The effect of PTH(1-34) on fracture healing during different loading conditions

Authors

  • Maria Ellegaard,

    Corresponding author
    1. Research Center of Ageing and Osteoporosis, Department of Medicine and Diagnostics, Copenhagen University Hospital, Glostrup, Denmark
    • Address correspondence to: Maria Ellegaard, MSc, Research Center of Ageing and Osteoporosis, Copenhagen University Hospital, Glostrup, Nordre Ringvej 69, DK-2600 Glostrup, Denmark. E-mail: maria.ellegaard.larsen@regionh.dk

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  • Tina Kringelbach,

    1. Research Center of Ageing and Osteoporosis, Department of Medicine and Diagnostics, Copenhagen University Hospital, Glostrup, Denmark
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  • Susanne Syberg,

    1. Research Center of Ageing and Osteoporosis, Department of Medicine and Diagnostics, Copenhagen University Hospital, Glostrup, Denmark
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  • Solveig Petersen,

    1. Research Center of Ageing and Osteoporosis, Department of Medicine and Diagnostics, Copenhagen University Hospital, Glostrup, Denmark
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  • Jens-Erik Beck Jensen,

    1. Department of Endocrinology, Copenhagen University Hospital, Hvidovre, Denmark
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  • Annemarie Brüel,

    1. Department of Biomedicine–Anatomy, Aarhus University, Aarhus, Denmark
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  • Niklas Rye Jørgensen,

    1. Research Center of Ageing and Osteoporosis, Department of Medicine and Diagnostics, Copenhagen University Hospital, Glostrup, Denmark
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  • Peter Schwarz

    1. Research Center of Ageing and Osteoporosis, Department of Medicine and Diagnostics, Copenhagen University Hospital, Glostrup, Denmark
    2. Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
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ABSTRACT

Parathyroid hormone (PTH) and PTH(1-34) have been shown to promote bone healing in several animal studies. It is known that the mechanical environment is important in fracture healing. Furthermore, PTH and mechanical loading has been suggested to have synergistic effects on intact bone. The aim of the present study was to investigate whether the effect of PTH(1-34) on fracture healing in rats was influenced by reduced mechanical loading. For this purpose, we used female, 25-week-old ovariectomized rats. Animals were subjected to closed midshaft fracture of the right tibia 10 weeks after ovariectomy. Five days before fracture, half of the animals received Botulinum Toxin A injections in the muscles of the fractured leg to induce muscle paralysis (unloaded group), whereas the other half received saline injections (control group). For the following 8 weeks, half of the animals in each group received injections of hPTH(1-34) (20 µg/kg/day) and the other half received vehicle treatment. Fracture healing was assessed by radiology, dual-energy X-ray absorptiometry (DXA), histology, and bone strength analysis. We found that unloading reduced callus area significantly, whereas no effects of PTH(1-34) on callus area were seen in neither normally nor unloaded animals. PTH(1-34) increased callus bone mineral density (BMD) and bone mineral content (BMC) significantly, whereas unloading decreased callus BMD and BMC significantly. PTH(1-34) treatment increased bone volume of the callus in both unloaded and control animals. PTH(1-34) treatment increased ultimate force of the fracture by 63% in both control and unloaded animals and no interaction of the two interventions could be detected. PTH(1-34) was able to stimulate bone formation in normally loaded as well as unloaded intact bone. In conclusion, the study confirms the stimulatory effect of PTH(1-34) on fracture healing, and our data suggest that PTH(1-34) is able to promote fracture healing, as well as intact bone formation during conditions of reduced mechanical loading. © 2013 American Society for Bone and Mineral Research.

Introduction

Fractures are a major burden not only for the individual patient but also for society. Therefore, the idea of pharmacological stimulation of fracture healing and prevention of nonunion fractures receives more and more attention in clinical practice. However, fracture healing is a complex process that can be influenced by several factors. The mechanical environment of the fracture is of great importance for the healing process and is determined by the degree of fixation, the type of fracture, and the mechanical loading applied to the fracture. The mechanical environment could potentially interact with a pharmacological treatment.[1]

Parathyroid hormone (PTH) is an 84-amino acid polypeptide produced and secreted by the parathyroid glands. The main physiological function of PTH is to maintain calcium levels of the blood by stimulating bone resorption as well as calcium uptake from the intestine and calcium reuptake from the kidneys. The biological activity is retained in the fragment containing the first 34 amino acids of the peptide (PTH(1-34)).

