Parathyroid hormone (PTH), an 84-amino-acid polypeptide, is an essential regulator of calcium and phosphate metabolism. The roles of PTH in mineral homeostasis are to increase serum calcium levels by enhancing gastrointestinal calcium absorption, increase renal calcium and phosphate reabsorption, liberate calcium from the skeleton in response to systemic needs, and participate in the regulation of vitamin-D metabolism1. Although the effects of this hormone are usually associated with bone resorption, the responses of osteoclasts to PTH are most likely mediated by means of osteoblastic activity and the receptors for parathyroid hormone are found on osteoblast membranes1. Indeed, while continuous exposure to PTH leads to an increase in osteoclast density and activity, intermittent exposure stimulates osteoblasts and results in increased bone formation in rats and humans2,3.
Clinical trials on the use of PTH (1-34) have shown increases in bone mass in osteoporotic men and increases in bone mineral density and reductions in the risk of vertebral and non-vertebral fractures in postmenopausal women4,5. In a study by Neer et al.4, a 40-μg dose in an average-sized adult human increased bone mineral density more than a 20-μg dose did, but it had similar effects on the risk of fracture and was more likely to produce side effects. Hence, it appears that only relatively low doses of this therapeutic agent are tolerated in patients.
Recent studies have described a potential role for PTH and PTH-related peptides (PTHrp) in other musculoskeletal tissues. Vortkamp et al.6 and Lanske et al.7 showed that the PTH/PTHrp receptor mediates the effects of Indian hedgehog and PTH or PTHrp on chondrocyte differentiation. Other reports have demonstrated a role for PTH/PTHrp receptor mutations in the pathogenesis of various chondrodysplasias8. Those studies have suggested that PTH-signaling plays an important role in a variety of mechanisms of bone formation, including endochondral ossification.
Because of the observed skeletal effects of intermittent administration of PTH (1-34), several animal studies have been conducted on the use of various doses of PTH (1-34) in orthopaedic applications. In a study with use of parathyroidectomized rats, PTH administration was shown to enhance early fracture-healing9. In a number of recent reports, doses ranging from 10 to 200 μg/kg in rat models of normal fracture-healing were found to be associated with substantial increases in both mechanical and histological properties10-12. In other studies, in which models of impaired bone metabolism were used, PTH analogs were shown to reverse the inhibition of bone-healing in ovariectomized rats13 and in rabbits treated with corticosteroids14. In one report, PTH (1-34) was shown to increase bone ingrowth and pullout strength in porous metallic implants15.
These reports have suggested that PTH (1-34) may be a useful adjunct in the treatment of orthopaedic injuries and diseases. However, most of these studies used doses of the hormone that were much higher than those that would be tolerated in humans, and the experimental designs did not always include sufficient numbers of animals to make robust statistical statements. In order to critically evaluate the potential for this hormone to be tested in future clinical applications, we performed an appropriately powered investigation using a well-established model of fracture-healing in the rat with doses of this drug that were either higher than or equivalent to those used in animal studies of the treatment of osteoporosis16-18, and we tested the hypothesis that a once-daily subcutaneous injection of PTH (1-34) could enhance fracture-healing.
Materials and Methods
Two hundred and seventy Sprague-Dawley rats, each weighing approximately 450 g, were used for this study. The animals were weighed at the beginning and at the end of the experiment. Radiographs were made of the fracture sites both at the time of enrollment and at the time that the animals were killed. Recombinant human PTH (1-34) was provided by Eli Lilly (Indianapolis, Indiana). PTH (1-34) or a normal saline-solution vehicle was administered to the animals by daily subcutaneous injection adjacent to the scapulae. The animals were killed by carbon-dioxide asphyxiation.
There were three experimental groups: a control group, in which a normal saline-solution vehicle was injected, and two groups that were injected with either 5 or 30 μg/kg/day of PTH (1-34). The 5-μg/kg/day dose was selected on the basis of published data showing skeletal efficacy (increased bone mass) in male and female intact and gonadectomized Sprague-Dawley, Wistar, and Fischer-344 rats16-18. The 30-μg/kg/day dose was selected on the basis of previous fracture-healing studies showing substantial beneficial effects at this dose15,19. The 5-μg/kg/day dose has not been previously evaluated in fracture-repair studies. However, according to the United States Package Insert for Forteo (teriparatide)20, a 5-μg/kg dose in the rat produces systemic exposures (on the basis of the area under the curve) that are approximately three times greater than those that occur in patients given a 20-μg dose of the drug. Although this dose produces greater systemic exposures than those that occur in patients given the marketed dose of Forteo, the difference should be interpreted cautiously as rats and humans have important differences in skeletal physiological properties and in their responses to PTH21.
