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. Author manuscript; available in PMC: 2013 Sep 1.
Published in final edited form as: Bone. 2012 May 11;51(3):578–585. doi: 10.1016/j.bone.2012.05.003

Black bear parathyroid hormone has greater anabolic effects on trabecular bone in dystrophin-deficient mice than in wild type mice

Sarah K Gray a, Meghan E McGee-Lawrence b, Jennifer L Sanders a, Keith W Condon c, Chung-Jui Tsai d, Seth W Donahue e,*
PMCID: PMC3412940  NIHMSID: NIHMS378918  PMID: 22584007

1.1 Introduction

Boys with Duchenne muscular dystrophy (DMD) progressively lose muscle strength and between the ages of 12–15 years are usually confined to a wheelchair [1]. The condition is typically fatal by 20–25 years due to respiratory or cardiac muscle failure [2]. DMD induced muscular necrosis and fibrosis are treated with glucocorticoids (e.g., prednisone or deflazacort) [3, 4]. DMD patients treated with glucocorticoids demonstrate a 30–50% decrease in bone mineral content and bone mineral density in the lumbar spine, hip, and long bones of the lower limbs as compared with healthy age-matched controls [3, 5–8]. Greater losses occur in trabecular bone than cortical bone due to its greater surface area for remodeling activity [3]. Fracture risk increases as a result. One study found that 44% of 71 boys with DMD sustained at least one fracture; of boys over the age of 16 years, 67% sustained at least one fracture [6]. The majority of fractures occur in the proximal and distal femur in DMD patients [9–11].

Like boys with DMD, mdx mice lack dystrophin [12–14]. These animals exhibit myopathic lesions characteristic of those seen in DMD patients [15, 16]. Mdx mice display larger body sizes and limb muscles, though these muscles are heavily fibrosed and less capable of high muscular forces [17]. At 3 weeks of age, mdx femurs display ~40% reductions in trabecular area, trabecular area fraction, and trabecular thickness as compared to age-matched wild type mice. They show decreases in osteoblast surface and increases in osteoclast surface area, leading to increased resorption and decreased formation [18]. In 4 week old male mdx mice, the metaphysis of the tibia showed decreases in bone volume fraction of 35%, with a decrease in trabecular number and increase in trabecular spacing. One study found no difference between mdx and wild type cortical thickness in the femur at 4 months of age [17]. However, at 6 months, mdx mice have decreased cortical bone properties relative to wild type mice and increased osteoclast number [18]. Whole bone breaking force and deformation at fracture are reduced in mdx mice regardless of age [19]. At 24 months of age, mdx mice show greater declines (relative to wild type mice) in trabecular and cortical properties in the tibia than at 7 weeks of age [20].

Treatments for DMD-related osteoporosis have not been widely explored. Treating DMD patients with the bisphosphonate alendronate prevented further decreases in bone mineral density over a two year period [21]. The impaired osteoblast function seen in both DMD patients and mdx mice suggests that an anabolic treatment for osteoporosis (i.e., PTH therapy) would be an effective therapeutic for increasing bone mass in these patients. Indeed, PTH treatment leads to greater reductions in fracture risk than alendronate in patients with glucocorticoid-induced osteoporosis [22]. Our laboratory has been investigating a novel PTH analog linked to preservation of bone during disuse. Black bears (Ursus americanus) hibernate for 6 months of the year, and do not experience disuse-related osteoporosis [23–25]. Black bear parathyroid hormone (bbPTH) has been positively correlated to bone formation markers during hibernation and is implicated in the mechanism of bone preservation during disuse [23, 25, 26]. Furthermore, bbPTH 1–34 causes greater reductions in serum starved induced caspase activity in osteoblasts than hPTH 1–34 [27]. Thus, bbPTH may be well suited to improve bone mass in cases of dystrophin deficiency that impair mobility. We hypothesized that bbPTH treatment in mdx mice would demonstrate improved bone properties compared to vehicle treated mice, and that bbPTH treatment would restore mdx bone properties to wild type levels.

2.1 Materials and Methods

2.1.1 PCR cloning and sequencing of bbPTH

Genomic DNA was extracted from black bear whole blood samples using the GenomicPrep Blood DNA Isolation Kit (Amersham Biosciences, Piscataway, NJ). The genomic DNA was used for PCR amplification of PTH using consensus primers designed based on alignment of eight full-length mammalian PTH sequences available in GenBank including bovine (Bos taurus, AAA30749), cat (Felis catus, Q9GL67), dog (Canis familiaris, P52212), human (Homo sapiens, NP_000306), macaque (Macaca fascicularis, Q9XT35), mouse (Mus musculus, NP_065648), pig (Sus scrofa, NP_999566), and rat (Rattus norvegicus, NP_058740). PCR amplification was performed using 10–15 ng genomic DNA, 100 μM dNTPs, 0.2 μM each primer, and 1 unit REDTaq (Sigma, St. Louis, MO) in 20 μL reaction volume. PCR products were gel-purified using the UltraClean GelSpin Kit (MoBio Carlsbad, CA) and cloned into the pCRII vector using the TA cloning kit (Invitrogen, Carlsbad, CA). DNA sequencing was performed using the DTCS Quick Start kit and the CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA). Nucleotide sequences were searched against the GenBank protein database using BlastX [28] to confirm their putative identity as PTH. Multiple sequence alignment was performed by ClustalW version 1.82 [29]. The sequence obtained for mature black bear PTH (bbPTH; GenBank #GU563375) is shown in Figure 1.

Figure 1.

Figure 1

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Amino acid sequence of human PTH 1-84. Amino acid substitutions in bear PTH 1-84 are highlighted next to the corresponding amino acid in the human sequence.

Proteos (Kalamazoo, MI) recombinantly produced bbPTH 1–84 in E. coli. The purified protein were characterized by analytical HPLC and mass spectroscopy and subjected to quantitative amino acid analysis to determine exact protein concentration. Lyophilized protein aliquots were stored at -80 °C.

