Human parathyroid hormone (1–34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys

Bone 40 (2007) 1475 – 1482 www.elsevier.com/locate/bone

Human parathyroid hormone (1–34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys Takeshi Manabe a , Satoshi Mori a,⁎, Tasuku Mashiba a , Yoshio Kaji a , Ken Iwata a , Satoshi Komatsubara a , Azusa Seki b , Yong-Xin Sun a , Tetsuji Yamamoto a a

Department of Orthopedic Surgery, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa, Japan b Hamri Corporation, Tokyo, Japan Received 31 October 2006; revised 21 December 2006; accepted 18 January 2007 Available online 2 February 2007

Abstract Several studies in rats have demonstrated that parathyroid hormone accelerates fracture healing by increasing callus formation or stimulating callus remodeling. However the effect of PTH on fracture healing has not been tested using large animals with Haversian remodeling system. Using cynomolgus monkey that has intracortical remodeling similar to humans, we examined whether intermittent treatment with human parathyroid hormone [hPTH(1–34)] accelerates the fracture healing process, especially callus remodeling, and restores geometrical shapes and mechanical properties of osteotomized bone. Seventeen female cynomolgus monkeys aged 18–19 years were allocated into three groups: control (CNT, n = 6), low-dose PTH (0.75 μg/kg; PTH-L, n = 6), and high-dose PTH (7.5 μg/kg; PTH-H, n = 5) groups. In all animals, twice a week subcutaneous injection was given for 3 weeks. Then fracture was produced surgically by transversely cutting the midshaft of the right femur and fixing with stainless plate. After fracture, intermittent PTH treatment was continued until sacrifice at 26 weeks after surgery. The femora were assessed by soft X-ray, three-point bending mechanical test, histomorphometry, and degree of mineralization in bone (DMB) measurement. Soft X-ray showed that complete bone union occurred in all groups, regardless of treatment. Ultimate stress and elastic modulus in fractured femur were significantly higher in PTH-H than in CNT. Total area and percent bone area of the femur were significantly lower in both PTH-L and PTH-H than in CNT. Callus porosity decreased dose-dependently following PTH treatment. Mean DMB of callus was significantly higher in PTH-H than in CNT or PTH-L. These results suggested that PTH decreased callus size and accelerated callus maturation in the fractured femora. PTH accelerates the natural fracture healing process by shrinking callus size and increasing degree of mineralization of the fracture callus, thereby restoring intrinsic material properties of osteotomized femur shaft in cynomolgus monkeys although there were no significant differences among the groups for structural parameters. © 2007 Elsevier Inc. All rights reserved. Keywords: Parathyroid hormone; Fracture healing; Callus remodeling; Histomorphometry; Cynomolgus monkey

Introduction Drug treatment of osteoporosis can be classified according to the response of the bone. Drugs either stimulate bone formation or suppress bone remodeling. Injection of parathyroid hormone ⁎ Corresponding author. Fax: +81 87 891 2196. E-mail addresses: [email protected] (T. Manabe), [email protected] (S. Mori), [email protected] (T. Mashiba), [email protected] (Y. Kaji), [email protected] (K. Iwata), [email protected] (S. Komatsubara), [email protected] (A. Seki), [email protected] (Y.-X. Sun), [email protected] (T. Yamamoto). 8756-3282/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2007.01.015

(PTH) has potent anabolic effects on bones as a result of stimulation of bone formation in several animal models [1–4] and human [5–19]. In rat studies, PTH increases bone mass by thickening trabeculae [20–22] and by stimulating periosteal and endocortical bone formation [3,23]. In monkeys, PTH increases trabecular number rather than trabecular thickness [24] and stimulates endocortical bone formation [25]. A large clinical trial of postmenopausal women with osteoporosis has demonstrated that 21-month treatment with hPTH(1–34) reduces vertebral fractures by 65% and non-vertebral fractures by 54% [9]. Because osteoporotic patients are prone to fractures, clinicians often encounter situations of fractures occurring in