Animals in each group were killed at twenty-one, thirty-five, and eighty-four days after fracture, resulting in nine subgroups. Drug treatment was administered once daily, and all treatments were discontinued at thirty-five days. On the basis of the results in two previous studies in which the senior author (T.A.E.) was involved and that used outcome measures comparable with those in the present investigation11,12, a coefficient of variation in the data of 25% was used for power calculations. After accepting α and β errors of 5% for both the mechanical testing and histomorphometric analyses, the number of animals required for the study was calculated to be 270 and the number needed for each subgroup was calculated to be thirty (seventeen for measurements of biomechanical properties and callus dimensions, eight for microquantitative computed tomography, and five for histological and histomorphometric assessments).
Male Sprague-Dawley rats (Harlan Bioproducts for Science, Indianapolis, Indiana) that weighed a mean (and standard deviation) of 449 ± 39 g and were approximately seven to nine months old were used for all experiments. The animals were individually housed at 22°C with free access to food (standard rat chow) and water on a twelve-hour light and dark cycle. Research was conducted in conformity with all federal and United States Department of Agriculture guidelines as well as a protocol approved by the Institutional Animal Care and Use Committee.
Closed, transverse mid-diaphyseal femoral fractures were produced as described by Bonnarens and Einhorn22. Briefly, fractures were generated with the use of a blunt guillotine after stabilization of the femur with an intramedullary pin. The animals were subjected to general anesthesia with a veterinary inhalation anesthesia machine with use of a mixture of isoflurane and oxygen. They were weighed on a digital scale and received an intramuscular injection of 0.2 mL of cefazolin and 0.1 mL of Buprenex (buprenorphine) into the left thigh. The left hind limb was shaved, swabbed with povidone iodine for disinfection, and draped. A median parapatellar skin incision was made, followed by a median parapatellar incision into the joint capsule, extending from the midline through the vastus medialis muscle to the patellar ligament insertion. Slow flexion of the knee and movement of the patella with forceps achieved lateral retraction of the patella on the extended knee. An intercondylar entry point for the insertion of the 0.045-in (0.114-cm) diameter Kirschner wire into the medullary space was made with use of a handheld drill. The wire was inserted until meeting resistance of the underlying greater trochanter. The wire was then slightly retracted, cut, reinserted, and buried under the knee cartilage surface. The operative site was closed with sutures, and the skin was stapled. Fractures were then produced, and radiographs were made to confirm pin placement and fracture configuration. Fractures that did not occur in the mid-diaphysis of the bone or that showed excessive comminution were excluded.
At the time that the animals were killed, the fractured limbs were once again radiographed and then disarticulated at the hip joint. Specimens were harvested and initially cleaned to remove muscle and soft tissue, with care taken not to scratch the bone. Callus dimensions were measured in the anterior-posterior and medial-lateral dimensions with use of an electronic caliper. Bones were then snap-frozen in liquid nitrogen and stored at -20°C (in preparation for biomechanical testing), fixed in 4% neutral buffered paraformaldehyde (in preparation for histological assessment), or kept in 75% ethanol (in preparation for tomographic analysis). Tissue was stored at 4°C before experimental assessment by the latter two procedures.
Bones were subjected to biomechanical testing to failure with use of a servo-actuated rapid-loading torsion-testing device at a rate of 10 N/mm/s. Specimens were thawed just prior to testing and were potted in aluminum blocks at either end in a lead alloy (Cerrobend; Cerro Metal Products, Bellefonte, Pennsylvania) that melts at a low temperature (158°F [70°C]). This method allows 1 cm of bone (i.e., 0.5 cm in each direction from the center of the fracture site) to be exposed. The applied moment and the angular deformation of the femora were measured and plotted. Values were obtained for shear modulus (stiffness) and maximum torque (torsional strength).
Microquantitative Computed Tomography
At the time that the animals were killed, the femora were immediately placed in 75% ethanol at 4°C in preparation for microquantitative computed tomographic analysis. The intramedullary pins were removed from the bones immediately before the tomographic analysis. Analysis with high-resolution microquantitative computed tomography was performed with use of an Enhanced Vision Systems computed tomography scanner (EVS, London, Ontario, Canada). Fracture calluses were imaged with 22.6-μm isotropic voxels. The volumetric images were mathematically projected to obtain 45.2-μm pixel projections. These projections were processed to obtain the mean bone mineral content per horizontal line in the projection and the mean bone mineral density for the entire callus.