2.1.2 cAMP Assay

The PKA/cAMP pathway is primarily responsible for PTH's anabolic effects on osteoblasts, and previous work suggests that small changes in the amino acid sequence of human PTH can produce a peptide that induces greater cyclic adenosine monophosphate (cAMP) production than the hormone's native version [30]. Thus, bbPTH peptide bioactivity was investigated via quantification of cAMP production. PTH receptor expression and PTH-stimulated cAMP production are temporally regulated during osteoblast differentiation [31, 32], where maximal PTH-stimulated cAMP production in MC3T3 cells occurs in cells incubated for 5–10 days in osteogenic medium; therefore, this time frame was chosen for our studies. MC3T3-subclone 4 cells were seeded at 10,000 cells/cm2 in 6 well plates in basal medium (α-MEM, 10% FBS, 1% penicillin-streptomycin). Cells were allowed to attach overnight, before medium was changed to osteogenic medium (basal medium supplemented with 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate) (Day 0). The medium was replaced with fresh osteogenic medium on Day 3, and the cAMP assay was performed on Day 5.

For the cAMP assay, the medium was aspirated and confluent cells were washed with PBS. The cells were stimulated with vehicle (1 mM acetic acid) or bbPTH (1–84) (10, 30, or 100 nM) in PBS containing 1 mM isobutylmethylxanthine (IBMX) for 10 min at 37°C with N = 3 wells per treatment. At the end of the treatment period, the PBS was collected and samples were prepared and assayed in triplicate without acetylation as per the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI). Cell culture supernatants were assayed directly; samples were diluted as necessary in cAMP EIA buffer.

2.1.3 Caspase-3/7 Activity Assay

We performed a caspase-3/7 assay because human PTH has been shown to have an anti-apoptotic effect on osteoblasts [31]. MC3T3-S4 cells were seeded at 20,000 cells/well in 96 well, tissue culture-treated white-walled plates. The cells were seeded in basal medium and allowed to attach overnight. The next day, the medium was aspirated, cells were washed with PBS, and then pretreated with vehicle (1 mM acetic acid) or bbPTH (1–84) (30 or 100 nM) in basal medium for 1 hr with N = 6 wells per treatment. At the end of the pretreatment period, the medium was aspirated, the cells were washed with PBS, and either basal medium (unstarved cells) or α-MEM alone (serum-starved cells) was added to each well. After 6 hours, the medium was aspirated, the cells were washed with PBS, and 50 μl of both PBS and a luminogenic caspase-3/7 substrate (Caspase-Glo 3/7 Assay, Promega, Madison, WI) were added to each well. After 1 hour, luminescence was quantified using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek, Winooski, VT). Luminescence was converted to international units (U) of caspase-3/7 activity using a standard curve generated with human recombinant caspase-3 (Enzo Life Sciences, Farmingdale, NY).

2.1.4 Animals

Twenty 4-week old male C57BL/10ScSn/DMD-mdx and 10 wild type control C57BL/10Sn mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were co-housed six per cage in a 12-h dark, 12-h light environment at 20°C. Mice were fed a standard rodent diet containing 0.95% calcium (Purnia LabDiet Autoclavable Rodent Diet #5010) and given water ad libitum. All mice were euthanized at 10 weeks of age, after 6 weeks of PTH treatment, using carbon dioxide asphyxiation. Three mice (1 mdx bbPTH, 1 mdx vehicle and 1 wild type vehicle) died during the study and were not included in subsequent data analysis. This study was approved by the Michigan Tech Animal Care and Use Committee.

2.1.5 PTH Treatment

Mice were given daily subcutaneous injections of bbPTH 1-84 or an acidic vehicle solution. Vehicle injections were prepared with 0.15 M NaCl and 0.001 N HCl. bbPTH solutions were prepared by dissolving PTH in the acidic saline solution. Mice were injected once daily, 5 times per week with 28 nmol/kg bbPTH 1-84. After six weeks of daily injection, mice were euthanized and their long bones were removed. At 1 and 4 days prior to sacrifice, mice were given calcein injections to label bone formation surfaces (10 mg/kg).

2.1.6 Isolation, Culture, and Mineralization of Bone Marrow Stromal Cells (BMSCs)

Bone marrow was flushed from the humeri of vehicle- and bbPTH-treated wild type and mdx mice. Cells were pooled by treatment group. Cells were seeded into 6 well plates (107 cells/well) in osteogenic culture medium (α-MEM, 20% FBS, 1% antibiotic-antimycotic, 1% non-essential amino acids, 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate). BMSCs were identified by adherence; after 72 hrs, non-adherent cells were removed. Media changes continued every 3 days. BMSCs were cultured in osteogenic medium for 21 days to promote calcified matrix production. At the end of this period, BMSCs were fixed (10% neutral-buffered formalin) and stained (2% alizarin red). Calcified matrix was quantified as the percentage of alizarin red-positive area over total well area using image analysis software (Bioquant Osteo, Nashville, TN).

2.1.7 Trabecular Bone Properties

Proximal tibial and distal femoral metaphyses were scanned using micro-computed tomography (μCT) (SCANCO 35, SCANCO Medical, Switzerland) to determine trabecular properties. The scan region started 0.5 mm from the physis and was 100 slices (0.7 mm) thick. From these data, trabecular number (Tb.n), trabecular spacing (Tb.sp), trabecular thickness (Tb.th), and bone volume/tissue volume (BV/TV) were calculated. Dynamic histomorphometry was evaluated in the same region of the proximal tibia by determining the distance between calcein labels on the trabecular surface and the lengths of single- and double-labeled surfaces using Bioquant Osteo software (Nashville, TN). Static histomorphometry was evaluated by measuring osteoid surface, osteoid thickness, and eroded surface in slides stained in von Kossa with MacNeal's tetrachrome stain. Osteoblast and osteoclast surfaces were measured in decalcified proximal tibia segments stained in toluidine blue and tartrate-resistant acid phosphatase (TRAP).

2.1.8 Cortical Bone Properties

The right femoral diaphysis was embedded in methylmethacrylate. A cross-section was cut at the midpoint using an Isomet diamond wafering blade (Beuhler, Lake Bluff, IL), ground to a thickness of <90 μm and mounted on a microscope slide. Cross-sections were digitized at 40× magnification using a SPOT Insight camera (Diagnostic Instruments, Sterling Heights, MI). A custom macro and Scion Image software (Scion Corp., Frederick, ME) were used to quantify cross-sectional properties including medio-lateral (Iml), anterio-posterior (Iap), and maximum (Imax) moments of inertia. Mineral apposition rate was determined by measuring the distance between calcein labels using Bioquant software. Mineralizing surface was determined by measuring the ratio of single- and double-labeled surfaces to the total cortical surface. Cortical thickness was measured as the average distance between the endosteal and periosteal surfaces.