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patients under drug treatment for osteoporosis. Therefore, it is important to examine the effects of anti-osteoporotic agents on fracture healing. In our previous studies using a rat fracture model, anti-resorptive agents such as bisphosphonates and selective estrogen receptor modulator (SERM) [26–29] delayed callus remodeling but did not impair restoration of mechanical strength of the fractured bone. On the other hand, when we examined the effect of intermittent PTH treatment on fracture healing using rat model, we demonstrated that PTH accelerated fracture healing through stimulation of callus remodeling and formation of new cortical shell [30]. Other studies using rat fracture model also indicated acceleration of fracture healing by intermittent PTH treatment [31–36]. The rat is a convenient, useful model to examine fracture healing. However, this model does not necessarily reflect the fracture healing process that occurs in humans because rats do not normally have intracortical remodeling. Larger animal models with the Haversian remodeling system similar to humans would provide more precise information of the fracture healing process following drug treatment. The aim of this study was to test whether intermittent treatment with human parathyroid hormone [hPTH(1–34)] accelerates fracture healing process, especially callus remodeling, and restores geometrical shapes and mechanical properties of osteotomized bone using cynomolgus monkey. Materials and methods Animals Seventeen female cynomolgus monkeys (Macaca fascicularis) aged 18–19 years (Siconbrec Inc. Makati, Philippines) with normal menstrual cycle were housed in a room maintained at 22 to 30 °C and a 12-hour light–dark cycle, with free access to water and restricted access to standard monkey breeder pellets (Republic Flour Mills Corp., Laguna, Philippines) of 100 g/day. All animal procedures were conducted in accordance with National Institutes of Health (NIH) guidelines, following a protocol approved by the Kagawa University Animal Study Committee.

Experimental design After one-month acclimatization, all monkeys were allocated based on their body weights into three groups: control group (CNT, n = 6), low-dose PTH group (PTH-L, n = 6), and high-dose PTH group (PTH-H, n = 5). Animals in CNT group were given subcutaneous injection of 0.9% saline vehicle. The remaining two groups were given subcutaneous injection of human parathyroid hormone (1–34) (Asahi Kasei Corp., Tokyo, Japan) at a dose of 0.75 μg/kg/day (PTH-L group) or 7.5 μg/kg/day (PTH-H group). These doses are 0.6 or 6 times, respectively, the clinical dose for human osteoporosis treatment. Injection was initiated 3 weeks before experimental fracture and performed twice a week until sacrifice. Body weights were measured weekly and doses were adjusted accordingly (Fig. 1).

Fracture surgery Fracture was produced surgically in all animals. After 3-week pretreatment, a transverse osteotomy were performed at the midshaft of the right femur using a fined-toothed circular microsaw (Kiso Power Co., Osaka, Japan) under general anesthesia with a 2:1 mixture (10 mg/kg, i.m.) of ketamine hydrochloride (Troy Laboratories Pty. Limited, Smithfield, Australia) and xylazine hydrochloride (Troy Laboratories Pty. Limited, Smithfield, Australia). The fracture was repositioned and fixed tightly with a stainless plate (Osteo-mini ACP plate,

Fig. 1. Experimental protocol. CNT group: vehicle treatment, PTH-L group: treatment with hPTH(1–34) at 0.75 μg/kg, PTH-H group: treatment with hPTH (1–34) at 7.5 μg/kg, respectively, before and after fracture surgery. Subcutaneous injection was performed 2 times a week. Stryker, Michigan, USA). Unrestricted ambulation was allowed after recovery from general anesthesia, and dosing was continued until sacrifice.

