PTH-treated Group Research Articles

Disruption of the aldehyde dehydrogenase 2 gene increases the bone anabolic response to intermittent PTH treatment in an ovariectomized mouse model.

Aldehyde dehydrogenase 2 (ALDH2) is the enzyme that oxidizes the acetaldehyde produced by alcohol metabolism. This variant not only affects the response to alcohol but is also associated with several diseases, such as esophageal cancer, myocardial infarction, and particularly osteoporosis. In our previous study, we reported that compared to wild-type (WT) mice, Aldh2 knockout (KO) mice naturally have a strong bone formation ability, and high expression of parathyroid hormone receptor (PTHR1) in osteocytes. The effect of the Aldh2 gene on bone metabolism in response to intermittent PTH treatment is unknown. The purpose of this study was to clarify the effect of the Aldh2 gene on the bone anabolic response to intermittent PTH treatment in ovariectomized mice. Female KO and WT mice were ovariectomized at 8weeks of age. At 14weeks of age, the KO and WT mice were divided into vehicle-treated (Veh) and PTH-treated (PTH) groups (i.e., the WT-Veh, WT-PTH, KO-Veh and KO-PTH groups). PTH (1-34) and vehicle were subcutaneously administered to each group at a dose of 40μg/kg body weight (BW) five times per week for 4weeks. Micro-CT showed that the bone volume (BV), trabecular number (Tb.N), connectivity density (Conn.D), and cortical thickness (Ct.Th) values in the KO-PTH mice were significantly higher than those in the KO-Veh mice. Histomorphometric analysis showed that the BV, Tb.N, and mineral apposition rate (MAR) values in the KO-PTH group were significantly higher than those in the KO-Veh group. The mRNA expression level of PTHR1 in the KO-PTH group was significantly increased and that of p21 in the KO-PTH group was significantly decreased compared with the levels in the KO-Veh group. The expression of PTHR in osteocytes from the KO-PTH group was also significantly increased compared with that in osteocytes from the KO-Veh group. Furthermore, cell cultures revealed that the ALP+CFU-f/total CFU-f percentage was significantly higher in the KO-PTH group than in the KO-Veh group. We concluded that in ovariectomized Aldh2 KO mice, the bone anabolic response to intermittent PTH treatment was significantly enhanced compared to that in WT mice, which may be mediated by the high expression level of PTHR1.

Quality of life in hypoparathyroidism.

Hypoparathyroidism is a rare endocrine disorder where deficiency (or lack of effect) of parathyroid hormone results in disordered mineral metabolism leading to hypocalcemia and hyperphosphatemia. Many patients with this disorder have physical, emotional and cognitive complaints suggestive of impaired quality of life (QOL). Several recent studies have demonstrated that hypoparathyroid patients treated with calcium and vitamin D (conventional therapy) have reduced QOL compared to either suitable controls or general population. QOL has also been studied during treatment with PTH1-84, which has been FDA approved in the USA as an adjunct to calcium and vitamin D in patients not adequately controlled on conventional therapy. In open label studies, PTH therapy has resulted in dramatic improvements in SF-36 scores. In placebo-controlled, double-blinded studies the effect of PTH on QOL has been less striking. In one such study there was no improvement, possibly due to high incidence of hypercalcemia in the PTH-treated group. In contrast, in the pivotal trial leading to PTH1-84 approval, were hypercalcemia was less common, SF36 scores improved in the PTH-treated but not in the placebo group although between-group differences were not statistically significant. In the same study, patients who started with lower baseline QOL scores had greater improvements, suggesting that patients with poor wellbeing may be particularly suitable for this new therapy. In the future, it will be necessary to develop disease specific instruments to better define the nature of QOL impairments in this disease. In addition, further studies should focus on the possible relationship between biochemical variables and QOL parameters during treatment with different regimens.

