Human parathyroid hormone (1–34) accelerates the fracture healing process of woven to lamellar bone replacement and new cortical shell formation in rat femora

Human parathyroid hormone (1–34) accelerates the fracture healing process of woven to lamellar bone replacement and new cortical shell formation in rat femora

Bone 36 (2005) 678 – 687 www.elsevier.com/locate/bone Human parathyroid hormone (1–34) accelerates the fracture healing process of woven to lamellar ...

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Bone 36 (2005) 678 – 687 www.elsevier.com/locate/bone

Human parathyroid hormone (1–34) accelerates the fracture healing process of woven to lamellar bone replacement and new cortical shell formation in rat femora Satoshi Komatsubaraa, Satoshi Moria,T, Tasuku Mashibaa, Kiichi Nonakab, Azusa Sekic, Tomoyuki Akiyamaa, Kensaku Miyamotoa, Yongping Caoa, Takeshi Manabea, Hiromichi Norimatsua a

Department of Orthopedic Surgery, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa, Japan b Section of Pharmacology, Department of Hard Tissue Engineering, Division of Bio-Matrix, Graduate School, Tokyo Medical Dental University, Tokyo, Japan c Hamri Corporation, Tokyo, Japan Received 26 September 2004; revised 29 January 2005; accepted 3 February 2005 Available online 17 March 2005

Abstract This study aimed to test whether intermittent treatment of human parathyroid hormone [hPTH(1 – 34)] disturbs or accelerates the fracture healing process using rat surgical osteotomy model. One hundred five, 5-week-old SD rats were allocated to vehicle control (CNT) and four PTH groups; 10 and 30 Ag/kg of hPTH(1 – 34) treatment before surgery (P10, P30), and treatment before and after surgery (C10, C30). All animals were given subcutaneous injections three times a week for 3 weeks. Then, fractures were produced by transversely cutting the midshaft of bilateral femora and fixing with intramedullary wire. Human PTH(1 – 34) treatment was continued in C10 and C30 groups until sacrifice at 3, 6, and 12 weeks after surgery. The femora were assessed by peripheral quantitative computed tomography, three-point bending mechanical test, and histomorphometry. Total cross-sectional area was not significantly different among all groups at any time point. At 3 weeks after surgery, the lamellar bone/callus area was significantly increased in C10 and C30 groups compared to the other groups. At 6 weeks, remodeling of woven bone to lamellar bone in the callus was almost complete in all groups. At 12 weeks, percent new cortical shell area was significantly higher in C10 and C30 groups compared to the other groups, and the ultimate load in mechanical testing was significantly higher in C30 group than in CNT, P10, and P30 groups. Intermittent PTH treatment at 30 Ag/kg before and after osteotomy accelerated the healing process as evidenced by earlier replacement of woven bone to lamellar bone, increased new cortical shell formation, and increased the ultimate load up to 12 weeks after osteotomy. D 2005 Elsevier Inc. All rights reserved. Keywords: Parathyroid hormone; Fracture healing; Callus remodeling; Histomorphometry; New cortical shell

Introduction Intermittent injections of parathyroid hormone (PTH) have potent anabolic effects on bones [1– 4]. PTH increases vertebral bone mass and mechanical strength in normal rats, both young and old, and reverses ovariectomy-induced decreases in bone mass and mechanical strength of vertebral T Corresponding author. Fax: +81 87 891 2196. E-mail address: [email protected] (S. Mori). 8756-3282/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.02.002

bodies [5– 10]. Histologically, PTH accelerates bone formation and increases trabecular bone mass by thickening trabeculae [5,11,12]. Although many effective anti-resorptive agents are available, PTH is the only anabolic agent for the treatment of osteoporosis. In a recent large-scale clinical trial of postmenopausal women with osteoporosis, recombinant human parathyroid hormone (1– 34) reduced vertebral fractures by 65% and non-vertebral fractures by 54% [13]. Since osteoporotic patients are prone to fractures, the clinicians