Histological and Histomorphometric Analyses
At the time that the animals were killed, the femora were fixed for four days in 4% paraformaldehyde and phosphate-buffered saline solution at 4°C. After fixation, the bones were rinsed in sterile phosphate-buffered saline solution and decalcification was carried out with use of 14% EDTA (pH, 7.2-7.4) (Boston BioProducts, Ashland, Massachusetts) for three to four weeks on a shaker at 4°C (the EDTA solution was changed once per week).
The intramedullary pins were removed from the bones before embedding and sectioning. With use of a scalpel, the femora were divided into two halves by identifying the center of the callus and making a cross-sectional cut (the fracture center was determined with the aid of the radiographs that had been made during the fracture procedure); another two cuts were placed 5 mm proximal and distal to the center of the fracture. The resulting two specimens of bone represented the entire fracture callus.
The specimens were embedded vertically into a paraffin mold. Eight sections every 500 μm were stained with safranin O-fast green for histological and histomorphometric analysis. Consecutive sections in each series were also stained for the presence of tartrate-resistant acid phosphatase (TRAP) to determine osteoclast density. Each of these sections was photographed with a light microscope (Olympus BX51; Olympus America, Melville, New York) attached to a digital camera kit (CoolSNAP-Pro; Media Cybernetics, Silver Spring, Maryland). Each photograph was made at 1.25 times magnification and was downloaded into an Image-Pro Plus program (version 22.214.171.124 for Windows; Media Cybernetics). An area of interest was created by outlining the outside periphery of the cortical bone, excluding muscle, soft tissue, and periosteum. With use of a color-match program, cartilage appeared red, bone appeared yellow, and void spaces appeared green. For all measurements, the mean values were calculated for the individual bones and then were used to create group means, standard deviations, and standard errors of the means for each of the groups.
The number of TRAP-positive cells was determined in serial sections adjacent to those that were processed for the quantification of cartilage and bone. Photomicrographs (10.5 × 7.8 mm) made with the light microscope were captured at ten times magnification, as described above, and six images for each slide section were downloaded into the Image-Pro Plus program. The total number of fields counted per time-point per group was forty-two. With use of a color-match program, the osteoclasts from each photograph were quantified with a spot filter with use of predefined pixel values that defined the lower and upper dimensions for the spots that were counted. The numbers from the two adjacent photomicrographs were combined, and the means were calculated for the specimens.
Histomorphometric measurements for the assessment of the fracture calluses included callus diameter, cartilage volume, total osseous tissue volume, void space, and osteoclast density (Table I). These static parameters were developed in a separate investigation (unpublished data23), and their derivation, nomenclature, and methods of measurement were based on standard units and nomenclatures accepted by the American Society for Bone and Mineral Research24,25.
The effects of experimental treatments on callus dimensions, bone mineral content, bone mineral density, torsional strength, stiffness, and histomorphometric measurements were evaluated with a two-way analysis of variance with use of SPSS software (release 11.0.1; SPSS, Chicago, Illinois). When either the treatment group effect or the interaction was significant at p < 0.05, further analyses to determine which groups differed from each other were performed with use of the Tukey method26.
The initial assessment of fracture-healing was performed with qualitative analysis of plain radiographs made at the time that the animals were killed. By day 21, the animals in the group treated with 30 μg/kg/day of PTH (1-34) showed osseous bridging over the fracture site, a finding not observed in the controls. In addition, radiopacity was increased compared with that seen in the controls. By day 35, the radiopacity observed in both PTH-treated groups was much greater than that seen in the controls. There also appeared to be more bone in the medullary spaces surrounding the pin in the femora treated with either dose of PTH (Figs. 1 and 2-A). The increased radiopacity is consistent with a faster progression toward a stable callus during treatment with PTH.
Bone Mineral Content and Density
The qualitative observations of the radiographic responses to PTH (1-34) noted above were confirmed by quantitative analysis of bone mineral content and density. Representative volumetric images of fracture calluses that were projected and quantitated with microquantitative computed tomography, and the values obtained for bone mineral content and density are shown in Figure 2-B. These graphs show that, by day 21, both bone mineral content and bone mineral density in the group treated with 30 μg/kg/day of PTH (1-34) were significantly increased compared with controls (p < 0.008 and p < 0.001, respectively). In addition, by day 35, bone mineral content and bone mineral density had increased significantly, compared with the controls, for both the group treated with 5 μg/kg/day of PTH (1-34) (p < 0.022 and p < 0.05, respectively) and the group treated with 30 μg/kg/day (p < 0.001 for both). By day 84, both parameters remained significantly elevated (p < 0.017 and p < 0.023, respectively) in the animals treated with the higher dose of PTH in comparison with the controls (Table II, Fig. 2-B).