Effect of high-frequency loading and parathyroid hormone administration on peri-implant bone healing and osseointegration

The objective of this study is to examine the effect of low-magnitude, high-frequency (LMHF) loading, and anti-osteoporosis medications such as parathyroid hormone (PTH) and bisphosphonates on peri-implant bone healing in an osteoporosis model, and to assess their combined effects on these processes. Thirteen-week-old ovariectomized rats (n = 44) were divided into three groups: PTH, alendronate, and saline. After 3 weeks of drug administration, titanium implants were inserted into the tibiae. Each group was subdivided into two groups: with or without LMHF loading via whole-body vibration (50 Hz at 0.5 g, 15 min per day, 5 days per week). Rats were killed 4 weeks following implantation. Removal torque test, micro-CT analyses (relative gray (RG) value, water = 0, and implant = 100), and histomorphometric analyses (bone-to-implant contact (BIC) and peri-implant bone formation (bone volume/tissue volume (BV/TV))) were performed. Removal torque values and BIC were significantly differed by loading and drug administration (ANOVA). Post hoc analysis showed that PTH-treated groups were significantly higher than the other drug-treated groups. BV/TV was significantly enhanced by PTH administration. In cortical bone, RG values were significantly increased by loading. In trabecular bone, however, RG values were significantly increased by PTH administration. These findings suggest that LMHF loading and PTH can act locally and additively on the bone healing process, improving the condition of implant osseointegration.

Correlations Between Changes in Undercarboxylated Osteocalcin and Muscle Function in Hypoparathyroidism.

BackgroundMuscles and bones are interconnected. Recent studies suggest that undercarboxylated osteocalcin from bone may affect muscle mass and strength. There are, however, no prospective human data on this relationship.MethodsWe previously treated patients with hypoparathyroidism with intact Parathyroid Hormone (PTH) or placebo in a six-month randomized, placebo-controlled trial and demonstrated a marked increase in undercarboxylated osteocalcin (ucOC) in the PTH-treated group. We therefore investigated if this increase correlated with changes in muscle mass, strength or function. Primarily, the muscle mass using Dual energy X-ray Absorptiometry (DXA) was measured and the maximal voluntary isometric muscle strength at the upper and lower extremities, using dynamometry, was assessed. Furthermore, repeated chair stands test, Timed Up and Go test were performed and postural stability using a stadiometer was assessed. Finally, the relationship between change in ucOC or the ratio of the changes in ucOC and total OC (ucOC%/OC%) and different measures of muscle function were analyzed, using regression analyses.ResultsThe findings indicated that ucOC%/OC% was positively and significantly associated with percentage change in max force production during elbow extension (β = 0.28, P = 0.034), however, all other associations were non-significant.ConclusionsGiven the number of statistical tests that were carried out, our one significant finding may represent a false positive. Thus the results do not support the role of ucOC in muscle function in humans with hypoparathyroidism. Our results are inconsistent with previous data from a human cross-sectional study; however, cross-sectional studies, do not allow for inference of causality. The analyses should be repeated in larger, randomized trials including healthy individuals.

PTH modulation of NCC activity regulates TRPV5 Ca2+ reabsorption.

Since parathyroid hormone (PTH) is known to increase transient receptor potential vanilloid (TRPV)5 activity and decrease Na(+)-Cl(-) cotransporter (NCC) activity, we hypothesized that decreased NCC-mediated Na(+) reabsorption contributes to the enhanced TRPV5 Ca(2+) reabsorption seen with PTH. To test this, we used mDCT15 cells expressing functional TRPV5 and ruthenium red-sensitive (45)Ca(2+) uptake. PTH increased (45)Ca(2+) uptake to 8.8 ± 0.7 nmol·mg(-1)·min(-1) (n = 4, P < 0.01) and decreased NCC activity from 75.4 ± 2.7 to 20.3 ± 1.3 nmol·mg(-1)·min(-1) (n = 4, P < 0.01). Knockdown of Ras guanyl-releasing protein (RasGRP)1 had no baseline effect on (45)Ca(2+) uptake but significantly attenuated the response to PTH from a 45% increase (6.0 ± 0.2 to 8.7 ± 0.4 nmol·mg(-1)·min(-1)) in control cells to only 20% in knockdown cells (6.1 ± 0.1 to 7.3 ± 0.2 nmol·mg(-1)·min(-1), n = 4, P < 0.01). Inhibition of PKC and PKA resulted in further attenuation of the PTH effect. RasGRP1 knockdown decreased the magnitude of the TRPV5 response to PTH (7.9 ± 0.1 nmol·mg(-1)·min(-1) for knockdown compared with 9.1 ± 0.1 nmol·mg(-1)·min(-1) in control), and the addition of thiazide eliminated this effect (a nearly identical 9.0 ± 0.1 nmol·mg(-1)·min(-1)). This indicates that functionally active NCC is required for RasGRP1 knockdown to impact the PTH effect on TRPV5 activity. Knockdown of with no lysine kinase (WNK)4 resulted in an attenuation of the increase in PTH-mediated TRPV5 activity. TRPV5 activity increased by 36% compared with 45% in control (n = 4, P < 0.01 between PTH-treated groups). PKC blockade further attenuated the PTH effect, whereas combined PKC and PKA blockade in WNK4KD cells abolished the effect. We conclude that modulation of NCC activity contributes to the response to PTH, implying a role for hormonal modulation of NCC activity in distal Ca(2+) handling.