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must face the situation of fracture occurring in patient under treatment. Therefore, it is getting important to know the effects of agent for osteoporosis treatment on fracture healing. We have previously reported the effect of anti-resorptive agents such as bisphosphonates and selective estrogen receptor modulator (SERM) on fracture healing [14 –17]. These agents delay callus remodeling although restoration of mechanical strength of fractured bone is not impaired. Although several studies have reported the effect of intermittent treatment of PTH on fracture healing [18 – 23], the durations of these experiments were no longer than 8 weeks. However, our previous studies have demonstrated that the fracture healing process of rat femur is not completed at 6 weeks after fracture, thus longer-term observation is required to evaluate the entire process of fracture healing. Furthermore, no geometrical analyses of fracture callus have ever been performed. The aim of this study was to test whether intermittent treatment of human parathyroid hormone [hPTH(1– 34)] disturbs or accelerates the fracture healing process, especially callus remodeling, geometrical changes, and mechanical properties. With respect to clinical relevance, this study may answer the question whether hPTH(1 – 34) treatment should be stopped or continued when fracture occurs in an osteoporotic patient under PTH treatment.

Materials and methods Materials Female Sprague – Dawley rats (n = 105; 4 weeks of age; Japan SLC, Inc., Hamamatsu, Japan) were acclimated for 1

679

week to local vivarium conditions (24 T 2-C and 12-h light– dark cycle). During the experimental period, animals were housed in cages (988 cm2 in floor area and 18 cm in height) and allowed free access to water and pelleted commercial rodent diet (Oriental Yeast Co., Tokyo, Japan). The experimental protocol was approved by the Kagawa University Animal Study Committee. Powdered form of hPTH(1 – 34) (Asahi Kasei Corporation, Tokyo, Japan) was dissolved in vehicle (acidified saline containing 2% heat-inactivated young rat sera) to 10 Ag/ml and 30 Ag/ml. PTH was administered subcutaneously, and the concentrations were adjusted such that the injection volume was the same in all animals. Experimental design The animals were randomly allocated to five groups; one vehicle control group (CNT) and four treatment groups, with the same mean body weight (Fig. 1). Treatment groups were divided based on the dose and mode of treatment. Two doses (10 and 30 Ag/kg) of hPTH(1 – 34) were given as pretreatment before fracture (P10 and P30 groups) or treatment before and after fracture (C10 and C30 groups). All animals were given subcutaneous injection of hPTH(1 – 34) (10 or 30 Ag/kg) or vehicle three times a week for 3 weeks. PTH dosage used in this study is about 10 or 30 times the clinical dosage for human osteoporosis treatment. Then surgical transverse osteotomy was performed in the bilateral femora. After surgery, hPTH(1 – 34) treatment was stopped in P10 and P30 groups but continued in C10 and C30 groups three times a week until sacrifice. Vehicle administration was also continued

Fig. 1. Experimental protocol. CNT group: vehicle control treatment; P10 and P30 groups: pretreatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before fracture surgery; C10 and C30 groups: treatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before and after fracture surgery. Subcutaneous injection was performed 3 times a week.

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until sacrifice. The animals were sacrificed at 3, 6, and 12 weeks after surgery. Femoral fracture model Femoral osteotomy and fixation were performed in the same manner as previously reported [14 –17]. Briefly, a transverse osteotomy was made at the midshaft of the femur and intramedullary fixation was performed using a stainless wire (diameter, 1.5 mm). The fracture fragments were contacted and stabilized. Wire was cut on the surface of the intercondylar groove to avoid restriction of motion of the knee joint. Unrestricted activity was allowed after recovery from anesthesia. Evaluation All animals were double fluorescent-labeled subcutaneously with calcein (6 mg/kg; Wako, Ltd., Osaka, Japan) 7 and 2 days before sacrifice. Right after the animals were sacrificed, the bilateral femora were collected. The intramedullary wires were extracted without difficulty from the fractured femora. After all the femora were dissected free of soft tissue, anteroposterior soft radiographs were taken (30 kVp, 2 mA, 30 s; SOR-40; Sofron, Tokyo, Japan). The left femora were frozen at 80-C wrapped in gauze soaked in isotonic saline until peripheral computed tomography (pQCT) measurement. After thawing at room temperature, the left femora were scanned by pQCT (Norland/Stratec XCT Research SA; Stratec Medizintechnic GmbH, Pforzheim, Germany). The bones were placed horizontally inside a glass tube and scanned using voxel size 0.12 mm. The scan line was adjusted using the scout view of the pQCT system. For analysis, a constant threshold of 464 mg/cm3 was used to separate the bone area from the marrow. The volumetric total bone mineral density (BMD, mg/cm3) of the fracture

