Yoshio Kaji

Effect of Bisphosphonate (Incadronate) on Fracture Healing of Long Bones in Rats

This study was designed to test whether bisphosphonates disturb the process of fracture healing. Female Sprague‐Dawley 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.

Long-Term Effect of Incadronate Disodium (YM-175) on Fracture Healing of Femoral Shaft in Growing Rats

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 μg/kg and 100 μg/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.

Suppressed Bone Turnover by Long-Term Bisphosphonate Treatment Accumulates Microdamage but Maintains Intrinsic Material Properties in Cortical Bone of Dog Rib†

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.

Long-Term Treatment of Incadronate Disodium Accumulates Microdamage but Improves the Trabecular Bone Microarchitecture in Dog Vertebra

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. Twenty‐nine 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.

Concentration of bisphosphonate (Incadronate) in callus area and its effects on fracture healing in rats

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 μg/kg and 100 μg/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.

Preadministration of Incadronate Disodium Can Prevent Bone Loss in Rat Proximal Tibial Metaphysis When Induced by Hindlimb Immobilization by Bandage

The purpose of this study is to determine whether short-term preadministration of bisphosphonates prevents bone loss in rat proximal tibial metaphysis when induced by hindlimb immobilization by bandage. Six-month-old female Sprague Dawley rats were injected with incadronate disodium (YM-175, 10 micrograms/kg) or vehicle, three times per week for 2 weeks (YM or V groups). Then, the left hindlimb was fixed to the abdomen with a bandage (V-B, YM-B groups), or only the abdomen was bandaged as control (V-SHM, YM-SHM groups), for 4 weeks. The animals were subsequently killed and left proximal tibiae were processed undecalcified for quantitative histomorphometric evaluation. Immobilization-induced cancellous bone loss resulted not only from increased percent eroded surface area but also from decreased percent labeling surface and bone formation rate in V-B compared with V-SHM animals. In contrast, preadministration of YM-175 decreased percent eroded surface significantly and prevented the loss of cancellous bone mass in YM-B compared with V-B animals. Cancellous bone mass was neither increased nor decreased by preadministration of YM-175 in YM-SHM animals. Our results suggest that preadministration of bisphosphonates is effective in prevention of bone loss at the tibial metaphysis when induced by hindlimb immobilization in rats.

IL-4, but not vitamin D3, induces monoblastic cell line UG3 to differentiate into multinucleated giant cells on osteoclast lineage

The formation of multinucleated giant cells (MGCs) from monocytes/macrophages is controlled by various cytokines, the roles of which are not fully understood. Both interleukin (IL)‐4 and 1α,25(OH)2 vitamin D3 (D3) are known to induce MGC formation from monocytes/macrophages. D3 is also known as a stimulator of osteoclast formation in the presence of stroma cells, and IL‐4 as an inhibitor. Previously, we showed that IL‐4‐induced MGCs from monocytes/macrophages expressed tartrate resistant acid phosphatase (TRAP) activity and hydroxyapatite‐resorptive activity in the presence of M‐CSF without stroma cells. In this study, we examined the effects of D3 and/or IL‐4 on MGC formation and the characteristics of these MGCs using a monoblastic cell line (UG3), to elucidate the involvement of these factors in osteoclast development without stroma cells. D3‐induced MGCs showed none of the markers of osteoclasts, such as TRAP activity, calcitonin receptor (cal‐R) expression, hydroxyapatite‐resorptive activity, and bone‐resorptive activity. A low concentration of D3 synergistically stimulated IL‐4‐induced TRAP‐positive MGC formation, whereas a high concentration of D3 inhibited it. When IL‐4 was added on day 7 of the 2‐week culture with D3, TRAP positivity reached maximum. On the other hand, delayed addition of D3 on day 7 of culture did not increase the TRAP positivity. Although the fusion rate increased during the first week of the 2‐week culture in the presence of D3, it increased further in the second week following the addition of IL‐4 on day 7. Furthermore, IL‐4‐induced, or IL‐4‐ and D3‐induced MGCs differentiated into functional osteoclasts with bone‐resorptive activity following coculture with osteoblastic cells, whereas D3‐induced MGCs did not acquire bone‐resorptive activity even after coculture with osteoblastic cells in the presence of D3. These findings suggest that IL‐4 initiates osteoclast development of UG3 cells, although stroma cells were necessary for development of functional osteoclasts. On the other hand, D3 had only a “supportive” effect on this differentiation. IL‐4 and direct contact with stroma cells may regulate different stages in the multistep process of osteoclastogenesis of UG3 cells. J. Cell. Physiol. 182:214–221, 2000.