Evaluations Anterior–posterior and lateral radiographs of the fractured femora were taken using an Acoma Portable X-ray Unit (20 mA, 60 Kvp, 0.4 s; PX-20N, Acoma X-ray Industry Co., Ltd. Tokyo, Japan), immediately after fracture surgery and repeated every 4 weeks until sacrifice to check the fracture alignment and fixation. The animals were sacrificed at 26 weeks after surgery. All animals were double-labeled intravenously with calcein (8 mg/kg, Dojin Chemical Institute, Kumamoto, Japan) at 22 and 9 days before sacrifice. After necropsy, femora were excised and dissected free of soft tissues. The stainless plate and screws were removed carefully so as not to damage the callus. Anterior–posterior soft radiographs of femora were taken and the femora were frozen at − 80 °C wrapped in gauze soaked in isotonic saline until microcomputed tomography (micro-CT) scanning. After thawing at room temperature, the femora were scanned by micro-CT (25 μA, 70 kV, 1.8 W; NX-LCP-C80, Nittetsu Elex Co. Ltd. Tokyo, Japan). The bones were placed horizontally on the table. A hundred slices were scanned both at the proximal and distal sides of the fracture site. Then a sagittal image of the fracture was reconstructed. After micro-CT scanning, the femora were tested for mechanical properties by a three-point bending method using a material testing machine (MZ500S, Maruto, Tokyo, Japan). A femur was placed on two lower support bars (40 mm apart) with the anterior surface facing upward. The fracture plane was centered at the loading point, and load was applied at a rate of 10.0 mm/min until breakage. From the load versus displacement curve, the structural mechanical properties of the fractured bone were determined as ultimate load (maximum force that the specimen sustained), stiffness (the slope of the linear portion of the load vs. deformation curve before failure), and work to failure (the area under the load vs. deformation curve before failure). These structural parameters depend on both intrinsic material properties and geometry. The load vs. displacement data were normalized to obtain intrinsic material properties such as ultimate stress, elastic modulus, and toughness, which are independent of cross-sectional size and shape. Ultimate stress was calculated from the ultimate load by the following formula: Ultimate stress ¼ 0:125TUltimate loadTLTb=I; where L is the length between supports, b is the width of the femur in anteroposterior direction, and I is the cross-sectional moment of inertia. Elastic modulus was calculated as: Elastic modulus ¼ StiffnessTL3 =48I

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Fig. 2. Soft X-ray and micro-CT images. Fracture lines are not visible in all groups. Periosteal callus is considerably smaller in PTH-H than in CNT.

Toughness was calculated from energy absorption as: Toughness ¼ 0:75Twork to failureTb2 =LI Cross-sectional moment of inertia (I) was calculated with the assumption that femoral cross-section is elliptically shaped: I ¼ ðp=64ÞTðab3  ða  2tÞðb  2tÞ3 Þ; where a is the width of the mediolateral direction, and t is the average cortical thickness. Average cortical thickness was calculated from the histomorphometric thickness measurements made in each of four quadrants of the femora section using a digitizing system. After mechanical testing, the proximal segments of fracture were fixed in 10% cold neutral buffered formalin for 3 days, decalcified in 10% EDTA at 4 °C for 4 weeks, and then embedded in paraffin. Within 500 μm from the original fracture line, 5-μm cross-sections were cut for tartrate-resistant acid phosphatase (TRAP) staining. The presence of osteoclasts was determined by TRAP activity using a leukocyte acid phosphatase kit (Sigma Chemical Co., St. Louis, MO, USA). The distal segments of the fracture were fixed in 70% ethanol, stained in Villanueva bone stain (Polysciences, Inc., Warrington, PA), dehydrated in graded ethanol, defatted in acetone, and embedded in methyl methacrylate. Within 500 μm from the original fracture line, 100-μm undecalcified transverse sections were cut using a diamond microtome saw (RM2065, Leica Instruments GmbH, Nussloch, Germany) for contact microradiographs (μFX-1000; Fujifilm, Tokyo, Japan). Then specimens were ground to 30 μm in thickness for histomorphometric measurement. Histomorphometric analysis was performed using a semi-automated digitizing image analyzer. The system consisted of a light or epifluorescent microscope and a digitizing pad coupled with computer and histomorphometric software (System Supply CO., Nagano, Japan). Total cross-sectional area (T.Ar; mm2), medullary area, callus area, and porosity and thickness (CTh; μm) were measured at ×40 magnification, and bone area (B.Ar = T.Ar − M.Ar) was calculated. Percent bone area (%B.Ar = B. Ar × 100 / T.Ar; %), percentage callus area (%Cl.Ar = Cl.Ar × 100 / T.Ar; %) and

porosity of callus (%) were also calculated. Single labeled surface, double labeled surface, and the interlabeling width were measured in the callus. Mineral apposition rate (MAR; μm/day), mineralizing surface (MS/BS; %), and bone formation rate (BFR/BS; mm3/mm2/year) were calculated. Osteoclast measurements including osteoclast number (N.Oc; #) and derived parameters (N.Oc/Cl.Ar; #/mm2) were performed at ×100 magnification in the callus. The right 9th rib was assigned for conventional intracortical histomorphometry. A 3-cm sample taken 2 cm proximal to the osteochondral junction was prepared as well as the undecalcified femoral specimen. For each spe-