Calluses from the animals treated with 30 μg/kg/day of PTH (1-34) showed significant increases compared with the controls with respect to stiffness (p < 0.05) at day 21 and in torque strength at all three time-points tested (p < 0.05, p < 0.006, and p < 0.02, respectively) (Table II, Fig. 3).
Histological and Histomorphometric Analyses
Analysis of variance revealed no significant differences among any of the treatment groups on any day or at any time with respect to callus diameters (Table II). Qualitative assessment of histological sections of the calluses showed an increase in the amount of cartilage (red-staining tissue) at day 21 and a reduction at day 35 after fracture in animals treated with 30 μg/kg/day of PTH (1-34) compared with controls. This was accompanied by an appreciable increase in the amount of osseous tissues at these time-points, suggesting a possible enhancement of chondrogenesis and an acceleration of endochondral resorption and bone formation with the higher dose of PTH (Fig. 4-A). Quantitative histomorphometric assessments confirmed these observations, demonstrating a significant increase, in comparison with the controls, with respect to cartilage volume in animals treated with 30 μg/kg/day of PTH (1-34) at day 21 after fracture (p < 0.05) and a significant decrease in this parameter in animals treated with either the 5-μg/kg or 30-μg/kg dose at day 35 (p < 0.05 for both). Significant increases in total osseous tissue volume were also measured at both doses (p < 0.022 and p < 0.008, respectively) and significant reductions were observed in void space with both doses (p < 0.026 and p < 0.009, respectively) compared with controls (Table II, Fig. 4-B). Measurements of void space showed that the volume of bone marrow, hematopoietic elements, and unstained void volume was inversely proportional to the total osseous tissue volume, suggesting that, as the newly formed bone in the callus was remodeled, more trabecular bone was produced. No significant differences were noted in osteoclast density with either dose of PTH at any time-point measured, indicating that the overall effect of intermittent administration of this drug on fracture-healing is an acceleration of cartilage-remodeling and bone formation but not an increase in bone resorption (Table II, Fig. 4-B).
Asystemic therapy for the enhancement of bone repair would be a major advance in treatment. The recent demonstration of the safety and efficacy of Forteo (PTH [1-34]; teriparatide) as a bone formation agent for the treatment of osteoporosis in postmenopausal women and in men provides a rationale for its potential use in the treatment of other skeletal conditions. On the basis of this advance in clinical therapeutics, the body of data demonstrating a role for PTH-signaling in cartilage development, and the results from studies of the effects of high doses of PTH on experimental fracture-healing in animals, this investigation was undertaken to test the hypothesis that lower doses of PTH, which produce systemic exposures in patients equivalent to between three and eighteen times the currently marketed dose of Forteo, could enhance experimental fracture-healing.
The doses of PTH used in this study were selected on the basis of published data showing skeletal efficacy with 5 μg/kg in male and female, intact and gonadectomized, Sprague-Dawley, Wistar, and Fischer-344 rats16-18. The higher dose was selected on the basis of previous fracture-healing studies that have shown substantial beneficial effects at approximately 30 μg/kg15,19. One prior study12 investigated the use of 10 μg/kg of PTH but did not include a histological or histomorphometric analysis of the calluses, and the number of animals treated was not powerful enough to detect a therapeutic effect. A 5-μg/kg dose may be more relevant to the clinically available dose used to treat osteoporosis21. Although this dose is still three times higher than the currently marketed dose based on the area-under-the-curve calculations, important differences in skeletal physiologic properties and metabolism in rats and humans provide a rationale for use in preclinical investigations21.