The Effect of Treatment With PTH on Undercarboxylated Osteocalcin and Energy Metabolism in Hypoparathyroidism.

Undercarboxylated osteocalcin (ucOC) has been shown to affect glucose metabolism in mice. We recently randomized patients with hypoparathyroidism to treatment with PTH or placebo and demonstrated a marked increase in total osteocalcin. To investigate whether there was a similar increase in ucOC and whether that increase affected glucose metabolism. A 24-week randomized, placebo-controlled trial. Ambulatory patients in a research facility. Sixty-two patients aged 31-78 years with hypoparathyroidism, of which 58 completed the trial. 100 μg/d of PTH (1-84). Change in ucOC. ucOC increased by 1185.0 ± 814.4% (mean ± SD) in the PTH-treated group and by 69.3 ± 79.4% in the placebo group (P < 10(-50)). In addition, body weight decreased by 1.1 ± 4.0% in the treatment group and increased 0.8 ± 2.5% in the placebo group (P = .04). Glucose, adiponectin, leptin, homeostasis model of assessment for insulin resistance, total body fat mass, or truncal fat did not change significantly. In addition, the number of hypercalcemic episodes per patient was 3.7 ± 2.9 (mean ± SD) in the PTH-treated group but only 0.2 ± 0.6 in the placebo group (P < .001). Moreover, there was a significant and negative correlation between the change in ucOC and change in body weight (P = .004) or change in total body fat mass (P = .03), and a negative but nonsignificant correlation between the number of hypercalcemic episodes and percentage change in body weight (r = -0.32; P = .1). Change in ucOC did not significantly correlate with changes in other parameters. An explanation for the weight loss may be subtle hypercalcemia in PTH treatment inhibiting appetite. Our data do not support a role for ucOC in energy metabolism in humans.

Prevention of Atrophic Nonunion by the Systemic Administration of Parathyroid Hormone (PTH 1–34) in an Experimental Animal Model

Recombinant human parathyroid hormone (PTH 1-34) has been previously shown to enhance fracture healing in animal models. Here, we sought to determine whether the systemic administration of PTH 1-34 is effective in preventing atrophic fracture nonunion in a murine, surgical nonunion model. We used an established reproducible long-bone murine fracture nonunion model by generating a midshaft femur fracture, followed by fracture distraction using an intramedullary pin and custom metallic clip to maintain a fracture gap of 1.7 mm. Mice were randomized to receive either daily intraperitoneal injections of 30 μg/kg PTH 1-34 for 14 days or saline injections. At 6 weeks after the procedure, radiographic and histologic assessment of fracture healing was performed. At 6 weeks after surgery, the group treated with PTH showed higher rates of bony union (50% vs 8%; P < 0.05) as assessed by radiographic analysis. Mean gap size was also significantly lower in the PTH group (1.42 vs 0.36 mm in the control group; P < 0.05). Histologic analysis of atrophic nonunions in the control group revealed a persistent fracture gap with intervening fibrous tissue. In contrast, healed subjects in the PTH-treated group had cortical bridging with mature bone and relatively little callus, which is consistent with primary intramembranous ossification. Daily systemic administration of recombinant PTH 1-34 increased the rate of union in a mouse atrophic nonunion model. This may have important implications for the potential clinical role of PTH 1-34 in the treatment of atrophic fracture nonunions.