In experimental models of osteoporosis where bone loss is induced by ovariectomy, treatment with PTH(1-84) and PTH(1-34) once daily leads to increased osteoblastic activity resulting in recovery of bone mass and increase in mechanical strength of the bones,[2-5] which is in contrast to the effects of continuously administered PTH. Today, PTH is in clinical use for the treatment of severe osteoporosis, where it increases bone mineral density (BMD) and reduces the risk of fracture.[6, 7] Furthermore, PTH treatment has been shown to decrease back pains and increase the life quality in osteoporotic patients.[8, 9]

PTH has also been shown to promote fracture healing in several animal studies.[10] These studies indicate that PTH exerts its effect on several stages of the healing process. In the early stages, there is an increase in chondrocyte proliferation and differentiation as well as in cartilage production.[11, 12] In the later stages, there is an increase in osteoblast differentiation, bone formation, and remodeling of callus.[13] The effects of PTH on fracture healing are seen as increased callus area, increased callus BMD, and increased mechanical strength of the fracture.[14-16]

A few clinical studies also suggest some benefit of PTH treatment in relation to fracture healing, although these results are not as convincing as those observed in animal studies.[17, 18] One study investigated the effect of PTH(1-34) on fractures of the distal radius and found that PTH slightly reduced the time to cortical bridging; however, no effect on the functional outcome was observed.[18] In another study of fractures of the pubic bone, PTH(1-84) was found to reduce the time to healing and improve both functional outcome and pain score.[17] This could suggest that the effects of PTH might be side specific, e.g., weight bearing versus non-weight bearing.

It has been suggested that PTH and mechanical loading have synergistic effects on intact bone, and a number of studies have investigated the bone anabolic effects of PTH in intact bone in combination with mechanical loading.[19-25] In general, it is not completely clarified whether PTH and mechanical loading display additive or synergistic effects. There might also be compartment-specific effects.[20, 24] In studies regarding PTH treatment during conditions of disuse, there are also some conflicting results.[26-35] Some studies suggest that PTH is able to reduce bone loss resulting from disuse,[28, 29, 31] whereas other studies show that disuse attenuates the effects of PTH.[27, 34] Osteoprogenitor cells from unloaded rats have also been shown to be resistant to PTH stimulation.[30]

Gardner and colleagues investigated the effect of combined PTH treatment and mechanical loading in a mouse tibia osteotomy model. They found that callus mineral density and bone volume was significantly increased in response to combined treatment with PTH and loading. This was not the case for the individual treatments alone.[36]

It is highly relevant to explore whether a potential beneficial effect of PTH on fracture healing would be dependent on the mechanical environment of the fracture, and to the best of our knowledge, no studies of the effect of reduced mechanical loading on PTH-stimulated fracture healing have been published. The aim of the present study was therefore to investigate the effect of PTH on fracture healing under different loading conditions using a rat tibia fracture model, where unloading was induced by repeated injections of Botulinum Toxin Type A (Botox). Fracture healing was assessed by radiological measurements of callus area, dual-energy X-ray absorptiometry (DXA) analysis of callus BMD, callus histology, and measurements of biomechanical strength of callus.

Materials and Methods

Study design

Twenty-five-week-old female nulli para Sprague Dawley rats (Taconic, Lille Skensved, Denmark) were ovariectomized (OVX) to induce osteopenia. Four OVX animals and four sham-operated animals were euthanized at baseline. Ten weeks after ovariectomy, the animals were stratified according to body weight into four groups and subjected to unilateral standardized closed fracture of the right tibia. Animals in the Botox Vehicle group and Botox PTH group received Botox injection (im) in the right leg, and animals in the Control Vehicle group and Control PTH group received saline injections. The left leg remained untreated in all groups. For the following 8 weeks, animals in the Botox Vehicle group and Control Vehicle group were injected subcutaneously 5 days a week with vehicle (0.9% saline + 0.1% bovine serum albumin) and animals in the Botox PTH group and Control PTH group were injected subcutaneously 5 days a week with 20 µg/kg human PTH(1-34) (Bachem H-4835, lot 1027 193). The animals were euthanized by exsanguination by cardiac puncture 8 weeks after fracture.

Animals

Study protocols were performed in compliance with the bioethical guidelines for animal research and approved by the Danish Animal Experiments Inspectorate. Rats were housed in Macrolon type 4 cages (3 to 4 rats/cage) under controlled conditions after a 12-hour light and 12-hour dark cycle with light on at 6 a.m. The animals were fed ad libitum with standard Altromin and had free access to drinking water (domestic quality tap water). The animals were ovariectomized from the supplier at age 25 weeks. Four animals were sham operated. They were allowed full recovery before shipment (2 weeks after surgery).

Botox injections and digit abduction score

Botox (Allergan Inc., Irvine, CA, USA) was dissolved in 0.9% saline and diluted to a final concentration of 1 U/100 µL. The rats were injected in the hamstrings, the quadriceps, and the calf muscle of the right leg using a 27-gauge needle. Rats received 2.5 U Botox (0.5 U, 1 U, and 1 U, respectively) 5 days before fracture; 2 U (0.5 U, 0.5 U, and 1 U, respectively) 2 weeks after fracture; and 1.5 U (0.5 U, 0.5U, and 0.5 U, respectively) 6 weeks after fracture. To evaluate muscle paralysis, digit abduction score (DAS) assay was performed as described by Aoki and colleagues.[37] Rats were suspended briefly by the tail to elicit a startle response, in which the animal extends its hind limbs and abducts its hind digits. The degree of hind digit abduction was scored using a five-point scale (0 = normal to 4 = maximal).