2.1.9 Mechanical Properties

Bending properties of the left femur were determined by a 3-point bend test at a rate of 1 mm/min. The testing fixtures had a span of 10 mm and a radius of 1 mm. Mechanical testing was performed using an Instron test machine (Norwood, MA). Stress-strain plots were determined using asymmetric beam theory [33], and used to calculate ultimate stress and modulus of toughness. Load-displacement plots were used to determine ultimate load and energy to failure for each sample.

2.1.10 Mineral Content

Mineral content was determined through ashing. After mechanical testing, femoral diaphyses were cleaned of marrow, placed in a furnace at 100°C for 24 hours to remove all water, and weighed to obtain dry mass. Samples were returned to the furnace for 48 hours at 600°C to burn off all organic material. Bones were again weighed to determine relative mineral content, defined as the ratio of ash mass to dry mass.

2.1.11 Statistics

Bone properties and cell parameters were compared between bbPTH- and vehicle-treated animals using one-way ANOVA and Tukey's post hoc analysis.

3.1 Results

3.1.1 Cell Studies

All three concentrations of bbPTH increased cAMP production in MC3T3 cells compared to vehicle controls (p < 0.0001). No difference was observed between 10 nM bbPTH and 30 nM bbPTH, but 100 nM bbPTH increased cAMP production more than the lower doses (p < 0.0001) (Figure 2). Caspase 3/7 activity was reduced (p < 0.0001) in serum starved MC3T3 cells treated with both concentrations of bbPTH, with 100 nM bbPTH decreasing caspase 3/7 to unstarved control values (Figure 3).

Figure 2.

Figure 2

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bbPTH increased cAMP production in a dose-responsive manner (p < 0.0001).

Figure 3.

Figure 3

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bbPTH was antiapoptotic in serum starved MC3T3 cells (p < 0.0001). Groups with different letters are significantly different from one another.

3.1.2 Bone Marrow Stromal Cells

Alizarin red staining showed greater (p = 0.0021) mineralized matrix in BMSC cultures from vehicle-treated mdx mice than in cultures from bbPTH-treated mdx mice (Figure 4A). In wild type mice, the difference (p = 0.09) between vehicle and bbPTH treatments was not as large (Figure 4B).

Figure 4.

Figure 4

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Alizarin red stained mineralized matrix in 21 day BMSC cultures from the humeri of A) mdx and B) wild type mice. The percentage of well area stained for mineralized matrix was quantified.

3.1.3 Trabecular Bone

Trabecular bone volume fraction was lower in the distal femoral and proximal tibial metaphyses of vehicle-treated mdx mice compared to wild type mice (p = 0.0024) (Figure 5A). Treatment with bbPTH improved trabecular bone volume in both the distal femur and proximal tibia in both mdx and wild type mice, but to a much greater degree in mdx mice (Figure 6). The increase in femoral bone volume fraction in mdx mice with bbPTH treatment was 7-fold, compared to a 2-fold change in wild type mice (Figure 5A), with similar magnitude responses in the tibia (Table 1).

Figure 5.

Figure 5

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A) Bone volume fraction was lower in vehicle-treated mdx femurs compared to wild type femurs. Mdx femurs showed greater response to PTH treatment than wild type femurs. B) Vehicle-treated mdx mice had lower trabecular number in the femur compared to wild type mice (p = 0.0028). Trabecular number increased with PTH treatment in mdx mice (p < 0.0001), but not wild type (p = 0.2047). Mean values with standard error bars. Groups with different letters are significantly different (p < 0.05) from each other.

Figure 6.

Figure 6

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Micro-CT images of distal femoral metaphyses show compromised trabecular architecture in mdx mice compared to wild type mice and improved trabecular architecture with PTH treatment, particularly in mdx mice.

Table 1.

The distal femur and proximal tibia trabecular regions show 1) compromised trabecular architectural parameters in mdx mice vs. wild type; 2) an anabolic effect of bbPTH in mdx and wild type mice, with a stronger effect in mdx; 3) bbPTH treatment increased mdx trabecular microarchitecture beyond vehicle-treated wild type values. Average ± SE.

mdx Vehicle mdx bbPTH Wild Type Vehicle Wild Type bbPTH
Distal Femur μCT
BV/TV (%) 6.6 ±2.2† 41.3 ±2.2*†† 11.7 ±3.2 22.3 ±3.2*
Tb.Th (mm) 0.03 ±0.001† 0.05 ±0.001** 0.04 ±0.002 0.05 ±0.002
Tb.Sp (mm) 0.22 ±0.008 0.08 ±0.008**†† 0.19 ±0.001 0.18 ±0.001
Tb.N (1/mm) 4.5 ±0.4† 11.1 ±0.5**†† 5.1 ±0.7 5.7 ±0.7
Proximal Tibia μCT
BV/TV (%) 5.7 ±0.03 39.0 ±0.03**†† 9.2 ±0.04 20.6 ±0.04**
Tb.Th (mm) 0.032 ±0.001† 0.044 ±0.001**† 0.037 ± 0.001 0.044 ±0.001
Tb.Sp (mm) 0.23 ±0.02 0.11 ±0.02**† 0.20 ±0.03 0.22 ±0.02
Tb.N (1/mm) 4.3 ±0.8 10.5 ±1.8**†† 4.8 ±1.17 5.1 ±1.0
Proximal Tibia Dynamic Histomorphometry
MS/BS 0.19 ±0.03 0.19 ±0.03 0.20 ±0.04 0.15 ±0.04
MAR (μm/day) 2.66 ±0.09 2.81±0.09 2.31 ±0.15 2.97 ±0.15*
Proximal Tibia Static Histomorphometry
Osteoid Thickness (μm) 2.51 ±0.54 3.27 ±0.54 1.59 ±0.82 2.60 ±0.82
Osteoid Surface (%) 22.39 ±5.00 24.67 ±5.00 31.09 ±8.61 21.42 ±6.67
Bone Surface (mm) 17.38 ±3.86 48.48 ±3.86**† 23.29 ±5.79 34.57 ±5.79
Eroded Surface (%) 0.04 ±0.014 0.05 ±0.014 0.05 ±0.022 0.04 ±0.019
Osteoblast Surface/Bone Surface 0.10 ±0.04 0.32 ±0.05* 0.13 ±0.06 0.39 ±0.06
Osteoclast Surface/Bone Surface 0.13 ±0.01 0.04 ±0.02*† 0.16 ±0.02 0.09 ±0.02
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*

p < 0.05 vs. vehicle-treated;

**

p < 0.005 vs. vehicle-treated

†

p < 0.05 vs. wild type vehicle;

††

p < 0.005 vs. wild type vehicle

Vehicle-treated wild type mice demonstrated greater trabecular thickness in the distal femur and proximal tibia than mdx mice (p > 0.0212). Trabecular spacing (p > 0.7780), trabecular number (p > 0.9813), and apparent mineral density (p > 0.7733) were not different between vehicle-treated mdx and wild type mice.

