This study was designed to test whether bisphosphonates disturb the process of fracture healing. Female SpragueDawley rats were injected with either two doses of bisphosphonate (incadronate) (10 µg/kg and 100 µg/kg) or vehicle three times a week for 2 weeks. Right femora were then fractured and fixed with intramedullary wires. Incadronate treatment was stopped in pretreatment groups (P-10 and P-100 groups), while the treatment was continued in continuous treatment groups (C-10 and C-100 groups). Animals were sacrificed at 6 and 16 weeks after surgery. Soft X-ray of all fractured femora was taken. After mechanical testing, fractured femora were stained in Villanueva bone stain and embedded in methyl methacrylate. Cross-sections near fracture line were analyzed by microradiography and histomorphometry. Radiographic study showed that bony callus was present in all the fractures and incadronate treatment led to a larger callus, especially in C-100 group at both 6 and 16 weeks. Histologic study showed that the process of fracture healing in pretreatment groups was delayed at 6 weeks, but reached control level thereafter and showed same characteristics as in control at 16 weeks. Woven bony callus could still be seen in continuous treatment groups at 16 weeks. Mechanical study indicated that the ultimate load of C-100 group was slightly higher than the other treatment groups and control. The results suggest that pretreatment with incadronate did not affect fracture healing at 16 weeks after fracture. However, continuous incadronate treatment could lead to larger callus, but it delayed remodeling process during fracture healing, especially with high-dose treatment. (J Bone Miner Res 1999;14:969-979)

The aim of this study was to investigate the long-term effect of incadronate on fracture healing of the femoral shaft in rats. Female Sprague-Dawley 8-week-old rats were injected subcutaneously (sc) with either vehicle (V group) or two doses of incadronate (10 g/kg and 100 g/kg) three times a week for 2 weeks. Right femoral diaphysis was then fractured and fixed with intramedullary stainless wire. Just after fracture, incadronate treatment was stopped in pretreatment groups (P groups: P-10 and P-100) or continued in continuous treatment groups (C groups: C-10 and C-100). All rats were killed at 25 weeks or 49 weeks after surgery. Fractured femur was evaluated radiologically and mechanically and then stained in Villanueva bone stain and embedded in methyl methacrylate. Undecalcified cross-sections from the fracture area were evaluated microradiologically and histomorphometrically. Radiographic observation showed that the fracture line disappeared in all groups. Cross-sectional area in the C-100 group was the biggest among all groups and in the C-10 group was larger than that in the V group at 25 weeks. Histological and histomorphometric observations showed that the process of fracture healing was delayed under continuous treatment with incadronate as evidenced by the delay of both lamellar cortical shell formation and resolution of original cortex in C groups. Percent linear labeling perimeter, mineral apposition rate (MAR), and bone formation rate (BFR) in C groups significantly decreased compared with the other groups, indicating that the callus remodeling was suppressed under continuous treatment, especially with a high dose. Mechanical study showed that the stiffness and ultimate load of the fractured femur in the C 100 group were the highest among all groups at both 25 weeks and 49 weeks. In conclusion, this study showed that long-term continuous treatment with incadronate delayed the process of fracture healing of femur in rats, especially under high dose but it did not impair the recovery of mechanical integrity of the fracture. (J Bone Miner Res 2001;16:429-436)

Effects of long-term suppression of bone remodeling by bisphosphonate were investigated in cortical bone of dog rib. Although microdamage was accumulated, BMD was increased without increasing cortical bone area. Consequently, the intrinsic material properties were not reduced.Introduction: Recently, we have reported that long-term suppression of bone remodeling increases microdamage accumulation but is not necessarily associated with vertebral fragility because of compensated increase of bone mass and improved microarchitecture. This study aimed to investigate the effect of long-term suppression of bone remodeling by bisphosphonate on the degree of mineralization, accumulation of microdamage, and mechanical properties of cortical bone in the same dogs. Materials and Methods: Twenty-nine 1-year-old beagles (15 males, 14 females) were divided into three groups and treated daily with vehicle (CNT) or with incadronate at a dose of 0.3 (LOW) or 0.6 mg/kg/day (HIGH) orally for 3 years. After death, pQCT, histomorphometry, microdamage measurements, and three-point bending mechanical test were performed using the ninth rib. Results: Cortical BMD was increased in the incadronate-treated groups. Cortical activation frequency was suppressed by 82% and 70% in HIGH and LOW, respectively, compared with CNT, without impairment of mineralization. Microdamage accumulation was increased in both incadronate-treated groups. Although there were no significant differences in total and cortical area among the three groups, structural mechanical properties were significantly increased after incadronate treatment while intrinsic material properties were not changed in the incadronate-treated groups. Conclusion: This study suggests that long-term suppression of bone remodeling by bisphosphonate increases microdamage accumulation. However, this was not necessarily associated with a reduction of intrinsic material properties probably because of an increased degree of mineralization.