Table 1 Biochemical properties of fractured femur Group

CNT

PTH-L

PTH-H

Structural properties Ultimate load (N) 675 ± 86 605 ± 163 660 ± 138 Stiffness (N/mm) 10,346 ± 1440 9180 ± 1403 9100 ± 1624 Work failure 279,748 ± 61,776 245,058 ± 143,024 322,814 ± 197,984 (N/mm) Intrinsic material properties Ultimate stress 21.3 ± 4.0 24.2 ± 4.8 33.9 ± 8.0a,b (MPa) Young's modulus 7780.8 ± 1648.6 9204.3 ± 2254.2 11,415.7 ± 1284.0a (MPa) Toughness 369.8 ± 94.8 402.3 ± 211.2 693.6 ± 480.0 (MJ/m3) Cross-sectional 606.3 ± 154.8 577.5 ± 141.6 497.6 ± 101.1 moment of inertia (mm4) CNT group: vehicle treatment, PTH-L group: treatment with hPTH(1–34) at 0.75 μg/kg, PTH-H group: treatment with hPTH(1–34) at 7.5 μg/kg, respectively, before and after fracture surgery. Data are express as means ± SEM. a p < 0.01 vs. CNT, bp < 0.05 vs. PTH-L.

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Table 2 Bone histomorphometry in fracture callus Group

CNT 2

T.Ar (mm ) %B.Ar (%) %Cl.Ar (%) MAR (mm/day) BFR/BS (mm3/mm2/year) MS/BS (%) N.Oc/Cl.Ar (#/mm2) Porosity of callus (%) Callus thickness (μm)

106.7 ± 6.5 83.21 ± 6.03 46.3 ± 7.2 1.0 ± 0.3 0.06 ± 0.03 17.9 ± 4.5 0.5 ± 0.3 24.8 ± 6.7 862.2 ± 252.8

DMB measurement PTH-L 97.2 ± 14.9 70.26 ± 7.37b 46.1 ± 7.6 1.0 ± 0.1 0.06 ± 0.02 16.7 ± 5.4 0.3 ± 0.1 17.6 ± 6.6 848.9 ± 447.6

PTH-H c

88.9 ± 5.4 59.69 ± 6.07a,e 32.4 ± 6.7b,d 1.0 ± 0.4 0.05 ± 0.03 11.0 ± 4.7 0.2 ± 0.1c 8.00 ± 3.7a,e 497.9 ± 126.3

CNT group: vehicle treatment, PTH-L group: treatment with hPTH(1–34) at 0.75 μg/kg, PTH-H group: treatment with hPTH(1–34) at 7.5 μg/kg, respectively, before and after fracture surgery. T.Ar: total area, %B.Ar: percent bone area, %Cl.Ar: percent callus area, MAR: mineral apposition rate, BFR/BS: bone formation rare, N.Oc/Cl.Ar: number of osteoclast. Data are express as means ± SEM. a p < 0.001 vs. CNT, bp < 0.01 vs. CNT, cp < 0.05 vs. CNT, dp < 0.01 vs. PTH-L, e p < 0.05 vs. PTH-L.

cimen, a 30-μm-thick transverse section was obtained for histomorphometric measurement. Whole cross-sectional area of specimen was measured at a magnification of ×100. Some derived parameters measured or calculated for cortical bone have been described by Mashiba et al. [37]: total area (T.Ar; mm2), medullary area (Me.Ar; mm2), cortical area (Ct.Ar; mm2), percent cortical area (%Ct.Ar, %), osteoid thickness (O.Th, μm), mineral apposition rate (MAR; μm/ day), bone formation rate (BFR/Ct.Ar; mm3/mm2/year), activation frequency (Ac.f; #/mm2/year).