Prefabrication of Vascularized Bone Allograft in a Recipient Rat Using a Flow-through Vascular Pedicle, Bone Morphogenetic Protein, and Bisphosphonate.

We attempted to prefabricate vascularized bone allografts by implanting flow-through vascular bundles from recipient rats into transplanted bone allografts. We also applied bone morphogenetic protein (BMP) and bisphosphonate into the bone allograft to accelerate bone formation and inhibit bone resorption in the transplanted bone. After prefabrication, bone formation and resorption in the vascularized bone allograft were evaluated radiographically and histologically. We also attempted to transfer the prefabricated vascularized bone allograft onto the femur of recipient rats, and bone union between was subsequently assessed. Bone formation in the transplanted allograft was significantly stimulated with addition of BMP. However, bone resorption was also stimulated by BMP; this stimulated bone resorption caused by BMP was effectively inhibited with addition of bisphosphonate. The bone union rate between transplanted bone allografts and recipient femora was also stimulated by BMP. Bisphosphonate slightly delayed bone union but effectively protected the grafted bone from bone resorption caused by BMP. Our results suggest that prefabrication of vascularized bone allografts can be achieved in the recipient rat by implanting a flow-through vascular bundle from the recipient into the transplanted bone allograft. Combination treatment with BMP and bisphosphonate allows development of an ideal vascularized bone allograft.

Prefabrication of Vascularized Allogenic Bone Graft in a Rat by Implanting a Flow-Through Vascular Pedicle and Basic Fibroblast Growth Factor Containing Hydroxyapatite/Collagen Composite

Background Basic fibroblast growth factor (bFGF) is known to stimulate bone formation and angiogenesis. Hydroxyapatite/collagen composite (HAp/Col) is also known to have very strong bone conductive activity. In this study, prefabrication of vascularized allogenic bone (allo‐bone) graft was attempted in recipients by implanting vascular bundles from recipients into the transplanted allo‐bone graft. Furthermore, the effect of bFGF‐containing HAp/Col on the prefabricated vascularized allo‐bone graft was investigated. Methods In this study, 32 Sprague‐Dawley rats were used as donors, and bone grafts were collected from their femora. Thirty‐two Wistar rats (recipients) were divided into four groups, and the allo‐bone grafts were transplanted into the thigh region. In the experimental groups, one or both of the flow‐through saphenous vascular bundles and 100‐&mgr;g bFGF‐containing HAp/Col were implanted into the medullary cavity of the allo‐bone grafts. In the control group, neither was implanted. These rats were sacrificed at 4 weeks after transplantation, and bone formation, angiogenesis, and bone resorption in the transplanted allo‐bone grafts were evaluated histologically and genetically. Results Bone formation and angiogenesis in the transplanted allo‐bone graft were effectively stimulated by implanting vascular bundles or bFGF‐containing HAp/Col on both histological and genetic evaluations compared with the control group. The most significant stimulation was observed in the group in which both were implanted. Bone resorption was not stimulated in any group. Conclusion By implanting a flow‐through vascular bundle and bFGF‐containing HAp/Col, an ideal vascularized allo‐bone graft that had high bone formative and angiogenetic activities and did not stimulate bone resorptive activity was prefabricated.

Localized type Volkmann's contracture treated with tendon transfer and tension-reduced early mobilization: A case report.

Rationale: For localized type Volkmanns contracture, in which degeneration of the flexor digitorum profundus (FDP) muscle to one or two fingers and restriction of finger extension occur, dissection or excision of the affected muscle is usually recommended. However, these surgical procedures need relatively wide exposure of the muscle, because the FDP muscle is in the deep portion of the forearm. Patient concerns: In this report, the case of a 35-year-old woman with localized type Volkmanns contracture is presented. Her left forearm had been compressed with an industrial roller 4 months earlier, and severe flexion contracture of the long finger and mild flexion contracture of the ring finger developed gradually. Diagnoses: localized type Volkmanns contracture. Intervention: Five months after the injury, transection of the FDP tendon to the long finger and transfer of the transected tendon to the FDP tendon to the index finger was performed after adjusting the tonus of these two tendons using a small skin incision. This procedure was followed by a tension-reduced early mobilization technique in which a tension-reduced position of the tendon suture site was maintained by taping the long finger to the volar side of the index finger, and then immediate active range of motion (ROM) exercise was started. Outcomes: Within 9 weeks after surgery, full ROM had been regained. Lessons: Using the treatment procedure presented in this case report, a good clinical result was obtained in a minimally invasive manner.