Flt-1 tyrosine kinase-deficient homozygous mice result in decreased trabecular bone volume with reduced osteogenic potential

To clarify the role of Fms-like tyrosine kinase-1 (Flt-1) signaling in bone dynamics, we examined C57BL/6J mice, aged 6, 9 and 16 weeks, with disruption of the flt1 tyrosine kinase domain gene (flt1^TK−/−) and compared with age-matched wild-type (flt1^TK+/+) mice.

Dynamic histomorphometric analysis confirmed a significant decrease in the values of mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) in the trabecular bone of the proximal tibiae of flt1^TK−/− mice compared with those in flt1^TK+/+ mice. The value of trabecular bone volume (BV/TV) was also significantly reduced in flt1^TK−/− mice compared with that in flt1^TK+/+ mice. The values of osteoclast surface (Oc.S/BS) and osteoclast number (Oc.N/BS) in flt1^TK−/− mice were somewhat lower than those in flt1^TK+/+ mice. The values of bending load of the femur significantly decreased in flt1^TK−/− mice. In addition, serum osteocalcin significantly decreased in flt1^TK−/− mice compared with those in flt1^TK+/+ mice. Furthermore, there was a significant decreased mineralization of bone marrow stromal cultures from flt1^TK−/− mice.

These findings demonstrate that flt1^TK−/− mice show lower trabecular bone volume than flt1^TK+/+ mice, providing powerful evidence that vascular endothelial growth factor signal pathway through the Flt-1 tyrosine kinase domain could be implicated in osteoblast development.     

Gene therapy with human osteoprotegerin decreases callus remodeling with limited effects on biomechanical properties

Osteoprotegerin (OPG) is a naturally occurring protein, which prevents bone resorption by inhibition of osteoclastogenesis, function, and survival. Therefore, recombinant OPG may be an attractive drug in the treatment of chronic bone resorptive diseases such as osteoporosis. Gene therapy has the potential to achieve long-term treatment by delivering genes of anti-resorptive proteins to the recipient. The effects of OPG gene therapy on fracture healing have not been described previously.

The influence of OPG gene therapy on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. Intramuscular administration of adeno-associated virus (AAV) vector-mediated OPG resulted in increased levels of OPG in serum of approximately 100 ng/ml throughout the study period. Control animals with fractures received transduction with an AAV reporter gene construct (AAV-enhanced green fluorescent protein (eGFP)), and in this group serum OPG levels remained at baseline (<10 ng/ml). After 3 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (33%, P < 0.001). However, AAV-OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or apparent callus tissue material properties. After 8 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (31%, P < 0.001) compared with AAV-eGFP fractures. Furthermore, deposition of new woven bone at the fracture line of the original cortical bone was hampered (new woven bone present: in all AAV-eGFP animals, in 41% of AAV-OPG-treated animals, P < 0.001). AAV-OPG treatment also increased callus BMC (18%, P = 0.023) compared with AAV-eGFP fractures. AAV-OPG did not influence callus dimensions, structural strength of the fractures, or ultimate stress, whereas elastic modulus was reduced in the AAV-OPG groups (37%, P = 0.039). The experiment demonstrates that AAV-OPG gene therapy decreases the fracture remodeling, but this does not influence the structural strength of healing fractures.     

Systemic leptin administration alters callus VEGF levels and enhances bone fracture healing in wildtype and ob/ob mice