Fracture

Ten weeks after ovariectomy at age 35 weeks, unilateral standardized closed mid-diaphyseal fracture of the right tibia was performed (modified from Bak and Andreassen[38]). The rats were anesthetized with a subcutaneous injection of 0.2 mL/100 g of a 1:1 mixture of Hypnorm (fentanyl citrate 0.315 mg/mL and fluanisone 10 mg/mL) and Dormicum (midazolam 5 mg/mL) diluted 1:1:2 in distilled water. The right leg was shaved and disinfected. Closed intramedullary nailing was performed through the cortex of the proximal tibia using a 0.8-mm-thick Kirschner wire. The skin was closed with monofilament nylon sutures. Fractures were generated just above the tibiofibular junction by three-point bending in anteroposterior direction, using a custom-made fracture device. Animals were subjected to X-ray immediately after fracture to verify that mid-diaphyseal fractures had been produced. To relieve postoperative pain, all rats were treated with carprofen (Rimadyl 5 mg/kg) 3 days after surgery. Animals were single-housed until the day after surgery.

Radiology

Fracture healing was examined every second week by radiographs of the tibias using a Faxitron MX-20 small specimen X-ray system (Faxitron, Tucson, AZ). The rats were anesthetized before the procedure. Pictures were taken at magnification 1× (26 kv/11 seconds) and 3× (35 kv/12 seconds) in duplicate. External callus area was measured on X-rays using Osiri X software and calculated as the mean of right and left side of the external callus in duplicate (mean of four measurements per animal).

DXA

BMD, bone mineral content (BMC), and bone area measurements were performed using a Lunar Piximus densitometer (Lunar Corp., Madison, WI, USA) after 4 and 8 weeks. Right and left tibias and femurs were scanned. Each leg was measured in duplicate and the mean was calculated. BMD, BMC, and bone area of callus (including the original bone) was determined within a fixed region of interest (ROI) (w: 53 pixels and L: 42 pixels). ROI was placed with the fracture line in the center and the ends of the ROI perpendicular to the pin. Furthermore, BMD of the total femur was analyzed after 8 weeks to evaluate effects on intact bone.

Bone strength measurements

Biomechanical properties of the fracture site, the contralateral intact tibia, and right and left femur were examined ex vivo. Right and left hind limb was collected after sacrifice, cleaned for soft tissue, wrapped in saline-moistened gaze, and stored at −20°C until testing. Measurements were performed on a Lloyd material testing device LR50K (Lloyd Instruments, Fareham, UK). The intramedullary nail in the fractured leg was removed before testing. The femoral neck was subjected to compression test. The femoral and tibial diaphysis as well as the fracture was subjected to three-point bending test. For three-point bending test, the bones were placed on a cylinder (diameter 18 mm) with the fracture line or mid-femur in the center. Deflection was performed by lowering a third bar onto the fracture line using a constant speed of 2 mm per minute and a 500 N load cell. The intact left tibia was tested at a point corresponding to the fracture line of the right tibia. Load-deflection curves were generated and ultimate force was recorded as the maximum load applied until fracture, stiffness was calculated as the slope of the curve, and work to failure was calculated as area under curve.

Histomorphometry

Ten and 2 days before euthanization, animals were injected ip with 20 mg/kg (1 ml/100g) of calcein (Sigma, St. Louis, MO, USA). Five animals from each group were randomly selected for histomorphometric analysis and the right and left leg were stored in 70% ethanol at 4°C. The bones were dehydrated in a graded series of ethanol solutions and embedded in methyl methacrylate. The bones were sectioned longitudinally through the frontal plane (7 µm thick) using a Polycut E microtome and a tungsten-carbide knife. Sections were mounted on gelatine-coated slides and dried for 1 hour at 40°C before further treatment. Five sections from each tibia were stained with Goldner's trichrome, and five sections were left unstained for analysis of fluorochrome labels. Sections were analyzed using an Olympus BX51 microscope attached to an image analyzer (CAST-GRID system from Olympus Denmark 2000).

The following indices were measured in the intact proximal tibia: percentage of eroded surfaces (ES/BS), bone volume fraction (BV/TV), cortical thickness (Ct.th.), trabecular thickness (Tb.th.), percentage of mineralized bone surface (MS/BS), and mineral apposition rate (MAR).

BV/TV of the entire external callus and the mid-part of the fracture line (including only newly formed bone) was analyzed in stained sections. MAR was measured in the external callus and fracture line in unstained sections.