An in vivo model to study and manipulate the hematopoietic stem cell niche

AbstractBecause the microenvironment that supports hematopoietic stem cell (HSC) proliferation and differentiation is not fully understood, we adapted a heterotopic bone formation model as a new approach for studying the HSC microenvironment in vivo. Endogenous HSCs homed to tissue-engineered ossicles and individually sorted HSCs from ossicles were able to reconstitute lethally irradiated mice. To further explore this model as a system to study the stem cell niche, ossicles were established with or without anabolic parathyroid hormone (PTH) treatment during the 4-week course of bone development. Histology and micro–computed tomography showed higher bone area-to-total area ratios, thicker cortical bone and trabecular bone, significantly higher bone mineral density and bone volume fraction in PTH-treated groups than in controls. By an in vivo competitive long-term reconstitution assay, HSC frequency in the ossicle marrow was 3 times greater in PTH groups than in controls. When whole bone marrow cells were directly injected into the ossicles after lethal irradiation, the PTH-treated groups showed an enhanced reconstitution rate compared with controls. These findings suggest the residence of HSCs in heterotopic bone marrow and support the future use of this ossicle model in elucidating the composition and regulation of the HSC niche.

Effect of Intermittent Systemic Administration of Recombinant Parathyroid Hormone (1-34) on Mandibular Fracture Healing in Rats

To establish a rat mandibular fracture model and investigate the short- and long-term effects of recombinant parathyroid hormone (PTH 1-34) on mandibular fracture healing in rats. A controlled unilateral mandibular fracture was created surgically in 29 male Sprague-Dawley rats and then stabilized using an external fixation device. The rats were divided into 2 groups: 1 group received daily subcutaneous injections of 10 microg/kg of PTH(1-34) and 1 group served as the vehicle control. The rats were killed on postoperative days 7 and 21, and radiographic densitometry and histologic evaluation of new bone formation were performed. A novel unilateral mandibular fracture model was established that has significant differences from previously published models, both in the location of the osteotomy site and in the rigid external stabilization device. The PTH(1-34) treated rats showed a statistically significant difference (P < .05) in callous formation compared with the control animals. Radiographic densitometry evaluation of the injury site revealed an increase in bone density, apparent at day 7 in the experimental group. Visual inspection of the histologic sections stained with Masson's trichrome blue showed an apparent increase in new bone formation at 21 days in the PTH-treated group compared with the control group. Intermittent systemic administration of PTH(1-34) might enhance the healing of mandibular fractures in the early phase (7-day period). Long-term administration (21-day period) showed no statistically significant differences between the control and experimental group by radiographic densitometry.

Parathyroid hormone PTH(1–34) increases the volume, mineral content, and mechanical properties of regenerated mineralizing tissue after distraction osteogenesis in rabbits

Background and purpose Parathyroid hormone (PTH) has attracted considerable interest as a bone anabolic agent. Recently, it has been suggested that PTH can also enhance bone repair after fracture and distraction osteogenesis. We analyzed bone density and strength of the newly regenerated mineralized tissue after intermittent treatment with PTH in rabbits, which undergo Haversian bone remodeling similar to that in humans.Methods 72 New Zealand White rabbits underwent tibial mid-diaphyseal osteotomy and the callus was distracted 1 mm/day for 10 days. The rabbits were divided into 3 groups, which received injections of PTH 25 µg/kg/day for 30 days, saline for 10 days and PTH 25 µg/kg/day for 20 days, or saline for 30 days. At the end of the study, the rabbits were killed and the bone density was evaluated with DEXA. The mechanical bone strength was determined by use of a 3-point bending test.Results In the 2 PTH-treated groups the regenerate callus ultimate load was 33% and 30% higher, absorbed energy was 100% and 65% higher, BMC was 61% and 60% higher, and callus tissue volume was 179% and 197% higher than for the control group.Interpretation We found that treatment with PTH during distraction osteogenesis resulted in substantially higher mineralized tissue volume, mineral content, and bending strength. This suggests that treatment with PTH may benefit new bone formation during distraction osteogenesis and could form a basis for clinical application of this therapy in humans.