IntroductionLeptinʼs role in bone formation has been reported, however, its mechanism of affecting bone metabolism is remaining unclear. In this study, we aimed to test whether leptin has a positive effect on fracture healing through the possible mechanism of increasing vascular endothelial growth factor (VEGF) expression in callus tissue.MethodsStandardized femur fractures were created in leptin-deficient ob/ob and wildtype C57BL/6J mice, and recombinant mouse leptin or its vehicle (physiological saline) was administered intraperitoneally during the study. Body weight, radiological, histologic and immunoblotting analyses were performed at different stages of fracture healing.Key findingsThe results showed that leptin treatment led to lower rate of body weight change in both mice genotypes. Radiological and histological analyses showed that the experimental groups had better fracture healing at 14, 21 and 28 days compared to the control groups. Leptin-treated groups had significantly higher VEGF expression in callus compared with the control groups at 2 and 3 weeks post-fracture except normal mice at 2 weeks, and leptin-deficient mice had higher VEGF levels in calluses than normal mice at the same timepoint.ConclusionLow-dose systemically-administered leptin has a positive effect on promoting fracture healing during the latter stages in a clinically-relevant mouse bone fracture model, and increase callus VEGF levels.     

Dsteoporosis influences the middle and late periods of racture healing in a rat osteoporotic model

OBJECTIVE: To evaluate the influence of osteoporosis on the middle and late periods of fracture healing process through observing the histomorphological changes, bone mineral density and biomechanical properties in ovariectomized rats. METHODS: Eighty-four female SD rats of 4 months old were randomly divided into osteoporosis group and sham operation group, 42 in each. Rats in osteoporosis group were performed ovariectomy operation while those in sham operation group were given sham operation. A midshaft tibia fracture model was established 10 weeks after ovariectomy. Tibias were harvested 2, 4, 6, 12, 18 weeks after fracture for bone mineral density, histomorphological and biomechanical evaluation. RESULTS: Compared with the sham operation group, callus bone mineral density was 12.8%, 18.0%, 17.0% lower in osteoporosis group 6, 12, 18 weeks after fracture, respectively (P<0.05); callus failure load was 24.3%, 31.5%, 26.6%, 28.8% lower in osteoporosis group, and callus failure stress was 23.9%, 33.6%, 19.1%, 24.9% lower in osteoporosis group 4, 6, 12, 18 weeks after fracture, respectively (P<0.05). In osteoporosis group, endochondral bone formation was delayed, more osteoclast cells could be seen around the trabecula, and the new bone trabecula arranged loosely and irregularly. CONCLUSIONS: Osteoporosis influences the middle and late periods of fracture healing in the rat osteoporotic model. The impairment is considered to be the result of combined effects of prolonged endochondral calcification, high activated osteoclast cell and the deceleration of the increase in bone mineral density.     

Increased fracture callus mineralization and strength in cathepsin K knockout mice

Cathepsin K (CatK) is a cysteine protease, expressed predominantly in osteoclasts (OC) which degrades demineralized bone matrix. Novel selective inhibitors of CatK are currently being developed for the treatment of postmenopausal osteoporosis. Pharmacological inhibition of CatK reduces OC resorption activity while preserving bone formation in preclinical models. Disruption of the CatK gene in mice also results in high bone mass due to impaired bone resorption and elevated formation. Here, we assessed mid-shaft femoral fracture healing in 8–10 week old CatK knock-out (KO) versus wild type (WT) mice. Fracture healing and callus formation were determined in vivo weekly via X-ray, and ex vivo at days 14, 18, 28 and 42 post-fracture by radiographic scoring, micro-computed tomography (μCT), histomorphometry and terminal mechanical four point bend strength testing. Radiological evaluation indicated accelerated bone healing and remodeling for CatK KO animals based on increased total radiographic scores that included callus opacity and bridging at days 28 and 42 post-fracture. Micro-CT based total callus volume was similar in CatK KO and WT mice at day 14. Callus size in CatK KO mice was 25% smaller than that in WT mice at day 18, statistically significant by day 28 and exhibited significantly higher mineralized tissue volume and volumetric BMD as compared to WT by day 18 onward. Osteoclast surface and osteoid surface trended higher in CatK KO calluses at all time-points and osteoblast number was also significantly increased at day 28. Increased CatK KO callus mineral density was reflected in significant increases in peak load and stiffness over WT at day 42 post-fracture. Regression analysis indicated a positive correlation (r = 0.8671; p < 0.001) between callus BMC and peak load indicating normal mineral properties in CatK KO calluses. Taken together, gene deletion of cathepsin K in mice accelerated callus size resolution, significantly increased callus mineralized mass, and improved mechanical strength as compared to wild type mice.     