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This study aimed to investigate the effect of long-term suppression of bone resorption by bisphosphonate on the microstructure, accumulation of microdamage, and mechanical properties of trabecular bone. Twentynine 1-year-old beagles (15 males, 14 females) were divided into three groups. The control group (CNT) was treated daily with vehicle, and the other two groups were treated with incadronate at a dose of 0.3 mg/kg/day (LOW) or 0.6 mg/kg/day (HIGH) orally for 3 years. After death, the second thoracic vertebra was scanned with microcomputed tomography (micro-CT) and assigned to histomorphometric and microdamage measurements. The fourth lumbar vertebra was mechanically tested by compression. Incadronate concentration in bone was measured in the 11th thoracic vertebra. Micro-CT analysis demonstrated a platelike trabecular structure and increased concave surface of trabeculae in the thoracic vertebra of incadronate-treated groups. Three-year incadronate treatment significantly suppressed trabecular activation rates by 56% in LOW and 67% in HIGH without impairment of mineralization, and increased microdamage accumulation in both incadronate-treated groups. Trabecular bone volume was significantly increased in both LOW and HIGH groups, and vertebral strength was significantly increased in the HIGH group compared with the CNT group. However, intrinsic material properties such as normalized ultimate stress and normalized toughness were reduced in incadronate-treated groups. Incadronate concentration in bone was dose-dependent. This study suggests that long-term suppression of bone remodeling increases microdamage accumulation, but this is not necessarily associated with vertebral fragility because of compensated increase of bone mass and improved microarchitecture. (J Bone Miner Res 2003;18:512-520)

The aim of the present study was to investigate effects of incadronate on early stages of fracture healing and to detect its concentration in callus area (Ca.Ar). Rats were injected three times per week with either two doses of incadronate (10 g/kg and 100 g/kg) or vehicle for 2 weeks. Femora were then fractured and fixed and animals were divided into pretreatment (P-10 and P-100) and continuous treatment (C-10 and C-100) groups. Incadronate treatment was stopped in P-10 and P-100 groups but continued in C-10 and C-100 groups. Animals were killed at 2 weeks and 4 weeks after fracture. Results showed significantly large callus, compared with the control, only in C-100 group at 4 weeks but not at 2 weeks. Both linear labeled surface (LS) and eroded surface (ES) decreased significantly in C-10 and C-100 groups at 2 weeks and 4 weeks. Osteoclast number (N.Oc) decreased significantly in C-10 and C-100 groups at 2 weeks but increased slightly at 4 weeks. However, there was no significant difference in the above parameters in P-10 and P-100 groups at 4 weeks. Apoptotic osteoclasts were observed only in the C-100 group at 4 weeks. A time-course decrease in incadronate concentration was detected in P-10 and P-100 groups whereas an increase was observed in C-10 and C-100 groups. These findings suggest that larger callus under incadronate treatment may result from the inhibition of bone resorption, histological characteristics of callus may be correlated with incadronate concentration, and metabolism of incadronate in bone may be related to the rate of bone turnover.

Interleukin (IL)-6, IL-11, leukemia inhibitory factor, and oncostatin M similarly induce osteoclast formation in cocultures of osteoblastic cells and bone marrow cells. These cytokines share a common signal transducer, gp130, which forms a receptor complex with the specific receptor for each cytokine. To investigate the role of gp130 in osteoclast development, we examined bone tissues in gp130-deficient and wild-type newborn mice of the ICR background. Soft x-ray radiographs and microfocus x-ray computed tomographs revealed that bone marrow cavities were present in tibiae and radii of both wild-type and gp130-deficient mice. Microfocus x-ray computed tomography and histological examination demonstrated a decrease in the amount of trabeculae at the metaphysial region in tibiae and radii of the gp130-deficient mice compared with the wild-type mice. The number ofosteoclasts in gp130-deficient mice was about double that in the wild-type mice. There were no apparent differences in the distributions of alkaline phosphatase-positive osteoblasts and the osteoid surface on the trabecular bone at the metaphysial region between the wild-type and gp130-deficient mice. The volume of mineralized trabecular bones was also decreased at mandibulae, accompanied by the increased number of osteoclasts in gp130-deficient mice compared with the wild-type and heterozygous mice. These results suggest that the formation of osteoclasts is not solely dependent on gp130 signaling, at least during fetal development. The osteoclastic bone resorption in gp130-deficient mice may be caused by the functional redundancy of bone-resorbing hormones and cytokines other than those of the IL-6 family.

The effects of bisphosphonate treatment schedule on fracture healing have not previously been tested. We evaluated the effect of ibandronate dosing interval duration on healing following surgical “fracture” (osteotomy) using a rat femoral fracture model. Six-week-old rats (n = 160) underwent osteotomy and were then allocated into vehicle control (CNT) or an ibandronate treatment group: 5 μg/kg daily (DAY, 5 days/week), 75 μg/kg once every 3 weeks (I-3), 150 μg/kg once every 6 weeks (I-6), resulting in the same total ibandronate dose over the study. Rats were killed after 6 or 18 weeks. At 18 weeks, all fracture lines had disappeared in the CNT and I-6 groups; approximately 10% of fracture lines remained in the DAY and I-3 groups. Ibandronate-treated groups showed large callus areas around the fractures, which shrank between 6 and 18 weeks after surgery; the extent of shrinkage decreased with shorter dosing interval. In histomorphometry, callus remodeling was suppressed by ibandronate; this became more apparent at shorter dose intervals. The structural properties of osteotomized femora were increased in the DAY group compared with CNT, but intrinsic material properties reduced inversely and became closer to those of CNT in response to increased dosing interval. Ibandronate induced formation of large calluses around osteotomies but delayed woven bone remodeling into lamellar bone and reduced intrinsic material properties in a rat fracture model. Extending the dosing interval of intermittent ibandronate treatment appeared to reduce the suppression of callus remodeling caused by ibandronate, which would have delayed healing after osteotomy.