plane was calculated. The cross-sectional moment of inertia (CSMI; mm4) was also calculated. Immediately after the measurement of pQCT, a threepoint bending test was performed on the fracture site of the left femora using a load mechanical testing machine (MZ500S, Maruto, Tokyo, Japan). The femur was placed with its anterior surface facing upward on two lower support bars 15 mm apart, and the loading bar was positioned at the fracture site (anteroposterior position). A load was applied at a rate of 2.5 mm/min until breakage. The ultimate load (N; maximum force that the specimen sustained), stiffness (N/ mm; the slope of the linear portion of the load-deformation curve), and work to failure (N mm; the area under the loaddeformation curve prior to failure) were calculated from the load – deformation curve by a connected computer. As previously described, the load-displacement data were normalized to obtain intrinsic material properties such as ultimate stress (MPa), elastic modulus (GPa), and toughness (MJ/m3), which are independent of cross-sectional size and shape [24]. After mechanical testing, the left femora were repositioned, fixed in 70% ethanol, stained in Villanueva bone stain, dehydrated in increasing concentration of ethanol, defatted in xylene, and embedded in methyl methacrylate. Two 200-Am-thick cross-sections were cut using a diamond microtome saw (SP1600, Leica Inst., Nussloch, Germany) in an area within 500 Am from the original fracture line, and then ground to 100-Am thickness for contact microradiographs (15 kVp, 2 mA, 10 min; SRO-40, Sofron, Tokyo, Japan) and 30-Am thickness for histomorphometry. The right femora were fixed in 10% cold neutral buffered formalin for 3 days and then decalcified in 10% EDTA at 4-C for 4 weeks. The specimens were embedded in paraffin and 5Am-thick cross-sections were cut in an area within 500 Am from the original fracture line. The section was stained with tartrate-resistant acid phosphatase (TRAP) using a leukocyte acid phosphatase kit (Sigma, St Louis, MO, USA).

Fig. 2. Two 200-Am-thick cross-sections were cut in an area within 500 Am from original fracture line and then ground to 100-Am thickness for contact microradiogram and to 30-Am thickness for histomorphometry. Histomorphometric measurement was performed on whole area and further on four specified areas.

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Fig. 3. Soft X-ray radiographs of fractured femora. At 3 weeks after fracture, the fracture line is visible in all groups. At 6 weeks, most of the fracture lines are still visible. At 12 weeks, the fracture line is invisible in all groups. The size of callus is not different among groups at any time point.

Fig. 4. Contact microradiographs of cross-sectional specimens at the fracture plane. At 3 weeks after fracture, the callus has a mostly porous structure, with no difference in size among the groups. At 6 weeks, the callus appears dense and new cortical shell is apparent. At 12 weeks, the size of callus is reduced compared to 3 and 6 weeks after fracture, and thicker new cortical shells are observed especially in C10 and C30 groups.

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Table 1 Bone mineral density and cross-sectional moment of inertia Group 3 weeks after fracture BMD (g/cm3) CSMI (mm4) 6 weeks after fracture BMD (g/cm3) CSMI (mm4) 12 weeks after fracture BMD (g/cm3) CSMI (mm4)

CNT

P10

P30

C10

C30

738.3 T 40.2 34.4 T 12.7

699.3 T 27.5 14.5 T 4.8

698.3 T 30.4 27.0 T 7.6

674.3 T 21.5 34.5 T 7.3

712.4 T 31.0 21.2 T 7.4

863.3 T 26.7 27.2 T 2.0

813.2 T 24.7 43.6 T 11.1

870.7 T 46.7 31.7 T 9.7

904.2 T 27.5 27.4 T 6.7

875.0 T 38.4 46.9 T 12.2

981.4 T 8.1 23.6 T 5.7

977.2 T 31.8 16.2 T 1.6

1068.5 T 27.5a 16.7 T 1.6

1110.4 T 52.0a,b 16.7 T 4.2

1118.1 T 18.4a,b 18.1 T 3.0

CNT group: vehicle control treatment; P10 and P30 groups: pretreatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before fracture surgery; C10 and C30 groups: treatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before and after fracture surgery; BMD, bone mineral density; CSMI, cross-sectional moment of inertia. Data are expressed as means T SEM. a P < 0.05 vs. P10. b P < 0.05 vs. CNT.