Statistics

Differences between baseline sham and OVX was tested using Mann-Whitney U test. Data were tested for normality using the Kolmogorov-Smirnoff test. Main effects and interaction effect of PTH(1-34) treatment and unloading was analyzed by two-way ANOVA with Botox and PTH(1-34) treatment as fixed factors. Levene's test was used to test for equal variances between groups. In case of unequal variances (callus BMD week 8 and tibial stiffness) differences were considered significant if p ≤ 0.025 (α/2) as suggested by Keppel. Otherwise, differences were considered significant if p ≤ 0.05. Pairwise comparisons of groups were made using LSD post hoc tests. Data are presented as mean ± SEM. Histomorphometric data were analyzed using nonparametric statistics (Kruskal-Wallis tests followed by Mann-Whitney post hoc tests) because of the low number. Data are presented as median (25th, 75th percentiles). Differences were considered significant when p ≤ 0.05.

Results

Health and mobility

One animal died during anesthesia at study start, and 7 animals were excluded because of failure of pin insertion or fracture (n = 13 to 14 in each group). All groups suffered a minor weight loss during the first week after surgery (p < 0.001). At study end, Botox-treated animals had approximately 12% lower body weight compared with controls (p < 0.05) (data not shown). With the exception of weight loss, no adverse events were seen after Botox injections and no adverse events were seen after PTH(1-34) treatment. Paralysis was confirmed by digit abduction score, and animals remained paralyzed during the 8-week study period (data not shown).

Four OVX animals and 4 sham-operated animals were euthanized at baseline, and body weight and BMD were measured. Body weight was 25% higher in the OVX animals compared with the control animals (p = 0.021). No significant difference in BMD or BMC of the total femur could be detected; however, bone area of the total femur was significantly higher in the OVX animals compared with the sham animals (p = 0.02). If using ANCOVA for correction of differences in body weight, there was a 13% decrease in femoral BMD in the OVX animals compared with sham animals (ns) (data not shown).

Radiology

Representative X-rays are presented in Fig. 1. After 2 weeks, only very diffuse callus appeared on the X-rays, and measurement of callus area was not technically possible. Two-way ANOVA indicated that only unloading had an effect on callus area, and there was no interaction between unloading and PTH(1-34) treatment (13 to 14 animals per group). This was the case at all three time points. Callus area was significantly lower in the unloaded animals compared with control animals (p < 0.001). PTH(1-34) treatment only led to a nonsignificant increase in callus area in the control animals after 4 and 6 weeks, whereas after 8 weeks they were comparable (Fig. 2).

Figure 1.

Representative X-rays illustrating external callus development from baseline and 4 and 8 weeks after fracture. Arrow indicates fracture line.

Figure 2.

Radiological callus area measured after 4, 6, and 8 weeks of healing. Callus area was significantly reduced in the unloaded animals, whereas PTH(1-34) treatment had no significant effect on callus area. No interaction between PTH(1-34) and unloading was found. Data are presented as mean ± SEM (p < 0.05, n = 13–14).

DXA

Callus

There were significant effects on callus BMD and callus BMC after 4 and 8 weeks of both Botox and PTH(1-34) (4 weeks: 10 to 12 animals per group; 8 weeks: 12 to 14 animals per group). Furthermore, there was a significant interaction effect of Botox and PTH(1-34) on BMD and BMC at both time points (Fig. 3A, B).

Figure 3.

Bone mineral density (A) and bone mineral content (B) of the callus after 4 and 8 weeks. BMD and BMC of callus were reduced in unloaded animals, whereas PTH(1-34) increased callus BMD and BMC. Data are presented as mean ± SEM. Significant difference compared with control vehicle (*) or Botox vehicle (#) at the respective time point (p < 0.05; 4 weeks: n = 10–12; 8 weeks: n = 12–14).

Unloading significantly decreased callus BMD by 16% after 4 weeks and by 22% after 8 weeks (p = 0.002 and p < 0.001, respectively). PTH(1-34) increased callus BMD significantly in the unloaded animals after 4 weeks by 13% (p = 0.028) and after 8 weeks by 22% (p = 0.003) when compared with vehicle-treated unloaded animals. In the control animals, PTH(1-34) increased BMD by 31% after 4 and 39% after 8 weeks when compared with control vehicle group (p < 0.001 for both).

BMC increased significantly in both Botox and control animals after 4 weeks of PTH(1-34)-treament (p = 0.009 and p = 0.001, respectively). After 8 weeks, BMC was significantly increased only in the control animals (p < 0.001). BMC was significantly decreased in the Botox animals compared with controls after 4 and 8 weeks (p < 0.001 for both).