To evaluate the maturity of fracture callus, the degree of mineralization in bone (DMB) was measured in the callus area using a 100-μm transverse section obtained within 500 μm from the original fracture plane. A microfocus X-ray diffraction unit (μFX-1000; Fujifilm, Tokyo, Japan) was used, which provides images of much higher resolution than conventional soft X-ray because the focus of the X-ray beam is as small as 8 μm in the former but is 500–1000 μm in the latter. Quantitative digital microradiography allows the measurement of the density of mineral substance in microscopic volumes of bone tissue (i.e., degree of mineralization). For constant quantitative evaluation, the specimens were exposed with a calibration step-wedge on the same radiograph. The step-wedge was made of one to five layers of 30-μm bone substitute plastic material containing hydroxyapatite (HA) with a density of 0.95718 g/cm3 (BE-103; Kyoto Kagaku Co., Kyoto, Japan). The section and the calibration step-wedge were placed on a plastic sheet and attached to the X-ray source. An ultra-sensitive phosphor imaging plate (BAS-TR2025; Fujifilm, Tokyo, Japan) was placed 58 cm beneath the X-ray source. Digital microradiograph was taken under the conditions of exposure at 10 kVand 10 μA for 30 min. The exposed imaging plate was scanned using a bioimaging analyzer system (BAS-1800; Fujifilm, Tokyo, Japan) to obtain digital image data. Using this system, more accurate digital image data can be obtained directly without using or developing conventional film. DMB of the callus area distinguishable from the original cortical bone was quantified using a computerized method as follows. For semi-automated analysis of the gray level of microradiographs, we used the image analyzing software Multi Gauge Ver2.0 (Fujifilm, Tokyo, Japan) operated under Microsoft Windows. This software produces a calibration curve using the step-wedge readings and measures the gray level in arbitrary area of the field. DMB was measured at three hundred regions of interest placed on the mineralized callus tissue. The DMB was further adjusted by the thickness of each specimen and expressed as grams of hydroxyapatite per cubic centimeter of bone. Mean DMB of a callus and the distribution of DMB were obtained and analyzed [38]. As for the extent of heterogeneity of mineralization, the inter-quartile range (IQR), the

Fig. 3. Histological findings of the callus. (a) Under natural light, (b) under epifluorescent light. PTH-H has less porous structure compared to CNT. Osteoid and labeled surface are not increased in PTH treatment groups compared to CNT. The border between fracture callus and original cortices is less clear in PTH-H than in CNT.

T. Manabe et al. / Bone 40 (2007) 1475–1482 Table 3 Bone histomorphometry in rib cortical bone Group

CNT

PTH-L

PTH-H

T.Ar (mm2) Mc.Ar (mm2) Ct.Ar (mm2) %Cl.Ar (%) O.Th (μm) MAR (μm/day) BFR/Ct.Ar (mm3/mm2/year) Ac.f (#/mm2/year)

7.0 ± 0.9 1.8 ± 0.4 5.2 ± 1.0 74.5 ± 6.3 9.0 ± 2.4 0.5 ± 0.2 3.1 ± 2.3 5.3 ± 3.8

7.1 ± 1.9 2.4 ± 1.5 4.7 ± 0.8 68.6 ± 11.4 9.3 ± 4.4 0.6 ± 0.2 12.2 ± 12.1 7.6 ± 6.0

8.0 ± 1.4 2.5 ± 1.0 5.5 ± 0.6 69.7 ± 7.8 9.0 ± 1.2 0.8 ± 0.1a,c 12.0 ± 8.4 18.8 ± 14.6b

CNT group: vehicle treatment, PTH-L group: treatment with hPTH(1–34) at 0.75 μg/kg, PTH-H group: treatment with hPTH(1–34) at 7.5 μg/kg, respectively, before and after fracture surgery. T.Ar: total area, Me.Ar: medullary area, Ct.Ar: cortical area, %Ct.Ar: percent cortical area, O.Th: osteoid thickness, MAR: mineral apposition rate, BFR/BS: bone formation rare, Ac.f: activation frequency. Data are express as means ± SEM. a p < 0.01 vs. CNT, bp < 0.05 vs. CNT, cp < 0.05 vs. PTH-L. interval between 25 and 75 percentile, was obtained using statistical computing software StatView 5.0 (SAS Institute, Inc., Cary, NC, USA) for each animal based on the distribution of measured DMB values in callus.