Fractures in Geriatric Mice Show Decreased Callus Expansion and Bone Volume

Background

Poor fracture healing in geriatric populations is a significant source of morbidity, mortality, and cost to individuals and society; however, a fundamental biologic understanding of age-dependent healing remains elusive. The development of an aged-based fracture model system would allow for a mechanistic understanding that could guide future biologic treatments.

Questions/purposes

Using a small animal model of long-bone fracture healing based on chronologic age, we asked how aging affected (1) the amount, density, and proportion of bone formed during healing; (2) the amount of cartilage produced and the progression to bone during healing; (3) the callus structure and timing of the fracture healing; and (4) the behavior of progenitor cells relative to the observed deficiencies of geriatric fracture healing.

Methods

Transverse, traumatic tibial diaphyseal fractures were created in 5-month-old (n = 104; young adult) and 25-month-old (n = 107; which we defined as geriatric, and are approximately equivalent to 70–85 year-old humans) C57BL/6 mice. Fracture calluses were harvested at seven times from 0 to 40 days postfracture for micro-CT analysis (total volume, bone volume, bone volume fraction, connectivity density, structure model index, trabecular number, trabecular thickness, trabecular spacing, total mineral content, bone mineral content, tissue mineral density, bone mineral density, degree of anisotropy, and polar moment of inertia), histomorphometry (total callus area, cartilage area, percent of cartilage, hypertrophic cartilage area, percent of hypertrophic cartilage area, bone and osteoid area, percent of bone and osteoid area), and gene expression quantification (fold change).

Results

The geriatric mice produced a less robust healing response characterized by a pronounced decrease in callus amount (mean total volume at 20 days postfracture, 30.08 ± 11.53 mm³ versus 43.19 ± 18.39 mm³; p = 0.009), density (mean bone mineral density at 20 days postfracture, 171.14 ± 64.20 mg hydroxyapatite [HA]/cm³ versus 210.79 ± 37.60 mg HA/cm³; p = 0.016), and less total cartilage (mean cartilage area at 10 days postfracture, 101,279 ± 46,755 square pixels versus 302,167 ± 137,806 square pixels; p = 0.013) and bone content (mean bone volume at 20 days postfracture, 11.68 ± 3.18 mm³ versus 22.34 ± 10.59 mm³; p < 0.001) compared with the young adult mice. However, the amount of cartilage and bone relative to the total callus size was similar between the adult and geriatric mice (mean bone volume fraction at 25 days postfracture, 0.48 ± 0.10 versus 0.50 ± 0.13; p = 0.793), and the relative expression of chondrogenic (mean fold change in SOX9 at 10 days postfracture, 135 + 25 versus 90 ± 52; p = 0.221) and osteogenic genes (mean fold change in osterix at 20 days postfracture, 22.2 ± 5.3 versus 18.7 ± 5.2; p = 0.324) was similar. Analysis of mesenchymal cell proliferation in the geriatric mice relative to adult mice showed a decrease in proliferation (mean percent of undifferentiated mesenchymal cells staining proliferating cell nuclear antigen [PCNA] positive at 10 days postfracture, 25% ± 6.8% versus 42% ± 14.5%; p = 0.047).

Conclusions

Our findings suggest that the molecular program of fracture healing is intact in geriatric mice, as it is in geriatric humans, but callus expansion is reduced in magnitude.

Clinical Relevance

Our study showed altered healing capacity in a relevant animal model of geriatric fracture healing. The understanding that callus expansion and bone volume are decreased with aging can help guide the development of targeted therapeutics for these difficult to heal fractures.