Intact bone

In the right injected leg, two-way ANOVA did not reveal any significant interaction between Botox and PTH(1-34) (p = 0.702) (13 to 14 animals per group). Femoral BMD was significantly decreased by 25% after 8 weeks of unloading (p < 0.001), and PTH(1-34) significantly increased BMD by 25% in the Botox animals and 20% in the control animals (p < 0.001) (Fig. 4A). In the left non-injected leg, there was no effect of Botox (p = 0.924), whereas PTH(1-34) significantly increased BMD (p < 0.001) (Fig. 4B).

Figure 4.

Bone mineral density of the total femur in (A) the right injected leg and (B) the left noninjected leg after 8 weeks. Botox-induced unloading decreased BMD in the right leg, whereas no change was seen in the left leg. PTH(1-34) treatment increased BMD in both the unloaded leg and in controls. Data are presented as mean ± SEM. *Significant difference from control vehicle; # significant difference from Botox vehicle (p < 0.05, n = 13–14).

Biomechanical bone strength measurements

Callus

To evaluate the strength of the fracture after 8 weeks of healing, three-point bending tests were performed in 8 to 9 animals from each group. All bones fractured in the original fracture line. Ultimate force, stiffness, and work to failure of callus were calculated from load-displacement curves. Two-way ANOVA confirmed an effect of both Botox-induced unloading and PTH(1-34) treatment on ultimate force (p < 0.001 for both); however, no interaction between PTH and unloading was seen (p = 0.178). Ultimate force of callus was significantly reduced by 50% in the Botox animals compared with the control animals (p = 0.004), whereas PTH(-1-34) treatment increased ultimate force with approximately 63% in both Botox and control animals when compared with respective vehicle group. This reached statistical significance in the control animals only (p < 0.001), whereas it was borderline significant in the Botox animals (p = 0.051) (Fig. 5A).

Figure 5.

Biomechanical strength parameters of callus. Callus was subjected to three-point bending test, and (A) ultimate force, (B) stiffness, and (C) work to failure were recorded. Two-way ANOVA revealed significant effects of both Botox-induced unloading and PTH(1-34) treatment on callus strength; however, no interaction between the treatments was observed. *Significant difference from control vehicle in pairwise comparison (p < 0.05; n = 8–9). Data are presented as mean ± SEM relative to control vehicle.

Callus stiffness and work to failure revealed a similar pattern, and two-way ANOVA indicated that PTH(1-34) treatment also increased callus stiffness (p = 0.003) and work to failure (p = 0.039) significantly. Botox-induced unloading reduced work to failure of the callus significantly (p < 0.001), whereas the effect of stiffness was only borderline significant (p = 0.052) (Fig. 5B, C). No interaction between the effect of PTH(1-34) and Botox on either stiffness (p = 0.684) or work to failure (p = 0.135) was seen.

Intact bone

Two-way ANOVA revealed significant effects on ultimate force, stiffness, and work to failure of the right femoral shaft and femoral neck of PTH(1-34) treatment (7 to 9 animals per group). Significant effects of Botox on ultimate force of the femoral shaft and ultimate force and stiffness of the femoral neck were seen. No significant interaction effect was seen on any mechanical parameters (Table 1).

Table 1. Relative Changes in Biomechanical Strength Parameters of Intact Bone
 Mean ± SEM relative to controlTwo-way ANOVA p values
Control vehicleControl PTHBotox vehicleBotox PTHPTHBotoxBotox*PTHn
  • *Unequal variances; p level adjusted to 0.025.
Right leg
Femoral shaft
Ultimate force1.00 ± 0.021.14 ± 0.040.86 ± 0.041.13 ± 0.04<0.0010.0360.0988–9
Stiffness1.00 ± 0.031.14 ± 0.040.89 ± 0.031.12 ± 0.05<0.0010.1240.2298–9
Work to failure1.00 ± 0.041.12 ± 0.090.80 ± 0.081.23 ± 0.150.0080.6100.1178–9
Femoral neck
Ultimate force1.00 ± 0.051.17 ± 0.040.80 ± 0.061.00 ± 0.080.0030.0030.7627–9
Stiffness1.00 ± 0.031.06 ± 0.060.81 ± 0.051.02 ± 0.040.0110.0270.1357–9
Work to failure1.00 ± 0.091.62 ± 0.180.80 ± 0.091.36 ± 0.240.0010.5030.6787–9
Left leg
Femoral shaft
Ultimate force1.00 ± 0.051.15 ± 0.040.99 ± 0.021.15 ± 0.03<0.0010.8410.9328–9
Stiffness1.00 ± 0.061.17 ± 0.050.99 ± 0.031.21 ± 0.05<0.0010.7590.5928–9
Work to failure1.00 ± 0.051.00 ± 0.080.79 ± 0.051.04 ± 0.080.0610.2120.0758–9
Femoral neck
Ultimate force1.00 ± 0.071.17 ± 0.061.04 ± 0.051.26 ± 0.080.0050.3320.6577–9
Stiffness1.00 ± 0.121.19 ± 0.191.08 ± 0.031.30 ± 0.030.0730.4230.8947–9
Work to failure1.00 ± 0.101.16 ± 0.081.07 ± 0.111.33 ± 0.130.0170.5200.4597–9
Tibial shaft
Ultimate force1.00 ± 0.050.95 ± 0.020.91 ± 0.030.96 ± 0.030.8890.2970.1438–9
Stiffness1.00 ± 0.080.83 ± 0.030.80 ± 0.030.82 ± 0.040.122*0.029*0.039*8–9
Work to failure1.00 ± 0.050.88 ± 0.080.85 ± 0.071.04 ± 0.10.6160.9960.0558–9