Expression of Rho GDIα in rat osteoblasts intermittently exposed to parathyroid hormone in vitro and in vivo

To investigate the mechanism of the bone-forming effects of intermittent parathyroid hormone (PTH) administration and to search for novel molecules of bone anabolism via the PTH signaling pathway. Primary cultures of rat osteoblasts (ROBs) were divided into an intermittent PTH-treated group (Itm) and a control group (Ctr). Imitating the pharmacokinetics of intermittent PTH administration in vivo, the ROBs in the Itm group were exposed to PTH for 6 h in a 24-h incubation cycle, and the ROBs in the Ctr group were exposed to vehicle for the entire incubation cycle. The cells were collected at 6 h and 24 h of the final cycle, and the proteins in the Itm and Ctr groups were analyzed by two-dimensional electrophoresis (2-DE) coupled with peptide mass fingerprinting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to detect proteins that were differentially expressed. The proteins with the most significant changes in vitro were validated by immunohistochemistry (IHC) in a rat model. The proteomics analysis indicated that a total of 26 proteins were up- or down-regulated in the Itm group compared with the Ctr group at 6 h and 24 h; among these, 15 proteins were successfully identified. These proteins mainly belong to the cytoskeleton and molecular chaperone protein families, and most of these have anti-apoptotic effects in various cells. Rho GDP-dissociation inhibitor alpha (RhoGDIalpha) and vimentin were the most significantly changed proteins. Further studies by IHC showed that the expression of RhoGDIalpha in ROBs was significantly higher in PTH-treated sham-operated rats than in vehicle-treated sham-operated rats, but the difference was not significant between PTH-treated and vehicle-treated OVX rats. Vimentin expression was not changed in either PTH-treated sham-operated rats or PTH-treated OVX rats. Our research suggests that intermittent PTH treatment induces changes in expression of many proteins in ROBs in vitro, and it results in RhoGDIalpha up-regulation in ROBs both in vitro and in vivo when estrogen is present. This up-regulation of RhoGDIalpha may be one of the mechanisms underlying the synergistic bone-forming effect of PTH and estrogen.Acta Pharmacologica Sinica (2009) 30: 1001-1007; doi: 10.1038/aps.2009.60.

The Anabolic Response to Parathyroid Hormone Is Augmented in Rac2 Knockout Mice

PTH is the only currently available anabolic therapy for osteoporosis. In clinical practice, the skeletal response to PTH varies and because therapy is limited to 2 yr, approaches to maximize the therapeutic response are desirable. Rac2 is a small GTPase that is expressed only in hematopoietic tissue. Rac2(-/-) mice have a slight increase in bone mass and osteoclasts isolated from these animals have reduced basal resorptive activity and reduced chemotaxis. To evaluate the anabolic response to PTH in Rac2(-/-) mice, we treated 18 Rac2(-/-) and 17 control, age-matched wild-type animals once daily for 28 d with 80 ng/g body weight of h(1-34)PTH. Treatment resulted in significantly greater increments in spinal, femur, and total bone density in the Rac2(-/-) as compared with wild-type animals. Microcomputed tomography analysis demonstrated greater increases in trabecular thickness and cortical thickness in the knockout mice. Interestingly, histomorphometric analysis showed an equivalent increase in osteoblast and osteoclast number in response to PTH treatment in both groups of animals. However, as judged by changes in serum markers, the resorptive response to PTH was impaired. Thus, telopeptide of type 1 collagen was 15.9+/-6.9 ng/ml after PTH treatment in the knockout animals and 26.8+/-11.1 ng/ml in the PTH-treated wild-type group. In contrast, serum aminoterminal propeptide of type 1 collagen and osteocalcin were equivalent in both groups. We conclude that, in the genetic absence of Rac2, the anabolic response to PTH is increased. This appears to be due to attenuated resorptive activity of osteoclasts.

