Hiromichi Norimatsu

Raloxifene, estrogen, and alendronate affect the processes of fracture repair differently in ovariectomized rats.

We investigated the effects of inhibitors of bone resorption (estrogen, raloxifene, and alendronate) on the processes of fracture repair in ovariectomized (OVX) rats. One hundred forty female Sprague‐Dawley rats at 3 months of age were either OVX or sham‐operated and divided into five groups: sham control, OVX control, estrogen (17α‐ethynyl estradiol [EE2], 0.1 mg/kg), raloxifene (Rlx, 1.0 mg/kg), and alendronate (Aln, 0.01 mg/kg) groups. Treatment began immediately after the surgery. Four weeks postovariectomy, prefracture controls were killed and bilateral osteotomies were performed on the femoral midshafts and fixed with intramedullary wires. Treatment was continued and fracture calluses were excised at 6 weeks and 16 weeks postfracture for evaluation by X‐ray radiography, quantitative computed tomography (QCT,) biomechanical testing, and histomorphometry. At 6 weeks postfracture, Aln and OVX had larger calluses than other groups. Sham and OVX had higher ultimate load than EE2 and Rlx, with Aln not different from either control. Aln calluses also contained more mineral (bone mineral content [BMC]) than all other groups. By 16 weeks postfracture, OVX calluses were smaller than at 6 weeks and the dimensions for Aln had not changed. Aln had higher BMC and ultimate load than OVX, EE2, and Rlx. EE2 and Rlx had similar biomechanical properties, which were similar to sham. Interestingly, OVX and Aln animals were heavier than other groups at all time points; therefore, ultimate load was normalized by body weight to show no significant differences in strength of the whole callus between groups at either 6 weeks or 16 weeks postfracture. However, Aln strongly suppressed remodeling of the callus, resulting in the highest content of woven bone, persistent visibility of the original fracture line, and lowest content of lamellar bone, compared with other groups. Therefore, the larger Aln callus appeared to be a remarkable, morphological adaptation to secure the fracture with inferior material. In conclusion, OVX‐stimulated bone turnover resulted in the fastest progression of fracture repair that was most delayed with Aln treatment, consistent with marked suppression of bone resorption and formation activity. Estrogen and Rlx had similar effects that were generally similar to sham, indicating that mild suppression of bone turnover with these agents has insignificant effects on the progression of fracture repair.

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.

The effects of suppressed bone remodeling by bisphosphonates on microdamage accumulation and degree of mineralization in the cortical bone of dog rib.

We evaluated the effects of suppressed bone remodeling caused by bisphosphonate on microdamage accumulation and degree of mineralization of bone (DMB) for the dog rib in two independent studies. Study 1: 36 female beagles, 1-2 years old, were treated daily for 1 year with saline vehicle, risedronate at 0.5 mg/kg/day, or alendronate at 1.0 mg/kg/day. Study 2: 29 beagles, 1 year old, were given lactose, or incadronate at 0.3 mg/kg/day or 0.6 mg/kg/day for 3 years. In both studies, the ninth rib was harvested. Intracortical remodeling was significantly suppressed following either 1 year or 3 years of bisphosphonate treatment without impairment of primary mineralization, although the remodeling rate was obviously lower in study 2 than in study 1 because of the aging of animals. Microdamage accumulation was significantly increased following any bisphosphonate treatment in response to the extent of remodeling suppression. One-year treatment with risedronate or alendronate did not significantly affect the mean DMB or osteonal distribution based on DMB. In contrast, mean DMB was significantly increased following 3 years of incadronate treatments, and osteonal distributions based on DMB showed a dose-dependent shift toward the higher values in incadronate-treated animals when compared with controls. Our results demonstrated that DMB was increased following only 3 years but not 1 year of bisphosphonate treatment. This finding suggests that suppressed remodeling induced by long-term bisphosphonate treatment increased DMB by increasing the population of old, highly mineralized osteons; however, the expression of this phenomenon depends on duration of the treatment because the secondary mineralization is a very slow process.

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.

Vastus lateralis oxygenation and blood volume measured by near-infrared spectroscopy during whole body vibration.