In the left noninjected leg, no effect of Botox-induced unloading was observed on ultimate force, stiffness, or work to failure of the femoral shaft and femoral neck. PTH(1-34) treatment showed significant effects on ultimate force and stiffness of the femoral shaft and ultimate force and work to failure of the femoral neck. No significant effects on ultimate force, stiffness, or work to failure of the left tibia were found of PTH(1-34) treatment or Botox (Table 1).

Callus histology/histomorphometry

The reduced formation of external callus in the unloaded animals compared with controls was confirmed histologically. One of five samples from both the Botox vehicle and Botox PTH group showed no external callus formation. Callus formation was observed in all samples from the control animals. The callus of vehicle-treated control animals mainly consisted of small amounts of wovenlike bone surrounded mostly by fat and a bony cortex-like structure. In contrast, the callus of PTH(1-34)-treated control animals consisted of a dense structure of wovenlike bone. The same pattern was observed in the Botox animals, only with a smaller total amount of callus. Cartilage only appeared sparsely in the callus.

PTH(1-34) increased BV/TV in the external callus significantly in both the control animals (p = 0.014) and the Botox-treated animals (p = 0.021). There was no significant difference between vehicle-treated control animals and Botox animals (p = 0.773) (Fig. 6A). There was no overall significant difference in BV/TV in the fracture line between the groups (p = 0.16). Interestingly, there was a trend toward a PTH(1-34)-induced increase in BV/TV of the fracture line in the Botox-treated animals but not in the control animals (Fig. 6B). No overall significant difference in MAR was found in either the callus (p = 0.091) or the fracture line (p = 0.686) (Fig. 6C, D).

Figure 6.

Bone volume and mineral apposition rate (MAR) of the external callus (A, C) and newly formed bone in the fracture line (B, D). PTH(1-34) increased BV/TV in external callus independently of loading status. No overall significant differences between groups were seen in MAR in callus or BV/TV and MAR in the fracture line. ▾ represent values from each animal; numerals represent number of animals; dashed lines represent medians.

Histomorphometry of the intact proximal tibia

BV/TV was significantly reduced in the proximal tibia of the Botox group compared with control group (p = 0.009). PTH(1-34) treatment increased BV/TV significantly compared with the respective vehicle group in both Botox animals (p = 0.016) and control animals (p = 0.028) (Table 2). No change in Tb.th. after unloading was observed (p = 0.221). PTH(1-34) did not increase Tb.th. in the unloaded animals (p = 0.327), whereas Tb.th. was significantly increased in the PTH(1-34)-treated control animals compared with vehicle-treated control animals (p = 0.016). PTH(1-34) increased MS/BS in both the Botox animals (p = 0.014) and control animals (p = 0.014) compared with the respective vehicle-treated animals, whereas no effect on MAR was seen (p = 0.142, p = 0.389, respectively). Unloading showed no effects on MS/BS (p = 0.564) and MAR (p = 0.564). No overall significant differences in ES/BS (p = 0.245) and C.th. (p = 0.891) between the groups were observed.

Table 2. Histomorphometry Right Proximal Tibia
 Control vehicleControl PTHBotox vehicleBotox PTH
  1. Data are presented as median (25th; 75th percentiles). Unless otherwise denoted in the table (n), n = 5.
  2. p < 0.05 *versus control vehicle; #versus Botox vehicle.
ES/BS (%)11.5 (9.7; 16.2)11.6 (10.7; 13.6)15.1 (12.9; 16.0) (4)13.9 (11.8; 16.6)
BV/TV (%)12.9 (6.7; 15.8)20.7 (16.6; 30.8)*1.6 (0.3; 2,4)*7.1 (3.8; 11.6)#
Tb.th. (µm)54.2 (51.2; 57.5)67.8 (61.9; 72.2)*43.5 (39.6; 66,7) (4)57.7 (46.0; 64.2)
C.th. (µm)437.9 (361.2; 525.1)438.0 (408.7; 566.7)438.1 (407.1; 512.6)449.5 (441.4; 488.3)
Lb.S/BS (%)58.6 (54.0; 61.0) (4)70.9 (68.3; 75.5)*57.5 (46.9; 59.7) (4)66.8 (62.9; 73.3)#
MAR (µm/day)1.00 (0.87; 1.04) (4)1.05 (0.95; 1.09)0.78 (0.61; 1.12) (4)1.15 (1.11; 1.26)

