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ABSTRACT: Urokinase plasminogen activator (uPA) regulates a proteolytic cascade of extracellular matrix degradation that functions in tissue development and tissue repair. The development and remodeling of the skeletal extracellular matrix during wound healing suggests that uPA might regulate bone development and repair. To determine whether uPA functions regulate bone development and repair, we examined the basal skeletal phenotype and endochondral bone fracture repair in uPA-deficient mice. The skeletal phenotype of uPA knockout mice was compared with that of control mice under basal conditions by dual-energy X-ray absorptiometry and micro-CT analysis, and during femur fracture repair by micro-CT and histological examination of the fracture callus. No effects of uPA gene deficiency were observed in the basal skeletal phenotype of the whole body or the femur. However, uPA gene deficiency resulted in increased fracture callus cartilage abundance during femur fracture repair at 14 days healing. The increase in cartilage corresponded to reduced tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts in the uPA knockout fracture callus at this time, consistent with impaired osteoclast-mediated remodeling of the fracture cartilage. CD31 staining was reduced in the knockout fracture tissues at this time, suggesting that angiogenesis was also reduced. Osteoclasts also colocalized with CD31 expression in the endothelial cells of the fracture tissues during callus remodeling. These results indicate that uPA promotes remodeling of the fracture cartilage by osteoclasts that are associated with angiogenesis and suggest that uPA promotes angiogenesis and remodeling of the fracture cartilage at this time of bone fracture repair.
Journal of Bone and Mineral Metabolism 05/2013; · 2.27 Impact Factor
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ABSTRACT: The present study aimed to develop a rat model of biceps tenodesis and to assess the feasibility of a lentiviral (LV)-based bone morphogenetic protein (BMP) 4 in vivo gene transfer strategy for healing of biceps tenodesis.
A rat model of biceps tenodesis was developed with an interference-fit open surgical technique. A LV vector expressing a BMP4 gene or β-galactosidase (β-gal) control gene was applied to the bone tunnel and the tendon graft before its insertion into the bone tunnel. Osteointegration was assessed by histology and pull-out tensile strength was measured by a biomechanical test suitable for small rat biceps tendon grafts.
Neo-chondrogenesis was seen at the tendon-bone interface of LV-BMP4-treated but not control rats. The LV-BMP4-treated rats showed 32% (p < 0.05) more newly-formed trabecular bone at the tendon-bone junction than the LV-β-gal-treated controls after 3 weeks. However, the sites of neo-chondrogenesis and new bone formation in the LV-BMP4-treated tenodesis were highly spotty. Although the LV-BMP4 strategy did not promote bony integration of the tendon graft, it yielded a 29.5 ± 11.8% (p = 0.066) increase in improvement the pull-out strength of rat biceps tendons compared to the LV-β-gal treatment after 5 weeks.
Although the LV-BMP4 in vivo gene transfer strategy did not enhance osteointegration of the tendon graft, it yielded a marked improvement in the return of the pull-out strength of the tendon graft. This presumably was largely a result of the bone formation effect of BMP4 that traps or anchors the tendon graft onto the bony tunnel.