The daily administration of 30 μg/kg of PTH (1-34) in rats leads to a consistent enhancement in the biomechanical properties of the fracture calluses at all time-points compared with the controls. Maximum torque, a measurement of callus strength, showed significant elevations at days 21, 35, and 84 after the fracture even though the dosing was discontinued after day 35. Stiffness, on the other hand, was only significantly elevated at the early time-point and then returned to normal levels throughout the remainder of the study period. This is consistent with the known relationship between the biomechanical properties of fracture callus and fracture-healing as stiffness is a property acquired early in this process while strength is acquired later27. The lack of an effect on biomechanical properties with the 5-μg/kg dose may indicate a potential limitation of this dose in fracture-healing in this species, or it may indicate that the kinetics of fracture-healing are such that the rapid evolution of events does not accommodate the time needed for this dose to produce effects on the skeleton. Indeed, skeletal efficacy has been demonstrated in rats with use of this dose for longer periods of exposure. However, the increases in bone mineral density and bone mineral content on day 35 did demonstrate enhanced callus development with this dose. In addition, while biomechanical testing might seem to represent a more relevant outcome measure with respect to a potential clinical indication in fracture-healing, it is important to note that, in experimental settings, measurements of bone mineral density and content may be more sensitive to the effects of bone-formation agents because the variance in the data is smaller than that found with biomechanical measurements. Thus, a significant effect on bone mineral density and content, in conjunction with a trend for an increase in stiffness at thirty-five days and in strength at eighty-four days, suggests that a clinical effect of this drug on fracture-healing may even be possible.
PTH (1-34) is a small polypeptide hormone that binds to a G protein-coupled receptor on osteoblasts and signals to the nucleus through a cyclic AMP-dependent, and possibly a phosphokinase-C, pathway. While PTH has effects on both bone formation and resorption2,3, chronic elevations in serum PTH concentration predominantly stimulate bone resorption. Intermittent administration, on the other hand, leads to transient increases in serum PTH concentration and a predominantly osteogenic effect28. Although less is known about the role of PTH in cartilage metabolism, signaling through the PTH/PTHrp receptor in the growth plate has been shown to be essential in chondrocyte differentiation and maturation during skeletal growth6,7. Indeed, in this investigation, an increase in cartilage volume in the calluses at twenty-one days, in response to the administration of the higher dose of PTH, suggests an early effect on chondrocyte proliferation during the healing process. The subsequent decline in cartilage volume at day 35, at both the high and low doses, suggests a potential acceleration of endochondral ossification. This finding, in conjunction with the significant increases in bone volume and the reductions in void volume of the calluses at day 35 in response to both doses, suggests an overall enhancement, and possible acceleration, of the healing process. These findings are consistent with previous studies suggesting that PTH may coordinate several events during endochondral ossification, including the regulation of osteoblast proliferation, survival, and apoptosis, as well as osteoblastic signaling of osteoclast activity29. Indeed, histomorphometric studies have shown that administration of PTH for four weeks in mice leads to an inhibition of osteoblast apoptosis, resulting in prolongation and enhancement of bone formation30,31.
Another important aspect of this study is the failure to detect a significant increase in either callus diameter or osteoclast density at any time-point. These findings suggest that the predominant effect of PTH on fracture-healing is to improve the quality of the callus as opposed to simply promote its structural and mechanical properties through an increase in area. The findings of increased total osseous tissue volume and decreased void space support this observation. Moreover, while no significant effect on osteoclast density was detected at the time-points measured, the intermittent administration of PTH did result in an increased trend in this parameter, suggesting that some of the anabolic effects might have resulted from enhanced bone-remodeling. Indeed, the increases in torque strength at the 30-μg dose suggest that osteoclastic activity was maintained within the normal range and led to improvements in callus remodeling.
The findings of this report, in combination with those from prior studies, support the exploration of the clinical utility of this drug in human fracture-healing. As several clinical trials were conducted in the development of PTH (1-34) for the treatment of postmenopausal osteoporosis, concerns regarding safety and tolerability have largely been addressed. Although near-lifetime administration of PTH (1-34) has been shown to induce rare osteosarcomas in rats32,33, the potential use of PTH in the management of fractures would be for considerably shorter, self-limited periods. No osteosarcoma was observed in any of the multiple histological sections examined in this investigation.
The ability to accelerate fracture-healing in both young and elderly patients could improve clinical outcomes by leading to a shorter time of immobilization and a potential acceleration of rehabilitative programs. There would also be the secondary benefit of improving quality of life by accelerating the return to work, recreation, and activities of daily living. In osteoporotic patients who have sustained fractures, this drug may have the dual benefit of enhancing healing, overcoming any potential role of age or osteoporosis on the healing process34,35, and initiating a therapeutic program for the treatment of the underlying disease. Although these experimental results are encouraging, the potential use of PTH to enhance human fracture-healing should be formally tested in the clinical setting. ▪
A commentary is available with the electronic versions of this article, on our web site () and on our quarterly CD-ROM (call our subscription department, at 781-449-9780, to order the CD-ROM).
In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Eli Lilly and Company. In addition, one or more of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (Eli Lilly). No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.
Investigation performed at the Orthopaedic Research Laboratory, Boston University Medical Center, Boston, Massachusetts, and Eli Lilly, Indianapolis, Indiana
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