The proximal tibia in the left leg was analyzed to assess if Botox-induced unloading had any effect on the noninjected leg. No overall difference between groups in MAR (p = 0.134), ES/BS (p = 0.668), C.th. (p = 0.214) could be detected. No significant effects of unloading on BV/TV (p = 0.465), Tb.th. (p = 0.347), and MS/BS (p = 0.327) was observed. BV/TV, Tb.th., and MS/BS was significantly increased after PTH(1-34) treatment in both the Botox animals (p = 0.047, p = 0.047, and p = 0.009, respectively) and control animals (p = 0.028, p = 0.009, and p = 0.014) (Table 3).

Table 3. Histomorphometry Left Proximal Tibia
 Control vehicleControl PTHBotox vehicleBotox PTH
  1. Data are presented as median (25th; 75th percentiles). Unless otherwise denoted in the table (n), n = 5.
  2. p < 0.05 *versus control vehicle; #versus Botox vehicle.
ES/BS (%)11.7 (7.0; 13.7)10.2 (9.0; 13.6)11.5 (9.7; 13.6)10.2 (8.8; 10.4)
BV/TV (%)10.7 (6.3; 16.7)29.6 (19.0; 33.0)*7.0 (5.3; 14.2)23.0 (12.3; 26.8)#
Tb.th. (µm)65.9 (58.5; 67.0)82.6 (74.1; 86.8)*58.2 (53.5; 69.8)73.5 (71.0; 75.7)#
C.th. (µm)435.7 (368.1; 564.3)502.8 (326.3; 573.1)413.6 (339.7; 548.1)572.7 (458.7; 706.1)
Lb.S/BS (%)62.6 (60.9; 67.3) (4)71.9 (70.9; 74.8)*61.3 (57.1; 63.4)72.0 (68.4; 75.7)#
MAR (µm/day)0.90 (0.77; 0.91) (4)0.96 (0.91; 1.03)0.96 (0.92; 1.0)1.07 (0.92; 1.08)

Discussion

The present study showed that PTH(1-34) was able to promote fracture healing of tibial shaft fractures in normally loaded as well as unloaded rats. Furthermore, we confirmed the bone anabolic effects of PTH(1-34) on intact unloaded bone in our model.

We showed that unloading significantly reduced the amount of external callus formed after tibial midshaft fracture compared with control rats. This difference was significant after 4 weeks of fracture healing and the effect persisted throughout the study period of 8 weeks. Our data are in agreement with a recently published study by Hao and colleagues.[39] They found reduced callus formation and callus strength after Botox paralysis in a rat femoral midshaft osteotomy model. Furthermore, we found that BMD and BMC of the callus ROI decreased significantly in the unloaded animals compared with controls. However, histomorphometry did not reveal differences in bone volume of the external callus. This might suggest that the reduction in bone strength after unloading was the result of the large difference in the amount of callus formed between unloaded and normally loaded animals. Bone volume of the newly formed bone in the fracture line also tended to decrease in unloaded animals, which might also have contributed to reduced strength of the fracture. However, a larger sample number would be necessary to confirm this.

As expected, we found that PTH(1-34) treatment increased callus BMD and callus strength, confirming previous findings.[14, 15, 40] Radiological callus area was only slightly elevated after 4 weeks of bone healing, not reaching statistical significance. Increase in callus volume has been suggested in the literature to be one of the mechanisms of PTH-stimulated fracture healing. Andreassen and colleagues found that PTH(1-34) increased external callus volume after 40 days of healing, using the same type of fracture model,[14] whereas Alkhiary and colleagues found that PTH(1-34) was able to promote fracture healing without increasing the amount of external callus in a femoral fracture model.[15] The callus volume is, however, highly dependent on the time after fracture, which could explain discrepancies between studies with regard to this parameter. The increase in callus strength after PTH(1-34) treatment in our study could, therefore, be the result of an increased bone volume in the external callus, supported by our observation of increased callus BMD and BMC.