Histomorphometric analyses were performed with 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). Polarized light was applied to distinguish lamellar bone from woven bone. Total cross-sectional area (T.Ar, mm2), medullary area (M.Ar, mm2), and new cortical shell area were measured at 10 magnification, and bone area (B.Ar = T.Ar M.Ar, mm2) was calculated. Percent bone area (%B.Ar = B.Ar  100/T.Ar, %) and percent new cortical shell area (new cortical shell area/T.Ar, %) were also calculated. In these whole bone measurements, we defined callus area as newly formed bone tissue outside of the original cortex, medullary area as non-bone tissue inside of total cross-sectional area, and new cortical shell as thickened outer shell locating outside of original cortex. Further measurements were made at 100

magnification in the four specified areas, anterior, posterior, medial, and lateral aspects as previously described (Fig. 2) [14]. Callus area (Ca.Ar), callus surface (Ca.S), single-labeled surface (sLS), double-labeled surface (dLS), and interlabeling width (Ir.L.Wi) were measured. Diffuse labeled area was excluded from the measurement. Mineral apposition rate (MAR; Am/day), callus area based bone formation rate (BFR/Ca.Ar; %/ year), and percent lamellar area (Lamellar/Ca.Ar, %) were calculated. Osteoclast measurements were also performed at 100 magnification in the four standardized quarters. The number of osteoclast (N.Oc) was counted and then N.Oc/Ca.S (#/mm) was calculated. Statistical analysis Statistical computation of data was performed using the statistical package Stat View 5.0 (SAS Institute, Inc., Cary,

Table 2A Mechanical test (structural properties) Group 3 weeks after fracture Ultimate load (N) Stiffness (N/mm) Work to failure (N mm) 6 weeks after fracture Ultimate load (N) Stiffness (N/mm) Work to failure (N mm) 12 weeks after fracture Ultimate load (N) Stiffness (N/mm) Work to failure (N mm)

CNT

P10

P30

C10

C30

28.9 T 5.2 154.0 T 16.5 21.1 T 8.0

23.1 T 3.6 155.4 T 11.2 15.8 T 3.4

46.6 T 13.4 226.2 T 42.1 29.6 T 7.5

38.2 T 5.5 175.3 T 18.0 18.3 T 3.2

28.6 T 6.8 165.4 T 17.1 17.1 T 4.4

150.6 T 11.7 387.7 T 30.7 53.5 T 8.5

150.6 T 18.2 410.5 T 27.6 64.2 T 17.4

109.5 T 16.6 371.8 T 44.4 42.7 T 6.2

178.3 T 34.1 369.5 T 28.1 71.1 T 24.0

145.6 T 9.0 380.9 T 27.6 42.0 T 5.2

154.7 T 14.7 465.3 T 56.8 75.0 T 27.0

160.2 T 16.7 420.5 T 27.5 87.0 T 22.1

160.4 T 13.5 427.6 T 42.6 73.5 T 19.7

180.1 T 7.5 439.0 T 25.5 74.5 T 18.6

229.7 T 28.6 521.3 T 57.6 74.2 T 16.3

a,b,c

CNT group: vehicle control treatment; P10 and P30 groups: pretreatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before fracture surgery; C10 and C30 groups: treatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before and after fracture surgery. Data are expressed as means T SEM. a P < 0.05 vs. CNT. b P < 0.05 vs. P10. c P < 0.05 vs. P30.

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Table 2B Mechanical test (intrinsic material properties) Group 3 weeks after fracture Ultimate stress (MPa) Elastic modulus (GPa) Toughness (MJ/m3) 6 weeks after fracture Ultimate stress (MPa) Elastic modulus (GPa) Toughness (MJ/m3) 12 weeks after fracture Ultimate stress (MPa) Elastic modulus (GPa) Toughness (MJ/m3)

CNT

P10

P30

C10

C30

15.8 T 5.5 0.49 T 0.19 2.39 T 1.37

32.4 T 7.4 1.44 T 0.40 4.80 T 1.60

19.8 T 2.6 0.83 T 0.25 2.36 T 0.51

17.5 T 5.8 0.74 T 0.39 2.07 T 1.11

25.2 T 7.3 1.65 T 0.99 3.02 T 1.04

50.6 T 3.9 1.01 T 0.05 2.25 T 0.31

42.6 T 7.0 0.96 T 0.27 2.38 T 0.64

40.9 T 10.5 1.19 T 0.32 1.87 T 0.51

42.1 T 6.7 1.20 T 0.30 1.55 T 0.54

36.7 T 8.7 0.81 T 0.25 1.50 T 0.48

49.4 T 8.2 1.57 T 0.25 2.32 T 0.82

66.5 T 9.0 1.94 T 0.22 3.25 T 0.73

74.4 T 7.2 1.95 T 0.33 3.67 T 0.92

82.7 T 19.4 2.61 T 0.65 2.53 T 0.55

98.3 T 7.9 2.26 T 0.30 3.39 T 0.70

CNT group: vehicle control treatment; P10 and P30 groups: pretreatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before fracture surgery; C10 and C30 groups: treatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before and after fracture surgery. Data are expressed as means T SEM.

