus at day 17 post-fracture. Mice were injected locally with endostatin (2.5mg/kg/day, n=4) daily for 10days from day 4 to day 14 post-fracture. In A and B, angiogenesis and blood vessel formation were determined by IHC staining of vWF. Panel A shows IHC staining of vWF (stained brown) in fracture callus of a representative mouse each treated with lenti-gfp control vector or lenti-COX2 vector with or without the endostatin treatment. Bars=50μm. Panel B summarizes the blood vessel area determined by measuring the vWF-stained blood vessel area with the Osteometric system. Results are shown as mean±SEM (n=4 per treatment group). Panel C summarizes the effect of the endostatin treatment on the callus size measured at day 17 post-fracture of lenti-COX2-treated or lenti-gfp-treated control mice. Results are shown as mean±SEM (n=4 per treatment group). Panel D shows the effect of the endostatin treatment on the % TRAP stained area within the healing calluses at day 17 post-fracture. Left is a representative TRAP histochemical stained section of a healing callus at day 17 post-fracture of a mouse treated with lenti-COX2 alone or with the endostatin treatment. Bars=50μm. Right summarizes the % of TRAP-stained area per total callus area of fractures treated with lenti-COX2 or lenti-gfp with or without the endostatin treatment. Results are shown as mean±SEM, n=4 per treatment group. Panel E shows the relative lack of effect of the endostatin treatment on the relative osteocalcin IHC stained area with the fracture calluses of lenti-gfp or lenti-COX2-treated fracture at day 17 post-fracture. Bars=50μm. *P<0.05 by two-tailed Student's t-test in panels B, C, and D.
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11. Fig. 10
A proposed mechanism of COX2-induced acceleration of bony bridging of fracture gaps. In this model, Cox-2 increases the number of certain MSC subpopulations at the fracture site by increasing MSC recruitment and/or expansion primarily through increases local expression of Sdf1 and/or Cxcr4 during early phase of the fracture repair. At the same time, COX2 suppresses the chondrocytic differentiation of MSCs in part through suppression of Sox9 expression, leading to formation of smaller fracture callus. The COX2 gene therapy also enhances osteoblastic differentiation of MSCs to increase number of osteoblasts at the fracture site to promote woven bone formation during latter phases of the repair. Concurrently, COX2 promotes expression of VEGF-A/C and VEGFR-1/2 in cells at the fracture site that leads to increased angiogenesis and vascularization within the calluses, which results in increased recruitment of circulating hematopoietic stem cells and precursor cells that subsequently are induced to differentiation into functional osteoclasts/chondrocytes to mediate remodeling of the cartilaginous callus at later stages. During the intermediate stage of the repair, the combined actions of COX2 on reduction in cartilage formation, increase in woven bone formation, and increase in bony remodeling of cartilaginous calluses together convert the repair process to one that resembles more to intramembranous bone formation than endochondral bone formation. More importantly, the shortening of time involved in cartilage formation and remodeling will then result in substantial acceleration in the bony bridging of fracture gaps.
Bone  , DOI: ( /j.bone )
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