The Journal of Gene Medicine 09/2011; 13(10):511-21. · 2.48 Impact Factor
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08/2011; , ISBN: 978-953-307-541-9
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ABSTRACT: This study sought to determine the role of the pro-apoptotic gene, Bax, in fracture healing by comparing femoral fracture healing in Bax knockout (KO) and wild-type C57BL/6J (background strain) mice. Bax KO fractures were larger, had more bone mineral content, had approximately 2-fold larger cartilage area per callus area in the first and second weeks of fracture healing, and showed an increased osteoclast surface area in the third and fourth weeks of fracture healing compared to C57BL/6J fractures. The increased cartilage area in the Bax KO fracture callus was due to increases in number of both pre-hypertropic and hypertropic chondrocytes. TUNEL analysis showed no significant differences in the number of either chondrocyte or non-chondrocyte apoptotic cells between Bax KO and C57BL/6J fractures at 7 or 14 days post-fracture, indicating that the increased number of chondrocytes in Bax KO fractures was not due to reduced apoptosis. Analysis of expression of apoptotic genes revealed that although the expression levels of Bcl-2 and Bcl-xL were not different between the Bax KO and C57BL/6J mice at 7 or 14 days post-fracture, the expression of BH3-domain only Bak and "Bik-like" pro-apoptotic gene increased approximately 1.5-fold and approximately 2-fold, respectively, in Bax KO fractures at 7 and 14 days post-fracture, compared to C57BL/6J fractures, suggesting that up-regulation of the Bak and Bik-like pro-apoptotic genes in Bax KO mice might compensate for the lack of Bax functions in the context of apoptosis. Analysis by in vivo incorporation of bromodeoxyuridine into chondrocytes within the fracture tissues indicated a highly significant increase in chondrocyte proliferation in Bax KO fractures compared to C57BL/6J fractures at day 7. The increased expression of collagen 2alpha1 and 9alpha1 gene in Bax KO fractures during early healing was consistent with an increased chondrocyte proliferation. In conclusion, this study demonstrates for the first time that Bax has an important role in the early stage of fracture healing, and that the increased callus size and cartilage area in Bax KO fractures was due to increased chondrocyte proliferation and not to reduced apoptosis or increased chondrocyte hypertrophy. The unexpected effect of Bax deficiency on chondrocyte proliferation implicates a novel regulatory function for Bax on chondrocyte proliferation during fracture repair.
Bone 08/2008; 43(5):880-8. · 4.02 Impact Factor
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ABSTRACT: An in vivo gene therapy strategy was developed to accelerate bone fracture repair.
Direct injection of a murine leukemia virus-based vector targeted transgene expression to the proliferating periosteal cells arising shortly after fracture. Cyclooxygenase-2 (Cox-2) was selected because the transgene for its prostaglandin products that promote angiogenesis, bone formation and bone resorption, are all required for fracture healing. The human (h) Cox-2 transgene was modified to remove AU-rich elements in the 3'-untranslated region and to improve protein translation.
In vitro studies revealed robust and sustained Cox-2 protein expression, prostaglandin E(2) and alkaline phosphatase production in rat bone marrow stromal cells and osteoblasts transgenic for the hCox-2 gene. In vivo studies in the rat femur fracture revealed that Cox-2 transgene expression produced bony union of the fracture by 21 days post-fracture, a time when cartilage persisted within the fracture tissues of control animals and approximately 1 week earlier than the healing normally observed in this model. None of the ectopic bone formation associated with bone morphogenetic protein gene therapy was observed.
This study represents the first demonstration that a single local application of a retroviral vector expressing a single osteoinductive transgene consistently accelerated fracture repair.
The Journal of Gene Medicine 04/2008; 10(3):229-41. · 2.48 Impact Factor
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ABSTRACT: Sex-dependent differences were identified in the femoral bone parameters of male and female ob/ob (leptin knockout) mice compared with their C57BL/6 wild-type background strain. Total fat, lean weight and body weight were not different between adult male and female leptin knockout mice. However, leptin knockout males exhibited lower lean weights than C57BL/6 males. Peripheral quantitative computerized tomographic measurements at the femoral midshaft revealed that the normal differences in the periosteal circumference, endosteal circumference, total bone mineral content, and polar moment of inertia normally observed between adult male and female wild-type mice were lost between adult male and female ob/ob mice. Significant reductions in these bone parameters were seen in male ob/ob mice compared to male wild-type mice but not in female ob/ob mice compared to female wild-type mice. In prepubertal mice, there were no differences in phenotype and femoral bone parameters between males and females within any strain, suggesting sex hormone functions. Serum free testosterone levels were 5.6-fold higher in adult male ob/ob mice than in adult male C57BL/6 wild-type mice, and serum estradiol levels were 1.8- and 1.3-fold greater in adult male and female ob/ob mice, respectively, than in their wild-type counterparts. Androgen receptor gene expression was not different in femur-derived bone cells of male ob/ob mice compared with wild-type mice. The loss of sex-related differences in these bone parameters in adult male ob/ob mice might result from deficient signaling in the androgen signaling pathway and the fact that leptin functions are permissive for androgen effects on bone development.