Statistical analysis Statistical computation of data was performed using the statistical package StatView 5.0. Differences among treatment groups were tested by one-way analysis of variance (ANOVA). If a significant difference was obtained, differences between the means of two groups were tested by Fisher's protected least significant difference (PLSD). A p value less than 0.05 was considered significant.

Results Radiography Soft X-ray observation after sacrifice showed that the fracture line was invisible in all animals. Micro-CT revealed

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that the size of callus was larger in CNT compared with PTHtreated groups. Especially the callus in PTH-H was the smallest among three groups (Fig. 2). Mechanical test The ultimate load, stiffness, and work to failure in the femur did not differ significantly among the three groups. When these data were normalized by cross-sectional moment of inertia, the intrinsic material properties such as ultimate stress, elastic modulus, and toughness tended to be greater in both PTHtreated groups. Especially, ultimate stress was significantly higher in PTH-H than in CNT (p < 0.01) and PTH-L (p < 0.05). Elastic modulus was also significantly higher in PTH-H than in CNT (p < 0.01) (Table 1). Histomorphometry Total area (T.Ar) of the femur was significantly lower in PTH-H and PTH-L than in CNT, although the difference reached statistical significance only in PTH-H (p < 0.05). Percent bone area (%B.Ar) was significantly lower in both PTH-L and PTH-H than in CNT. Percent callus area (%Cl.Ar) was significantly lower in PTH-H than both PTH-L and CNT, but there was no significant different between PTH-L and CNT. Callus thickness tended to be lower in PTH-H than in both CNT and PTH-L, but not significantly different (Table 2). Mineral apposition rate (MAR) and bone formation rate (BFR/BS) did not differ significantly among the three groups (Fig. 3). Number of osteoclast (N.Oc/Cl.Ar) was lower in both PTH-H and PTH-L compared with CNT. Porosity of callus in PTH-H was 68% (p < 0.001) and 55% (p < 0.05) less than in CNT and PTH-L, respectively. In contrast, intracortical bone remodeling was accelerated following PTH treatment in the cortical bone of right 9th rib.

Fig. 4. Contact microradiographs at the fracture plane. PTH decreased the size and porosity of periosteal callus and increased degree of mineralization in callus dose-dependently.

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Table 4 Degree of mineralization in fracture callus

Mean DMB (g/cm3) IQR (g/cm3)

CNT

PTH-L

PTH-H

1.09 ± 0.26 0.25 ± 0.03

1.13 ± 0.24a 0.30 ± 0.03

1.42 ± 0.30a,b 0.34 ± 0.03

CNT group: vehicle treatment, PTH-L group: treatment with hPTH(1–34) at 0.75 μg/kg, PTH-H group: treatment with hPTH(1–34) at 7.5 μg/kg, respectively, before and after fracture surgery. DMB: degree of mineralization in bone, IQR: inter-quartile range. Data are express as means ± SEM. a p < 0.001 vs. CNT, bp < 0.001 vs. PTH-L.

MAR and Ac.f were significantly higher in PTH-H than in CNT although T.Ar, Me.Ar, Ct.Ar, %Ct.Ar, O.Th and BFR/Ct.Ar did not differ significantly among the three groups (Table 3). Mineralization of callus On contact microradiographs of cross-sectional specimens at the fracture plane, PTH-treated animal had more highly mineralized callus when compared with CNT (Fig. 4). Mean DMB was significantly higher in PTH-H than in CNT (p < 0.001) and PTH-L (p < 0.001) (Table 4). The DMB distribution curve showed a dose-dependent shift towards higher values in PTH-treated animals when compared with CNT (Fig. 5). IQR, the extent of heterogeneity of mineralization, tended to be higher in PTH-treated animals although this did not reach statistical significance. Discussion The primary aim of this study was to examine the effect of intermittent treatment with hPTH(1–34), a potent anabolic agent, on the fracture healing process using femoral osteotomy model of cynomolgus monkey that has the Haversian intracortical remodeling system similar to humans. Intracortical remodeling plays important roles in restoring structural and mechanical properties of fractured bone. Therefore, our simian fracture model is expected to provide more precise information