The purpose of this study was to investigate the effects of whole body vibration (WBV) on oxygenation of vastus lateralis muscle during squatting exercise. Eighteen male subjects [mean age, 27·3 ± 6·0 (SD) years; mean height, 171·8 ± 4·9 cm; mean weight, 64·4 ± 6·1 kg] performed squatting exercise on a vibration platform for 3 min with and without vibration, and changes in oxygenation of the vastus lateralis muscle were determined by near‐infrared spectroscopy. The muscle oxygenation levels and total haemoglobin and myoglobin levels (total Hb/Mb) decreased during squatting exercise with and without vibration. After exercise, the muscle oxygenation level and total Hb/Mb rapidly increased from the minimum value during exercise and remained constant for latter 10 min. The muscle oxygenation levels with vibration from 90 to 180 s after the start of squatting exercise were significantly lower than those without vibration. Total Hb/Mb with vibration from 90 s after the squatting exercise to 540 s were significantly higher than those without vibration. This study demonstrated that WBV exercise affects the oxygenation level of vastus lateralis muscle and reduces muscle oxygenation level compared to that with no WBV. Therefore, WBV exercise may be an efficient training stimulus for muscle deoxygenation.

Temporal progressive antigen expression in radial glia after contusive spinal cord injury in adult rats.

In the development of the CNS, radial glial cells are among the first cells derived from neuroepithelial cells. Recent studies have reported that radial glia possess properties of neural stem cells. We analyzed the antigen expression and distribution of radial glia after spinal cord injury (SCI). Sprague‐Dawley rats had a laminectomy at Th11‐12, and spinal cord contusion was created by compression with 30g of force for 10 min. In the injury group, rats were examined at 24 h and 1, 4, and 12 weeks after injury. Frozen sections of 20‐μm thickness were prepared from regions 5 and 10 mm rostral and caudal to the injury epicenter. Immunohistochemical staining was performed using antibodies to 3CB2 (a specific marker for radial glia), nestin, and glial fibrillary acidic protein (GFAP). At 1 week after injury, radial glia that bound anti‐3CB2 MAb had spread throughout the white matter from below the pial surface. From 4 weeks after injury, 3CB2 expression was also observed in the gray matter around the central canal, and was especially strong around the ependymal cells and around blood vessels. In double‐immunohistochemical assays for 3CB2 and GFAP or 3CB2 and nestin, coexpression was observed in subpial structures that extended into the white matter as arborizing processes and around blood vessels in the gray matter. The present study demonstrated the emergence of radial glia after SCI in adult mammals. Radial glia derived from subpial astrocytes most likely play an important role in neural repair and regeneration after SCI. GLIA 42:172–183, 2003.

Post-traumatic moderate systemic hypothermia reduces TUNEL positive cells following spinal cord injury in rat

Study design: A standardized animal model of contusive spinal cord injury (SCI) with incomplete paraplegia was used to test the hypothesis that moderate systemic hypothermia reduces neural cell death. Terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate [dUTP] nick-end labeling (TUNEL) staining was used as a marker of apoptosis or cell damage.Objective: To determine whether or not moderate hypothermia could have a neuroprotective effect in neural cell death following spinal cord injury in rats.Setting: Kagawa Medical University, Japan.Methods: Male Sprague–Dawley (SD) rats (n=39) weighing on average 300 g (280–320 g) were used to prepare SCI models. After receiving contusive injury at T11/12, rats were killed at 24 h, 72 h, or 7 days after injury. The spinal cord was removed en bloc and of examined at five segments: 5 and 10 mm rostral to the center of injury, center of injury, and 5 and 10 mm caudal to the center of injury. Rats that received hypothermia (32°C/4 h) were killed at the same time points as those that received normothermia (37°C/3 h). The specimens were stained with hematoxylin and eosin, and subjected to in situ nick-end labeling (TUNEL), a specific method for visualizing cell death in the spinal cord.Results: At 24 h postinjury, TUNEL positive cells (TPC) decreased significantly 10 mm rostral to center of injury in hypothermic animals compared to the normothermia group. At 72 h post-SCI, TPC also decreased significantly at 5 mm rostral, and 5 and 10 mm caudal to the lesion center compared to normothermic animals. At 7 days postinjury, a significant decrease of TPC was observed at the 5 mm rostral and 5 mm caudal sites compared to normothermic animals.Conclusion: These results indicate that systemic hypothermia has a neuroprotective effect following SCI by attenuating post-traumatic TPC.