Both trabecular number (Figure 5B) and trabecular thickness (Table 1) increased (p < 0.0001) with bbPTH treatment in mdx distal femurs and proximal tibias, but not in wild type mice. Trabecular spacing decreased (p < 0.0001) in mdx femurs, but not in wild type mice.

In the proximal tibia there was no difference in mineralizing surface between any groups (p = 0.7437). Trabecular mineral apposition rate increased with bbPTH in wild type mice (p = 0.0202), but there was no difference in mineral apposition rate between vehicle- and bbPTH-treatment in mdx mice (p = 0.6628). There were no differences in mineral apposition rate treatments between mdx and wild type mice in either treatment group (p > 0.245) (Table 1).

Despite the observed anabolic effect of bbPTH on trabecular bone volume fraction, no differences were found between groups in osteoid thickness (p = 0.4047) or osteoid surface (p = 0.966). However, bbPTH-treated mdx mice had increased osteoblast surface (p = 0.0435) and decreased osteoclast surface (p = 0.0336), compared to vehicle treatment. These changes likely contributed to the increased trabecular bone volume fraction that occurred with bbPTH treatment in mdx mice. Similar changes were seen in wild type mice treated with bbPTH, but the differences were not significant for osteoblast surface (p = 0.0711) or osteoclast surface (p = 0.1242) (Table 1). Normally, PTH treatment increases both osteoblast and osteoclast surfaces [34]. Increased osteoblast surface and decreased osteoclast surface, as seen in the bbPTH treated mdx mice, may be a feature unique to bbPTH.

3.1.4 Cortical Bone

No differences were found in femur length for either treatment or mouse type (p > 0.22). Mdx mice appeared to have an altered femoral cross-section, with a more prominent linea aspera than wild type mice, regardless of bbPTH treatment (Figure 7). No differences were detected between mdx and wild type, or bbPTH and vehicle-treated mice for cortical thickness (p = 0.2803), Imax (p = 0.4344), or Iap (p = 0.4492). However, moment of inertia about the bending axis (Iml) was greater in bbPTH-treated mdx mice than vehicle-treated mdx mice (p = 0.0097) (Table 2).

Figure 7.

Figure 7

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mdx femoral cross-sections (left) had a more prominent linea aspera than wild type mice (right). Images are femoral cross-sections of vehicle-treated animals, but are representative of all samples treated or untreated.

Table 2.

bbPTH did not change mechanical properties of cortical bone in mdx or wild type, or strongly affect geometric values. The only change in geometry was Iml, the moment about the bending axis in PTH-treated mdx animals. Average ± SE.

mdx Vehicle mdx bbPTH Wild Type Vehicle Wild Type bbPTH
Cortical Geometry
Cortical Thickness (μm) 440.2 ±25.2 446.7 ±25.2 363.2 ±37.8 369.1 ±37.8
Cortical Area (mm2) 0.96 ±0.06 1.06 ±0.06 0.85 ±0.10 0.93 ±0.10
Lml (mm4) 0.17 ±0.01 0.22 ±0.01** 0.19 ±0.02 0.21 ±0.02
Lap (mm4) 0.37 ±0.03 0.44 ±0.03 0.36 ±0.06 0.44 ±0.06
Lmax (mm4) 0.37 ±0.04 0.45 ±0.04 0.37 ±0.06 0.44 ±0.06
Endosteal Dynamic Histomorphometry
MAR (μm/day) 1.36 ±0.23 1.19 ±0.23 1.59 ±0.34 1.62 ±0.34
MS/BS 0.61 ±0.05 0.42 ±0.05 0.59 ±0.08 0.44 ±0.08
Periosteal Dynamic Histomorphometry
MAR (μm/day) 2.09 ±0.17 2.31 ±0.17 1.49 ±0.26 1.76 ±0.26
MS/BS 0.61 ±0.04 0.61 ±0.04 0.49 ±0.06 0.48 ±0.06
Ash Fraction
Ash Fraction 0.65 ±0.03 0.67 ±0.02 0.66 ±0.04 0.64 ±0.03
Mechanical Testing
Ultimate Force (N) 15.4 ±0.82 16.7 ±0.89 16.2 ±0.23 17.1 ±0.95
Energy to Failure (mJ) 15.0 ±2.1 17.1 ±2.3 10.0 ±1.0 13.3 ±2.6
Ultimate Stress(MPa) 179.7 ±11.2 147.7 ±11.3 166.3 ±18.4 162.0 ±18.4
Modulus of Toughness (mJ/mm3) 16.9 ±3.1 14.6 ±3.1 9.1 ±5.0 12.7 ±5.0
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**

p = 0.0097 vs. vehicle-treated

No differences were detected in cortical bone mineralizing surface on the fluorescently labeled endosteal surface, either when analyzed by quadrant, or by total endosteal area (p > 0.2579). Mineral apposition rate was not different between any groups, though bbPTH-treatment tended to decrease endosteal mineral apposition rate (p = 0.0776). Similarly, no differences were found between any groups for periosteal mineralizing surface on the calcein labeled periosteal surface (p > 0.1970). bbPTH-treated mdx mice tended to have greater periosteal mineral apposition than vehicle-treated controls (p = 0.0764), unlike wild type mice (p = 0.8890). No other differences were detected in cortical bone mineral apposition rate between groups (p > 0.1492) (Table 2).

Three-point bend testing of the femur showed no differences (p > 0.1314) between ultimate load or energy to failure between bbPTH- and vehicle-treated mice or between mdx and wild type mice. Normalizing by body weight had no affect (p > 0.1086). Similarly, no differences were found in ultimate stress (p = 0.9231) or modulus of toughness (p = 0.5541) (Table 2).