NC, USA). Differences among treatment groups were tested by one-way analysis of variance (ANOVA). If significant differences were indicated, 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 No difference in body weight was observed among all groups during the study period. The animals resumed normal activity within a few days after surgery. Of 105 animals that underwent fracture surgery, 11 were excluded

Table 3 Bone histomorphometry in fracture callus Group 3 weeks after fracture T.Ar (mm2) %B.Ar (%) Lamellar/Ca.Ar (%) MAR (Am/day) BFR/Ca.Ar (%/year) N.Oc/Ca.S (#/mm) 6 weeks after fracture T.Ar (mm2) % B.Ar (%) Lamellar/Ca.Ar (%) MAR (Am/day) BFR/Ca.Ar (%/year) N.Oc/Ca.S (#/mm) 12 weeks after fracture T.Ar (mm2) % B.Ar (%) Lamellar/Ca.Ar (%) MAR (Am/day) BFR/Ca.Ar (%/year) N.Oc/Ca.S (#/mm)

CNT

P10

P30

C10

C30

20.1 44.8 3.82 2.42 353.5 0.90

T T T T T T

2.3 1.9 2.11 0.86 153.2 0.22

19.9 43.9 1.75 3.13 234.2 0.98

T T T T T T

2.4 2.7 0.84 0.52 49.9 0.09

24.0 44.5 7.15 3.84 331.3 1.04

T T T T T T

2.5 2.0 2.45 0.15 42.5 0.09

24.2 47.5 25.38 3.88 524.3 1.02

T T T T T T

2.0 1.4 6.57a,b,c 0.19 75.4b 0.08

20.5 52.3 35.5 4.21 488.3 1.45

T T T T T T

22.6 46.6 98.0 4.17 818.2 0.76

T T T T T T

0.9 1.6 1.1 0.17 72.1 0.16

25.4 45.2 97.6 4.48 786.2 0.56

T T T T T T

2.5 1.4 0.6 0.24 116.8 0.14

24.4 49.5 98.0 4.45 714.4 0.53

T T T T T T

4.8 4.4 0.7 0.55 136.5 0.16

18.8 56.4 98.7 4.12 742.2 0.73

T T T T T T

2.2 5.7a,b 0.6 0.74 134.9 0.14

24.5 57.2 100 4.38 594.9 1.33

T 3.0 T 2.6a,b

20.5 45.0 100 3.64 500.8 0.39

T 1.8 T 2.1

17.0 52.7 100 3.25 371.2 0.29

T 1.6 T 5.1

18.8 50.8 100 3.31 374.8 0.20

T 8.4 T 1.8

16.5 61.0 100 3.33 330.2 0.35

T 1.1 T 3.2a,c

17.8 68.7 100 3.51 235.2 0.83

T 0.24 T 73.6 T 0.10

T 0.25 T 79.0 T 0.08

T 0.31 T 60.8 T 0.07

T 0.25 T 75.2 T 0.12

1.7 1.0a,b,c 4.94a,b,c 0.15 37.5b 0.19a,b,c,d

T 0.28 T 70.6 T 0.30a,b,c,d T 1.7 T 1.3a,b,c T 0.31 T 37.1 T 0.19a,b,c,d

CNT group: vehicle control treatment; P10 and P30 groups: pretreatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before fracture surgery; C10 and C30 groups: treatment with hPTH(1 – 34) 10 and 30 Ag/kg, respectively, before and after fracture surgery; T.Ar: total cross-sectional area; %B.Ar: percent bone area; Lamellar/Ca.Ar: percent lamellar area; MAR: mineral appositional rate; BFR/Ca.Ar: bone formation rate per unit callus area; N.Oc/Ca.S: number of osteoclasts per unit callus surface. Data are expressed as means T SEM. a P < 0.05 vs. CNT. b P < 0.05 vs. P10. c P < 0.05 vs. P30. d P < 0.05 vs. C10.