regarding human fracture healing process than animal models without Haversian remodeling system. To the best of our knowledge, this is the first study to evaluate the effect of PTH on fracture healing using a non-human primate. Our results demonstrated that, in cynomolgus monkeys, intermittent systemic administration of hPTH(1–34) accelerates the natural fracture healing process in a dose-dependent manner. This acceleration was featured by smaller callus size and higher degree of mineralization in the callus than the control, leading to significant increases in intrinsic material properties of the fractured femur shaft. No significant increases in bone formation parameters were observed following PTH treatment in the fractured femur of present study. A possible explanation for this phenomenon is that, because fracture healing is more nearly complete and the callus is well matured at 26 weeks in the PTH-treated groups, no increase in bone formation was detectable in this study. This is consistent with our finding of lower osteoclast number in high-dose PTH-treated group compared to control. Acceleration of fracture healing by PTH treatment observed in the current study is consistent with our previous study using rats, in which intermittent treatment with PTH accelerated the restoration of structural and mechanical properties in fractured femur, achieved by accelerated callus remodeling of woven bone to lamellar bone and formation of new cortical shell without significant change of callus size [30]. Thus, it is most likely that PTH shortens the natural fracture healing process by accelerating callus remodeling, regardless of the experimental animal species. Interestingly, this is in contrast to the findings that bisphosphonates suppress callus remodeling and delay natural fracture healing although the mechanical strength of fractured bone increased significantly as a result of greatly increased callus volume. The implication of these findings may be that callus volume represents the extent of fracture healing progression. A significant point of this study was to examine callus maturity by measuring the degree of mineralization of the fracture callus. Once fractured bone is primarily stabilized by

Fig. 5. DMB distribution. DMB distribution shifted higher following PTH treatment in a dose-dependent manner.

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rapid woven callus production, the size of the fracture callus shrinks as woven bone is remodeled to lamellar bone to restore the original morphology of the fracture bone. Although fracture callus becomes highly mineralized as it matures, the extent of mineralization in fracture callus has never been quantified in any previous animal fracture model. In the current study, the degree of mineralization in the periosteal fracture callus, which was distinguishable from the original cortical bone, was measured to evaluate callus maturity. Our results demonstrate that 6 months of PTH treatment dose-dependently increases the degree of mineralization of periosteal fractured callus. This finding also indicates that PTH shortens the natural fracture healing process by accelerating callus remodeling into more mature, dense callus. Thus, the evaluation of degree of mineralization in fractured callus may be quite useful to know the degree of maturation of the fracture callus. The secondary aim of this study was to answer the question of whether hPTH(1–34) treatment should be continued or stopped when fracture occurs in an osteoporotic patient under PTH treatment. Our results have demonstrated that continued PTH treatment before and after fracture accelerated the fracture healing process and restoration of mechanical strength compared to controls. These findings may encourage the clinician to continue PTH treatment after a fracture has occurred in an osteoporotic woman under treatment to shorten the treatment period for fracture. Because we do not have any mechanical data of intact femur in this study, we cannot provide the information how many percent of mechanical properties has been restored following fracture. This may be the subject of future study. Based on radiological, histomorphometric and biomechanical evaluation of fractured femur in cynomolgus monkeys treated with hPTH(1–34) for 6 months, we conclude that PTH accelerates the natural fracture healing process by shrinking callus size and increasing the degree of mineralization of the fracture callus, consequently increasing the intrinsic material properties of the fractured femur shaft. Our data support the clinical application of intermittent PTH treatment for osteoporotic women who sustained fractures as a means to shorten the treatment period for fractures. Acknowledgments The authors thank Mika Kawada and Yoshiko Fukuda for histological preparation and Asahi Kasei Co. for kindly supplying the hPTH(1–34). References [1] Gunness-Hey M, Hock JM. Increased trabecular bone mass in rats treated with human synthetic parathyroid hormone. Metab Bone Dis Res 1984;5:171–81. [2] Hock JM, Centrella M, Canalis E. Insulin-like growth factor I has independent effects on bone matrix formation and cell replication. Endocrinology 1988;122:2899–904. [3] Oxlund H, Ejersted C, Andreassen TT, Torring O, Nilsson MH. Parathyroid hormone (1–34) and (1–84) stimulate cortical bone formation both from periosteum and endosteum. Calcif Tissue Int 1993;53:394–9.

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