Mineral content in the femoral diaphysis, as determined by ash fraction, was not different for mdx vs. wild type or PTH vs. vehicle-treated animals (p = 0.8516) (Table 2).

4.1 Discussion

We cloned the gene for black bear PTH and found nine amino acid residues were different from human PTH 1–84. We recombinantly produced bbPTH 1–84 and found it activates cAMP production as has been previously reported for human PTH [35]. The cAMP/protein kinase A pathway is belived to be responsible for the majority of PTH induced increases in histological and serum indices of bone formation [36–38]. PTH also produces osteoanabolic activity via anti-apoptosis mechanisms in osteoblasts [39]. We also found that treating osteoblasts with bbPTH prior to serum starvation reduces caspase 3/7 activity. Previously we found bbPTH 1–34 causes greater reductions in serum starved induced caspase activity than hPTH 1–34 [27]. Here we demonstrate the ability of bbPTH 1–84 to have a greater osteoanabolic effect in dystrophin deficient mice than in wild type mice.

Though a strong anabolic response to bbPTH treatment (i.e., increased trabecular bone volume fraction) was observed in both mdx and wild type mice, the relative effect was much greater in mdx mouse bone. The lower mineralization in BMSC cultures from mdx mice treated with bbPTH raise the possibility that the majority of osteoblast precursors may have differentiated and been removed from the marrow to participate in bone formation during the course of treatment. Dystrophin deficiency alters calcium signaling and calcium reuptake in muscle cells, and calcium signaling is also an important biological mechanism in osteoblasts [40]. Thus, there is potential that the mdx mouse also features differences in calcium signaling in osteoblasts in response to PTH and mechanical loading [41]. This change, if it is occurring, may help explain the low bone volume in mdx mice and the differential response to PTH treatment between mdx and wild type mice. Mdx mice display a low bone mass phenotype at 3 weeks of age, which precedes the onset of muscle weakness [42]. Dystrophin deficiency may also affect primary spongiosa formation and the development of hypertrophic chondrocytes during development due to aberrant calcium signaling [43]. No connection between PTH and dystrophin has been established previously, but the marked difference in anabolic response to bbPTH in mdx as compared to wild type mouse trabecular bone raises the possibility that dystrophin or associated altered calcium signaling due to dystrophin deficiency could play a role in trabecular bone formation. However, surprisingly there is no difference in serum calcium concentrations between 2 month old mdx and wild type mice [44]. Because of the influence of PTH on calcium homeostasis, it will be important to quantify the effects of PTH on serum ionized calcium concentration in mdx mice in future studies. Bone losses attributed to the mdx phenotype may be due to decreased muscular force as well. Though mouse behavior was not markedly different by gross observation during this study, previous work has shown decreased muscle forces despite increased muscle size in mdx mice from ages 4 weeks to 24 months [17, 19, 20]. Notably, PTH has been shown to attenuate bone loss in disuse scenarios, such as hind limb suspension [45].

bbPTH increased bone volume fraction to a greater degree than has been observed in previous studies administering hPTH to mice, with a 91% increase in bone volume fraction in wild type mice in this study, compared to 14–38% increases in bone volume fraction in mice administered with high dose hPTH [46, 47]. Trabecular number was also increased in mdx bones, contributing to a large increase in bone volume fraction (Figure 5B). A possibility for the increase in trabecular number, since dystrophin deficiency leads to aberrant calcium signaling in muscle cells, and possibly other musculoskeletal cells, is that mdx mice have a greater number of hypertrophic chondrocytes or increased primary spongiosa since hypertrophic chondrocyte proliferation is regulated by a calcium signaling pathway [48]. Higher values of these parameters in mdx mice at the start of the PTH treatment period could lead to increases in trabecular number and bone volume fraction with PTH treatment [43, 49]. The substantial increase in bone surface in the bbPTH treated mice may explain the lack of change in mineralizing surface (MS/BS), which is normally seen with PTH treatment. Furthermore, in vitro data suggest that bbPTH has an anti-apoptotic effect in osteoblasts, which would increase osteoblast survival, consistent with the increased osteoblast surface we observed histologically in bbPTH treated mdx mice.

Cortical bone did not display the marked changes with bbPTH treatment that were observed in trabecular bone. This is similar to studies with PTH in other mouse models, which show approximately a 10% increase in cortical bone ultimate force with PTH [50, 51]. Our study showed a comparable increase in femoral ultimate force of approximately 8%, though the difference was not significant. PTH did not have the expected effect on endosteal mineralizing surface, possibly because the majority of osteoblast precursors were mobilized in the heavily affected trabecular bone. However, bbPTH did significantly increase medio-lateral moment of inertia in mdx mice. This is important because the medio-lateral axis is the primary bending axis in the femur [52]. This raises the possibility that bbPTH may have a moderate effect on mdx cortical bone, which may improve with longer duration treatment.

The potent osteoanabolic response to bbPTH in mdx trabecular bone is clinically relevant to DMD patients, as the majority of fractures occur in the highly trabecularized regions of bone including the distal femur, proximal tibia, and vertebrae [9–11, 53]. An increase in trabecular bone volume, as occurs with parathyroid hormone treatment, could greatly reduce fracture risk by increasing trabecular bone density in DMD patients [54]. Additionally, a longer duration PTH treatment in DMD patients could have beneficial effects on cortical bone. However, the potential of PTH to increase the risk of osteosarcoma in children and adolescents is a concern that would need to be addressed before a recommendation to use PTH clinically in DMD patients could be made. Osteosarcoma develops in approximately 4 out of one million males less than 24 years of age with a peak incidence of approximately 10 per million for ages 15–19 years [55], and PTH has been shown to increase the incidence of osteosarcoma in rats that were given high PTH doses for the majority of their lifetime [56]. The use of PTH in children with hypoparathyroidism is considered safe and effective for maintaining calcium homeostatsis and was found to promote normal skeletal development [57]. However, the potential for PTH to increase the risk of osteosarcoma in children and adolescents has not been studied. Decreasing fracture prevalence in DMD patients could improve overall quality of life by allowing greater mobility for a longer period of time, even with the use of glucocorticoids. Parathyroid hormone is effective in the treatment of glucocorticoid-induced osteoporosis [22, 58]. Therefore, treating DMD patients with parathyroid hormone could reduce the probability of fractures and prolong the amount of time these patients are independently mobile. Our data support the idea that bbPTH is an effective therapeutic to combat bone loss in cases of dystrophin deficiency. However, this study administered only high dose bbPTH. Future work comparing the dose responses of bbPTH and hPTH in mdx mice are needed to further elucidate the mechanisms by which PTH improves bone properties in the mdx model of Duchenne muscular dystrophy.