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because of technical failure in surgery (poor fracture fixation), death during or after surgery, or post surgical infection. Finally, 31, 31, and 32 animals were evaluated and analyzed at 3, 6, and 12 weeks after surgery, respectively. Radiology Soft X-ray observation showed that the sizes of fracture callus were similar among groups at all time points (Fig. 3). At 3 weeks after fracture, the fracture line was visible in all groups. At 6 weeks, most fracture lines were still visible. At 12 weeks, the fracture line was invisible in all groups. Contact microradiographs at 3 weeks after fracture revealed that the structure of callus was mostly porous, with no difference in size among groups (Fig. 4). At 6 weeks after fracture, the callus became dense and a new cortical shell became apparent. At 12 weeks after fracture, the size of callus was reduced compared to 3 and 6 weeks

after fracture, and thicker new cortical shells were observed especially in C10 and C30 groups. Peripheral quantitative computed tomography measurement The BMD of the fracture site was not significantly different among all groups at both 3 and 6 weeks after fracture. However, at 12 weeks after fracture, BMD was significantly higher in P30, C10, and C30 groups than in CNT group (Table 1). No significant difference in CSMI was observed among all groups at all time points after fracture. Mechanical test The ultimate load, stiffness, and work to failure were not significantly different among groups at both 3 and 6 weeks after fracture (Table 2A). However, at 12 weeks after fracture, the ultimate load in C30 group was significantly higher than those in CNT, P10, and P30 groups. Intrinsic material properties, such as ultimate stress, elastic modulus,

Fig. 5. Histological findings of the callus under epifluorescent light: at 3 weeks after fracture, the structure of callus was woven-like and most of surface was diffusely labeled. At 6 weeks after fracture, most of the callus was consisting of lamellar bone with linear labels, and new cortical shell was appeared. At 12 weeks after fracture, linear labels were found only in the internal surface of thickened, less porous new cortical shell.

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and toughness, were not significantly different among groups at all time points (Table 2B). However, at 12 weeks after fracture, ultimate stress in C30 group tends to increase compared to CNT group.

percent new cortical shell area in both C10 and C30 groups was significantly higher than those in CNT, P10, and P30 groups (Fig. 6).

Histology and histomorphometry

Discussion

Observation of the natural time course after fracture (CNT) showed that Lamellar/Ca.Ar increased dramatically at 6 weeks and reached 100% at 12 weeks after fracture (Table 3). New cortical shell (%New cortical shell area) appeared and increased in a time-dependent manner (Fig. 4). However, no changes in T.Ar and %B.Ar were observed. BFR/Ca.Ar was highest at 6 weeks during the experimental period (Fig. 5). In contrast, N.Oc/Ca.S showed maximum value at 3 weeks after fracture, and then tended to decrease with time. Following PTH treatment, %B.Ar was increased in C30 group at all time points and in C10 at 6 and 12 weeks after fracture. T.Ar was not significantly different among all groups in any time point. Lamellar/Ca.Ar was significantly higher in C10 and C30 groups than in CNT, P10, and P30 groups at 3 weeks after fracture. MAR was not significantly different among all groups at each time point. BFR/Ca.Ar was significantly higher in C10 and C30 groups than in P10 group at 3 weeks after fracture, but was not significantly different among groups at 6 and 12 weeks after fracture. N.Oc/Ca.S was significantly higher in C30 than in other groups at all time points. At 6 weeks after fracture, percent new cortical shell area was significantly higher in C10 group than in CNT, P10, and P30 groups. At 12 weeks after fracture,

The primary aim of this study was to examine the effect of intermittent treatment of hPTH(1 – 34), a potent anabolic agent, on the fracture healing process in a rat fracture model. Our results clearly showed that PTH accelerated the remodeling of woven bone to lamellar bone, the formation of new cortical shell, and the increasing of fractured bone strength. Many studies have confirmed that the rat fracture model is adequate to investigate the fracture healing mechanism and the effects of intervention factors on the healing process [14 –17,25– 27]. Histomorphometry and mechanical tests have shown that fracture healing is a closely regulated process that restores the structural geometry and the mechanical properties of the broken bone [28 –30]. Early in the fracture healing process, cartilage and woven bone are rapidly produced to stabilize the fracture. After the fractured bone is primarily stabilized, woven bone is remodeled to lamellar bone and a new cortical shell is formed. Eventually, the fractured bone is geometrically and mechanically restored to the original structure. The current study demonstrated that PTH increased the mechanical strength of fractured bone through accelerating the remodeling of woven bone to lamellar bone and formation of new cortical shell, without increasing callus size. The implication of these findings is that PTH

Fig. 6. Percent new cortical shell area. At 12 weeks after fracture, percent new cortical shell areas in both C10 and C30 groups are significantly higher than those in CNT, P10, and P30 groups.