Calcified Tissue International 07/2007; 80(6):374-82. · 2.38 Impact Factor
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ABSTRACT: Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire were introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes was restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms.
Bone 05/2006; 38(4):521-9. · 4.02 Impact Factor
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ABSTRACT: Molecular Therapy (2006) 13, S308|[ndash]|S308; doi: 10.1016/j.ymthe.2006.08.882
794. Retroviral-Based Gene Therapy with Cyclo-Oxygenase-2 Promotes Bony Union and Accelerates Fracture Healing in the Rat
Charles H. Rundle1, Donna D. Strong1, Shin-Tai Chen1, Matilda H.-C. Sheng1, Jon E. Wergedal1, K.-H. William Lau1 and David J. Baylink11Musculoskeletal Disease Center, JL Pettis VAMC, Loma Linda, CA
Molecular Therapy 04/2006; · 6.87 Impact Factor
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ABSTRACT: This study sought to develop an in vivo gene therapy to accelerate the repair of bone fractures. In vivo administration of an engineered viral vector to promote fracture healing represents a potential high-efficacy, low-risk procedure. We selected a murine leukemia virus (MLV)-based retroviral vector, because this vector would be expected to target transgene expression to the proliferating periosteal cells arising shortly after bone fracture. This vector transduced a hybrid gene that consisted of a bone morphogenetic protein (BMP)-4 transgene with the BMP-2 secretory signal to enhance the secretion of mature BMP-4. The MLV vector expressing this BMP-2/4 hybrid gene or beta-galactosidase control gene was administered at the lateral side of the fracture periosteum at 1 day after fracture in the rat femoral fracture model. X-ray examination by radiograph and peripheral quantitative computed tomography at 7, 14, and 28 days after fracture revealed a highly significant enhancement of fracture tissue size in the MLV-BMP-2/4-treated fractures compared to the control fractures. The tissue was extensively ossified at 14 and 28 days, and the newly formed bone exhibited normal bone histology. This tissue also exhibited strong immunohistochemical staining of BMP-4. Additional control and MLV-BMP-2/4-treated animals each were monitored for 70 days to determine the fate of the markedly enhanced fracture callus. Radiographs showed that the hard callus had been remodeled and substantial healing at the fracture site had occurred, suggesting that the union of the bone at the fracture site was at least as high in the BMP-4-treated bone as in the control bone. There was no evidence of viral vector infection of extraskeletal tissues, suggesting that this in vivo gene therapy for fracture repair is safe. In summary, we have demonstrated for the first time that a MLV-based retroviral vector is a safe and effective means of introducing a transgene to a fracture site and that this procedure caused an enormous augmentation of fracture bone formation.
Bone 07/2003; 32(6):591-601. · 4.02 Impact Factor
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ABSTRACT: The spatial and temporal expression domains of the fibroblast growth factor receptor genes were examined in the healing rat femur fracture by in situ hybridization. Fibroblast growth factor receptor gene expression was detected in diverse fracture tissues throughout healing. Fibroblast growth factor receptor 1 and 2 expression was present throughout fracture repair, in the early proliferating periosteal mesenchyme, in the osteoblasts during intramembranous bone formation, and in the chondrocytes and osteoblasts during endochondral bone formation. Fibroblast growth factor receptor 3 expression colocalized with fibroblast growth factor receptor 1 and 2 expression in the chondrocytes and osteoblasts beginning at 10 days of healing, and persisted throughout endochondral bone formation. Fibroblast growth factor receptor 3 recapitulated its expression in fetal skeletal development, suggesting that it has a similar function in the control of endochondral bone growth during fracture repair. Fibroblast growth factor receptor 4 expression was not observed at any time. The extensive colocalized expression of the fibroblast growth factor receptors in healing indicates that fibroblast growth factor regulation of fracture callus maturation is extensive, and accurate identification of the receptor isoforms is necessary to establish the functions of fibroblast growth factor family members in fracture repair.
Clinical Orthopaedics and Related Research 11/2002; · 2.53 Impact Factor