PTH(1-34) did not increase external callus area in the unloaded animals. Interestingly, we found a significant interaction between the effects of unloading and PTH(1-34) treatment on callus BMD and callus BMC. Although PTH(1-34) increased BMD and BMC in the unloaded animals, the increase was not as profound as in normally loaded animals when compared with the respective vehicle group (22% versus 39% increase after 8 weeks). Our DXA measurements of callus BMD and BMC included both the external callus as well as a part of the diaphyseal bone. As external callus was significantly reduced in the unloaded animals in our study, diaphyseal bone constituted a relatively larger part of the callus ROI analyzed compared with the normally loaded animals. The observed interaction effects of PTH(1-34) and unloading on callus BMD and BMC might, therefore, reflect a differential effect of PTH(1-34) on intact diaphyseal bone and callus, rather than an actual synergistic effect on bone healing. Andreassen and colleagues found that PTH(1-34) had more profound effects on callus BMC of tibial fractures compared with BMC of a diaphyseal segment of the contralateral intact tibia.[14] Unfortunately, we did not measure BMD and BMC at a corresponding region of the contralateral intact tibia, which could assist in differentiating the effects of PTH(1-34) on callus and intact bone. However, we found no significant effect on ultimate force of the left tibia, suggesting only minor effects of PTH(1-34) on intact bone in this region. The above theory was further supported by histomorphometry data, which indicated that PTH(1-34) was able to increase bone volume in the external callus in the unloaded animals to a comparable level of that observed in the normally loaded animals. Although bone strength was generally higher in both the vehicle- and PTH(1-34)-treated control animals compared with unloaded animals, the relative increase in ultimate force after PTH(1-34) treatment was similar in the loaded and unloaded animals (63% increase) when compared with their respective vehicle. This observation also supports that PTH(1-34) is able to promote fracture healing in unloaded animals as well.

It has previously been suggested that one of the mechanisms of PTH-stimulated fracture healing is increased chondrocyte proliferation and cartilage formation in the early stages of healing.[11, 12] As the unloaded fractures only formed small amounts of external callus and PTH(1-34) nevertheless increased callus strength, it could suggest that PTH(1-34) is able to promote fracture healing with minimal involvement of endochondral ossification. Measurements of bone volume in the fracture line suggested, although not significantly, that PTH increased bone volume in the fracture line of the unloaded animals, whereas no effect was seen in the normally loaded animals. Investigation using, e.g., µCT could be used to explore this further.

In our study, no significant interaction between PTH(1-34) and unloading on intact bone were observed, and 20 µg/kg/day PTH(1-34) increased BMD, BMC, and bone strength parameters independent of loading status. In fact, ultimate force of the femoral shaft was increased to a similar level in unloaded and loaded animals. This was confirmed by histomorphometry of the proximal intact tibia, which showed that PTH(1-34) increased bone volume and mineralizing surface significantly. This indicated increased bone formation in both normally loaded and unloaded animals. Trabecular thickness was only significantly increased in the loaded animal. There was, however, a trend toward an increase in the unloaded animals as well.

Our study is limited by the low number of animals used for the histomorphometric measurements. Furthermore, µCT of callus might have provided additional information regarding underlying effects of PTH during different loading conditions and/or types of healing. Another limitation is that only one time point was chosen. Because PTH has been shown to influence at different stages in the process of healing, it would have been desirable if animals were euthanized at different time points. This could potentially have revealed any differences occurring in the early stages of healing and might also have clarified potential differences in the progress through the stages of healing. Moreover, we only used diaphyseal fractures in this study setup. Osteoporotic fractures, which are a major clinical challenge, often occur in regions rich in cancellous bone. It would, therefore, have been highly relevant to investigate the same parameters in fractures of a cancellous bone region, e.g., the proximal tibia. This would, however, require an open fracture and more advanced fixation techniques. When using closed diaphyseal fractures, there is a minimum of injury to the surrounding soft tissue. Furthermore, the closed diaphyseal fracture is the model most widely used in this type of study and is, therefore, well characterized.

In a clinical perspective, our study indicates that PTH(1-34) might be beneficial in the enhancement of not only bone formation but also fracture healing during condition of mechanical unloading e.g. prolonged bed rest. However, further clinical studies of PTH and fracture healing are needed to confirm the findings in humans.

This is, to our knowledge, the first study to investigate the effects of PTH(1-34) on fracture healing during unloading. We found that PTH(1-34) treatment (20 µg/kg/day) for 8 weeks was able to promote fracture healing of closed diaphyseal tibial fractures in both normally loaded and unloaded rats, when assessed by callus area, callus BMD/BMC, histology, and callus strength. In conclusion, our study suggests that the bone anabolic effect of PTH(1-34) on fracture healing and intact bone persists during mechanical unloading.

Disclosures

JEBJ has served as a consultant or advisor or has been on the speaker's bureau for Eli Lilly, Takeda, Nycomed, Amgen Inc., Novartis, and MSD. All other authors state that they have no conflicts of interest.

Acknowledgments

This study was kindly supported by The Danish Council for Independent Research, Medical Sciences grant 09-063453 and Region Hovedstadens Forskningsfond.

Authors' roles: Study design: ME, PS, and NRJ. Study conduct: ME, TK, JEBJ, AB, SS, and SP. Data interpretation: ME, PS, and NRJ. Drafting manuscript: ME. Revising manuscript: PS, NRJ, TK, SS, SP, JEBJ, and AB. All authors approved final version of manuscript. ME takes responsibility for the integrity of the data analysis.

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