Highlights

  • Black bear PTH is a potent osteoanabolic agent in the dystrophin deficient mdx mouse

  • Black bear PTH is more potent in mdx mice than in wild type mice

  • Black bear PTH increases osteoblast surface and decreases osteoclast surface in mdx mice

Acknowledgements

The authors gratefully acknowledge Dr. Mike Vaughan for the black bear blood samples, Matt Nelsen and Yinan Yuan for assistance with bbPTH cloning, and funding from Aursos Inc. and NIH (DK078407).

Footnotes

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References

  • [1].Nair KP, Vasanth A, Gourie-Devi M, Taly AB, Rao S, Gayathri N, Murali T. Disabilities in children with Duchenne muscular dystrophy: a profile. J Rehabil Med. 2001;33:147–9. doi: 10.1080/165019701750300591. [DOI] [PubMed] [Google Scholar]
  • [2].Bianchi ML, Mazzanti A, Galbiati E, Saraifoger S, Dubini A, Cornelio F, Morandi L. Bone mineral density and bone metabolism in Duchenne muscular dystrophy. Osteoporos Int. 2003;14:761–7. doi: 10.1007/s00198-003-1443-y. [DOI] [PubMed] [Google Scholar]
  • [3].Bianchi ML, Morandi L, Andreucci E, Vai S, Frasunkiewicz J, Cottafava R. Low bone density and bone metabolism alterations in Duchenne muscular dystrophy: response to calcium and vitamin D treatment. Osteoporos Int. 2010;22:529–39. doi: 10.1007/s00198-010-1275-5. [DOI] [PubMed] [Google Scholar]
  • [4].Straathof CS, Overweg-Plandsoen WC, van den Burg GJ, van der Kooi AJ, Verschuuren JJ, de Groot IJ. Prednisone 10 days on/10 days off in patients with Duchenne muscular dystrophy. J Neurol. 2009;256:768–73. doi: 10.1007/s00415-009-5012-y. [DOI] [PubMed] [Google Scholar]
  • [5].Soderpalm AC, Magnusson P, Ahlander AC, Karlsson J, Kroksmark AK, Tulinius M, Swolin-Eide D. Low bone mineral density and decreased bone turnover in Duchenne muscular dystrophy. Neuromuscul Disord. 2007;17:919–28. doi: 10.1016/j.nmd.2007.05.008. [DOI] [PubMed] [Google Scholar]
  • [6].Larson CM, Henderson RC. Bone mineral density and fractures in boys with Duchenne muscular dystrophy. J Pediatr Orthop. 2000;20:71–4. [PubMed] [Google Scholar]
  • [7].Aparicio LF, Jurkovic M, DeLullo J. Decreased bone density in ambulatory patients with duchenne muscular dystrophy. J Pediatr Orthop. 2002;22:179–81. [PubMed] [Google Scholar]
  • [8].Crabtree NJ, Roper H, McMurchie H, Shaw NJ. Regional changes in bone area and bone mineral content in boys with duchenne muscular dystrophy receiving corticosteroid therapy. J Pediatr. 2009;156:450–5. doi: 10.1016/j.jpeds.2009.09.010. [DOI] [PubMed] [Google Scholar]
  • [9].Hsu JD, Garcia-Ariz M. Fracture of the femur in the Duchenne muscular dystrophy patient. J Pediatr Orthop. 1981;1:203–7. doi: 10.1097/01241398-198110000-00012. [DOI] [PubMed] [Google Scholar]
  • [10].Hsu JD. Extremity fractures in children with neuromuscular disease. Johns Hopkins Med J. 1979;145:89–93. [PubMed] [Google Scholar]
  • [11].Gray B, Hsu JD, Furumasu J. Fractures caused by falling from a wheelchair in patients with neuromuscular disease. Dev Med Child Neurol. 1992;34:589–92. doi: 10.1111/j.1469-8749.1992.tb11489.x. [DOI] [PubMed] [Google Scholar]
  • [12].Ryder-Cook AS, Sicinski P, Thomas K, Davies KE, Worton RG, Barnard EA, Darlison MG, Barnard PJ. Localization of the mdx mutation within the mouse dystrophin gene. EMBO J. 1988;7:3017–21. doi: 10.1002/j.1460-2075.1988.tb03165.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].King WM, Ruttencutter R, Nagaraja HN, Matkovic V, Landoll J, Hoyle C, Mendell JR, Kissel JT. Orthopedic outcomes of long-term daily corticosteroid treatment in Duchenne muscular dystrophy. Neurology. 2007;68:1607–13. doi: 10.1212/01.wnl.0000260974.41514.83. [DOI] [PubMed] [Google Scholar]
  • [14].Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 1989;244:1578–80. doi: 10.1126/science.2662404. [DOI] [PubMed] [Google Scholar]
  • [15].Bulfield G, Siller WG, Wight PA, Moore KJ. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci U S A. 1984;81:1189–92. doi: 10.1073/pnas.81.4.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Call JA, McKeehen JN, Novotny SA, Lowe DA. Progressive resistance voluntary wheel running in the mdx mouse. Muscle Nerve. 2010;42:871–80. doi: 10.1002/mus.21764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Montgomery E, Pennington C, Isales CM, Hamrick MW. Muscle-bone interactions in dystrophin-deficient and myostatin-deficient mice. Anat Rec A Discov Mol Cell Evol Biol. 2005;286:814–22. doi: 10.1002/ar.a.20224. [DOI] [PubMed] [Google Scholar]
  • [18].Rufo A, Del Fattore A, Capulli M, Carvello F, De Pasquale L, Ferrari S, Pierroz D, Morandi L, De Simone M, Rucci N, Bertini E, Bianchi ML, De Benedetti F, Teti A. Mechanisms inducing low bone density in Duchenne muscular dystrophy in mice and humans. J Bone Miner Res. 2011;26:1891–903. doi: 10.1002/jbmr.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Anderson JE, Lentz DL, Johnson RB. Recovery from disuse osteopenia coincident to restoration of muscle strength in mdx mice. Bone. 1993;14:625–34. doi: 10.1016/8756-3282(93)90084-n. [DOI] [PubMed] [Google Scholar]