Electronic supplementary material

The online version of this article (doi:10.1007/s11999-014-3829-x) contains supplementary material, which is available to authorized users.     

Osteoporosis influences the middle and late periods of fracture healing in a rat osteoporotic model

Osteoporosisischaracterizedbydecreasedbonemass, increasedbonefragilityinducedbydemolitionofbonemicrostructureandincreasedsusceptibilitytofracture. Currentstudiesmainlyfocusonthepreventionoffracture. However, theinfluenceofosteoporosisonthefracturehealingremainspoorlyunderstoodandcontroversial.Inourpreviousstudy, wehaveevaluatedtheeffectofosteoporosisontheearlyperiodoffracturehealing, andfoundthatosteoporosisinfluencesthequantityandqualityofcallusduringtheearlyperiodofracturehealing.1 Incurrent…     

Bax deficiency in mice increases cartilage production during fracture repair through a mechanism involving increased chondrocyte proliferation without changes in apoptosis

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 geometry and density in the skeleton of pre-pubertal gymnasts and school children

We have studied the differences between the peripheral and axial skeleton of pre-pubertal gymnasts and controls. We hypothesised that compared to controls, gymnasts would have larger and stronger radius and tibia diaphyses with greater bone mineral content and larger cross-sectional muscle area. At the distal metaphyseal sites of the radius and tibia, gymnasts would have greater bone cross-sectional area and total and trabecular volumetric bone mineral density (vBMD). Differences between the lumbar spine, total body and body composition in gymnasts versus controls were also studied. Peripheral quantitative computed tomography (pQCT) was used to measure bone geometry, density and muscle of the peripheral skeleton; dual energy X-ray absorptiometry (DXA) for total body and axial measurements.

Eighty-six pre-pubertal children, 44 gymnasts (mean age 9.0 years, range 5.4–11.9 years) and 42 controls (mean age 8.8 years, range 5.6–11.9 years) were studied. Eighty-four children were Caucasian, one child was mixed race, one Chinese. Data were adjusted for age, sex and height. Differences in the effect size between sexes were also tested.

At the 50% radius diaphysis gymnasts had larger bones (9.2%, p = 0.0054) with greater cortical area (8.2%, p = 0.022) and stress strain index (surrogate measure of bone strength) than controls (13.6%, p = 0.015). The effect size was different between males and females for cortical thickness (p = 0.03). At the 65% tibia diaphysis, gymnasts had greater cortical area (5.3%, p = 0.057) and thickness (6.2%, p = 0.068) than controls; consequently, bone strength was 5.4% higher (p = 0.14). There were no significant differences in cortical volumetric bone mineral density (vBMD) at the radius or tibia diaphysis between the groups. There was a difference in effect size for tibia muscle cross-sectional area between the sexes (p = 0.035). At the distal radius and tibia total and trabecular vBMD was greater (Total: radius 17%, p < 0.0001, tibia: 5.7%, p = 0.0053; trabecular: radius 21%, p < 0.0001, tibia 4.5%, p = 0.11). Bone size was not different in gymnasts compared to controls

Lumbar spine BMC (12.3%, p = 0.0007), areal bone mineral density (aBMD) (9.1%, p = 0.0006) and bone mineral apparent density (BMAD) (7.6%, p = 0.0047) were greater in gymnasts but vertebral size was not significantly different. Likewise, total body BMD (3.5%, p = 0.0057) and BMC (4.78%, p = 0.085) were greater in gymnasts but there were no differences in skeletal size.

These data suggest site-specific differences in how the pre-pubertal skeleton develops in response to the repetitive loading it experiences when participating in regular gymnastics. At diaphyseal sites these differences are predominantly in the bone and muscle geometry and not density. Conversely, at trabecular sites, the differences are increased density rather than geometry.

In conclusion, the present study has demonstrated skeletal differences between gymnasts and controls. These differences appear to be site and sex specific.