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accelerates the natural fracture healing process. The most striking observation of the present study was the early appearance and accelerated formation of new cortical shell following intermittent PTH treatment, which is the most important determinant to restore mechanical strength after fracture. In contrast, bisphosphonate treatment suppresses new cortical shell formation although the amount of newly formed cortical shell has never been quantified [14 –17]. 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 demonstrated that (1) pretreatment of PTH before fracture did not affect fracture healing; (2) continued PTH treatment before and after fracture accelerated the fracture healing process and restoration of mechanical strength. These findings suggest that the clinician may continue PTH treatment after fracture. Several studies have focused on the effect of intermittent treatment of PTH on the fracture healing process up to 8 weeks after fracture [18 – 23]. Andreassen et al. [20] reported that daily hPTH(1 –34) treatment at 200 Ag/kg, the highest dose that can avoid woven bone formation under non-fracture conditions, [31,32] enhanced callus formation and mechanical strength in rat tibial shaft at both 20 and 40 days of healing. However, they found no significant effects of PTH(1 – 34) at 60 Ag/kg. Holzer et al. [21] reported that daily treatment of PTH(1 – 34) at 80 Ag/kg increased callus area and strength at 3 weeks after fracture, but the PTH doses of these studies were much higher than the clinical dose used in osteoporosis treatment. Nakajima et al. [22] reported that daily treatment with low dose (10 Ag/kg/day) hPTH(1 –34) in rats increased the number of osteoclasts and expression of ALP and osteocalcin mRNA within 3 weeks after femoral fracture, and also increased the mechanical strength of the callus at 6 weeks. Since fracture healing is not yet completed at 8 weeks after fracture, longer-term observation is needed to evaluate the remodeling process of fracture callus. Furthermore, dynamic histomorphometry or geometrical analyses of fracture callus have never been performed in previous studies. No significant increases in bone formation parameters following PTH treatment were observed at any time point in the present study. There are several possible explanations for this phenomenon. First, in the early phase of fracture healing, bone formation activities are increased because of callus formation and remodeling. In such a condition, the additional bone forming effect of PTH may have been masked. Second, histomorphometric bone formation parameters are calculated based on linear fluorescent labeling of the mineralization front. However, rapid woven bone production in the early phase of fracture healing is mainly associated with diffuse fluorescent labeling. This condition may have caused the difficulty in detecting increased bone formation activity induced by PTH especially in the early phase of fracture healing. Finally, accelerated geometrical adaptation of fracture callus in the PTH-treated group at 12 weeks after

fracture, as evidenced by higher ratios of new cortical shell, suggests nearer completion of fracture healing compared to CNT. This may cause reduced bone formation activity. There are some limitations of this study. First, our fracture model was a surgically produced osteotomy model, different from a closed fracture model which is similar to most clinical fractures. However, we have valued to make the consistent fracture line, which is important for precise evaluations both mechanically and histologically. Second, we have used three-point bending mechanical test for the evaluation of mechanical properties of fracture callus, because we desired to evaluate the exact point of fracture plane. However, our mechanical results derived from threepoint bending may have been influenced by the nonhomogeneity of the material, such as fracture callus, directly beneath the indenter. We conclude that (1) intermittent treatment of hPTH(1 – 34) at 10 or 30 Ag/kg three times a week had no adverse effect on fracture healing in rat femur; (2) 3 weeks pretreatment with PTH before fracture had no significant effect on fracture healing; (3) intermittent PTH treatment at 10 or 30 Ag/kg before and after fracture accelerated the fracture healing process as evidenced by replacement of woven bone by lamellar bone and increased new cortical shell formation; and (4) the mechanical strength of the remodeled bone was significantly increased at 12 weeks after fracture following intermittent PTH treatment at 30 Ag/kg.

Acknowledgments The authors thank Mika Kawada and Yoshiko Fukuda for histological preparation and Asahi Kasei Co. for kindly supplying the hPTH(1 – 34).

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