  • [20].Novotny SA, Warren GL, Lin AS, Guldberg RE, Baltgalvis KA, Lowe DA. Bone is functionally impaired in dystrophic mice but less so than skeletal muscle. Neuromuscul Disord. 2011;21:183–93. doi: 10.1016/j.nmd.2010.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Hawker GA, Ridout R, Harris VA, Chase CC, Fielding LJ, Biggar WD. Alendronate in the treatment of low bone mass in steroid-treated boys with Duchennes muscular dystrophy. Arch Phys Med Rehabil. 2005;86:284–8. doi: 10.1016/j.apmr.2004.04.021. [DOI] [PubMed] [Google Scholar]
  • [22].Saag KG, Zanchetta JR, Devogelaer JP, Adler RA, Eastell R, See K, Krege JH, Krohn K, Warner MR. Effects of teriparatide versus alendronate for treating glucocorticoid-induced osteoporosis: thirty-six-month results of a randomized, double-blind, controlled trial. Arthritis Rheum. 2009;60:3346–55. doi: 10.1002/art.24879. [DOI] [PubMed] [Google Scholar]
  • [23].Donahue SW, McGee ME, Harvey KB, Vaughan MR, Robbins CT. Hibernating bears as a model for preventing disuse osteoporosis. J Biomech. 2006;39:1480–8. doi: 10.1016/j.jbiomech.2005.03.030. [DOI] [PubMed] [Google Scholar]
  • [24].McGee-Lawrence ME, Wojda SJ, Barlow LN, Drummer TD, Castillo AB, Kennedy O, Condon KW, Auger J, Black HL, Nelson OL, Robbins CT, Donahue SW. Grizzly bears (Ursus arctos horribilis) and black bears (Ursus americanus) prevent trabecular bone loss during disuse (hibernation) Bone. 2009;45:1186–91. doi: 10.1016/j.bone.2009.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].McGee-Lawrence ME, Wojda SJ, Barlow LN, Drummer TD, Bunnell K, Auger J, Black HL, Donahue SW. Six months of disuse during hibernation does not increase intracortical porosity or decrease cortical bone geometry, strength, or mineralization in black bear (Ursus americanus) femurs. J Biomech. 2009;42:1378–83. doi: 10.1016/j.jbiomech.2008.11.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Donahue SW, Galley SA, Vaughan MR, Patterson-Buckendahl P, Demers LM, Vance JL, McGee ME. Parathyroid hormone may maintain bone formation in hibernating black bears (Ursus americanus) to prevent disuse osteoporosis. J Exp Biol. 2006;209:1630–8. doi: 10.1242/jeb.02185. [DOI] [PubMed] [Google Scholar]
  • [27].McGee-Lawrence ME. Relative Anti-Apoptotic and Osteogenic Effects of Black Bear PTH 1-34 Compared to Human PTH 1-34. ASBMR Annual Meeting; Denver, CO. 2009. [Google Scholar]
  • [28].Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 2003;31:3497–500. doi: 10.1093/nar/gkg500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Reidhaar-Olson JF, Davis RM, De Souza-Hart JA, Selick HE. Active variants of human parathyroid hormone (1-34) with multiple amino acid substitutions. Mol Cell Endocrinol. 2000;160:135–47. doi: 10.1016/s0303-7207(99)00211-7. [DOI] [PubMed] [Google Scholar]
  • [31].Bellido T, Ali AA, Plotkin LI, Fu Q, Gubrij I, Roberson PK, Weinstein RS, O'Brien CA, Manolagas SC, Jilka RL. Proteasomal degradation of Runx2 shortens parathyroid hormone-induced anti-apoptotic signaling in osteoblasts. A putative explanation for why intermittent administration is needed for bone anabolism. J Biol Chem. 2003;278:50259–72. doi: 10.1074/jbc.M307444200. [DOI] [PubMed] [Google Scholar]
  • [32].Schiller PC, D'Ippolito G, Roos BA, Howard GA. Anabolic or catabolic responses of MC3T3-E1 osteoblastic cells to parathyroid hormone depend on time and duration of treatment. J Bone Miner Res. 1999;14:1504–12. doi: 10.1359/jbmr.1999.14.9.1504. [DOI] [PubMed] [Google Scholar]
  • [33].Levenston ME. Periosteal bone formation stimulated by externally induced bending strains. J Bone Miner Res. 1995;10:671–2. doi: 10.1002/jbmr.5650100422. [DOI] [PubMed] [Google Scholar]
  • [34].Wu X, Pang L, Lei W, Lu W, Li J, Li Z, Frassica FJ, Chen X, Wan M, Cao X. Inhibition of Sca-1-positive skeletal stem cell recruitment by alendronate blunts the anabolic effects of parathyroid hormone on bone remodeling. Cell Stem Cell. 7:571–80. doi: 10.1016/j.stem.2010.09.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Koh AJ, Beecher CA, Rosol TJ, McCauley LK. 3',5'-Cyclic adenosine monophosphate activation in osteoblastic cells: effects on parathyroid hormone-1 receptors and osteoblastic differentiation in vitro. Endocrinology. 1999;140:3154–62. doi: 10.1210/endo.140.7.6872. [DOI] [PubMed] [Google Scholar]
  • [36].Fujimori A, Cheng SL, Avioli LV, Civitelli R. Dissociation of second messenger activation by parathyroid hormone fragments in osteosarcoma cells. Endocrinology. 1991;128:3032–9. doi: 10.1210/endo-128-6-3032. [DOI] [PubMed] [Google Scholar]
  • [37].Rixon RH, Whitfield JF, Gagnon L, Isaacs RJ, Maclean S, Chakravarthy B, Durkin JP, Neugebauer W, Ross V, Sung W, et al. Parathyroid hormone fragments may stimulate bone growth in ovariectomized rats by activating adenylyl cyclase. J Bone Miner Res. 1994;9:1179–89. doi: 10.1002/jbmr.5650090807. [DOI] [PubMed] [Google Scholar]
  • [38].Hilliker S, Wergedal JE, Gruber HE, Bettica P, Baylink DJ. Truncation of the amino terminus of PTH alters its anabolic activity on bone in vivo. Bone. 1996;19:469–77. doi: 10.1016/s8756-3282(96)00230-x. [DOI] [PubMed] [Google Scholar]
  • [39].Jilka RL, Weinstein RS, Bellido T, Roberson P, Parfitt AM, Manolagas SC. Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest. 1999;104:439–46. doi: 10.1172/JCI6610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Zayzafoon M. Calcium/calmodulin signaling controls osteoblast growth and differentiation. J Cell Biochem. 2006;97:56–70. doi: 10.1002/jcb.20675. [DOI] [PubMed] [Google Scholar]
  • [41].Lansman JB, Franco-Obregon A. Mechanosensitive ion channels in skeletal muscle: a link in the membrane pathology of muscular dystrophy. Clin Exp Pharmacol Physiol. 2006;33:649–56. doi: 10.1111/j.1440-1681.2006.04393.x. [DOI] [PubMed] [Google Scholar]
  • [42].Nakagaki WR, Bertran CA, Matsumura CY, Santo-Neto H, Camilli JA. Mechanical, biochemical and morphometric alterations in the femur of mdx mice. Bone. 2010;48:372–9. doi: 10.1016/j.bone.2010.09.011. [DOI] [PubMed] [Google Scholar]
  • [43].Silbermann M, Kadar T. Quantitative changes in the cellular population of the growth plate of triamcinolone-treated mice. Acta Anat (Basel) 1977;98:396–400. doi: 10.1159/000144818. [DOI] [PubMed] [Google Scholar]
  • [44].Kikkawa N, Ohno T, Nagata Y, Shiozuka M, Kogure T, Matsuda R. Ectopic calcification is caused by elevated levels of serum inorganic phosphate in mdx mice. Cell Struct Funct. 2009;34:77–88. doi: 10.1247/csf.08039. [DOI] [PubMed] [Google Scholar]
  • [45].Moriyama I, Iwamoto J, Takeda T, Toyama Y. Comparative effects of intermittent administration of human parathyroid hormone (1-34) on cancellous and cortical bone loss in tail-suspended and sciatic neurectomized young rats. J Orthop Sci. 2002;7:379–85. doi: 10.1007/s007760200063. [DOI] [PubMed] [Google Scholar]
  • [46].Jilka RL, Almeida M, Ambrogini E, Han L, Roberson PK, Weinstein RS, Manolagas SC. Decreased oxidative stress and greater bone anabolism in the aged, when compared to the young, murine skeleton with parathyroid hormone administration. Aging Cell. 2010;9:851–67. doi: 10.1111/j.1474-9726.2010.00616.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [47].Ferrari SL, Pierroz DD, Glatt V, Goddard DS, Bianchi EN, Lin FT, Manen D, Bouxsein ML. Bone response to intermittent parathyroid hormone is altered in mice null for {beta}-Arrestin2. Endocrinology. 2005;146:1854–62. doi: 10.1210/en.2004-1282. [DOI] [PubMed] [Google Scholar]
  • [48].Li Y, Ahrens MJ, Wu A, Liu J, Dudley AT. Calcium/calmodulin-dependent protein kinase II activity regulates the proliferative potential of growth plate chondrocytes. Development. 2011;138:359–70. doi: 10.1242/dev.052324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Silbermann M, Kadar T. Age-related changes in the cellular population of the growth plate of normal mouse. Acta Anat (Basel) 1977;97:459–68. doi: 10.1159/000144767. [DOI] [PubMed] [Google Scholar]
  • [50].Washimi Y, Chen H, Ito A, Takao R, Uzawa T, Yamamoto Y, Yamada H, Shoumura S. Effect of intermittent treatment with human Parathyroid Hormone 1-34 in SAMP6 senescence-accelerated mice. J Endocrinol Invest. 2010;33:395–400. doi: 10.1007/BF03346610. [DOI] [PubMed] [Google Scholar]
  • [51].Zhou H, Iida-Klein A, Lu SS, Ducayen-Knowles M, Levine LR, Dempster DW, Lindsay R. Anabolic action of parathyroid hormone on cortical and cancellous bone differs between axial and appendicular skeletal sites in mice. Bone. 2003;32:513–20. doi: 10.1016/s8756-3282(03)00057-7. [DOI] [PubMed] [Google Scholar]
  • [52].Margolis DS, Lien YH, Lai LW, Szivek JA. Bilateral symmetry of biomechanical properties in mouse femora. Med Eng Phys. 2004;26:349–53. doi: 10.1016/j.medengphy.2003.11.002. [DOI] [PubMed] [Google Scholar]
  • [53].Bothwell JE, Gordon KE, Dooley JM, MacSween J, Cummings EA, Salisbury S. Vertebral fractures in boys with Duchenne muscular dystrophy. Clin Pediatr (Phila) 2003;42:353–6. doi: 10.1177/000992280304200408. [DOI] [PubMed] [Google Scholar]
  • [54].Fields AJ, Eswaran SK, Jekir MG, Keaveny TM. Role of trabecular microarchitecture in wholevertebral body biomechanical behavior. J Bone Miner Res. 2009;24:1523–30. doi: 10.1359/JBMR.090317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [55].Mirabello L, Troisi RJ, Savage SA. International osteosarcoma incidence patterns in children and adolescents, middle ages and elderly persons. Int J Cancer. 2009;125:229–34. doi: 10.1002/ijc.24320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [56].Jolette J, Wilker CE, Smith SY, Doyle N, Hardisty JF, Metcalfe AJ, Marriott TB, Fox J, Wells DS. Defining a noncarcinogenic dose of recombinant human parathyroid hormone 1-84 in a 2-year study in Fischer 344 rats. Toxicol Pathol. 2006;34:929–40. doi: 10.1080/01926230601072301. [DOI] [PubMed] [Google Scholar]
  • [57].Winer KK, Sinaii N, Reynolds J, Peterson D, Dowdy K, Cutler GB., Jr. Long-term treatment of 12 children with chronic hypoparathyroidism: a randomized trial comparing synthetic human parathyroid hormone 1-34 versus calcitriol and calcium. J Clin Endocrinol Metab. 2010;95:2680–8. doi: 10.1210/jc.2009-2464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [58].Weinstein RS, Jilka RL, Almeida M, Roberson PK, Manolagas SC. Intermittent parathyroid hormone administration counteracts the adverse effects of glucocorticoids on osteoblast and osteocyte viability, bone formation, and strength in mice. Endocrinology. 2010;151:2641–9. doi: 10.1210/en.2009-1488. [DOI] [PMC free article] [PubMed] [Google Scholar]

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