Effect of Recombinant Human Parathyroid Hormone (1-84) on Vertebral Fracture and Bone Mineral Density in Postmenopausal Women with Osteoporosis

Recombinant human parathyroid hormone (1-84) (PTH) increases bone mass and strength and improves bone quality by stimulating new bone formation. To determine the safety of PTH and its effect on the incidence of vertebral fractures in postmenopausal women with osteoporosis. 18-month, randomized, double-blind, placebo-controlled, parallel-group study. 168 centers in 9 countries. 2532 postmenopausal women with low bone mineral density at the hip or lumbar spine. Women received 100 mug of recombinant human PTH or placebo daily by subcutaneous injection. All received calcium, 700 mg/d, and vitamin D3, 400 U/d. New or worsened vertebral fractures (primary outcome) and changes in bone mineral density and safety (secondary outcomes). 67.2% of patients who received at least 1 dose of the study drug completed the study. Parathyroid hormone reduced the risk for new or worsened vertebral fractures, but in sensitivity analyses, the magnitude of the reduction was changed with assumptions about fracture incidence in patients who did not complete the study (relative risk assuming no fractures, 0.42 [95% CI, 0.24 to 0.72] [P = 0.001]; relative risk assuming fracture incidence observed in all patients who completed the trial, 0.60 [CI, 0.36 to 1.00] [P = 0.05]; relative risk assuming fracture incidence observed in the placebo group, 0.62 [CI, 0.37 to 1.04] [P = 0.07]). Compared with placebo, mean bone mineral density increased at the spine by 6.9% (CI, 6.4% to 7.4%) and at the hip by 2.1% (CI, 1.7% to 2.5%) but decreased at the forearm in the PTH-treated group. Parathyroid hormone treatment increased the percentage of participants with hypercalciuria, hypercalcemia, and nausea by 24% (CI, 20% to 27%), 23% (CI, 21% to 26%), and 14% (CI, 11% to 16%), respectively, compared with placebo. Baseline serum PTH and vitamin D levels were not measured. Many patients discontinued the trial prematurely. Parathyroid hormone (1-84) reduced the overall risk for new or worsened vertebral fracture in postmenopausal women with osteoporosis. Hypercalciuria, hypercalcemia, and nausea were more common in women who took the drug. Although the magnitude of the reduction was sensitive to assumptions about fracture incidence in patients who did not complete the study, the findings suggest that PTH provides an alternative therapeutic option for fracture prevention.

Comparison of dried plum supplementation and intermittent PTH in restoring bone in osteopenic orchidectomized rats

Bone loss was confirmed after 90 days in 50 6-month-old male Sprague Dawley rats that were sham-operated or orchidectomized (ORX). In this study, we have shown that dried plum (DP) has potent effects on bone in terms of bone mass, microarchitecture, and strength in osteopenic male rats. Although these changes may be mediated through the suppression of bone resorption, the fact that the restoration in some of the bone structural and biomechanical parameter shares some similarities with parathyroid hormone (PTH) should not be overlooked. Further investigation is needed on a mechanistic level to clarify the influence of DP on bone metabolism. This study was designed to investigate the extent to which DP reverses bone loss in osteopenic ORX rats and to compare its effects to PTH. Fifty, 6-month-old male Sprague Dawley rats were sham-operated or ORX, and bone loss was confirmed after 90 days. The ORX groups were assigned to control (AIN-93M) diet, 25% DP diet, or PTH (80 microg/kg) for 90 days. DP induced an 11% increase in vertebral and femoral BMD compared to ORX-controls. BMD in the PTH-treated group was increased by 20.7% (vertebra) and 17.9% (femur). Vertebral trabecular bone volume (BV/TV) and number were increased by DP and trabecular separation was decreased compared to controls, which were similar to PTH. Alterations in trabecular bone of the femur were similar to those in the vertebra, but DP did not restore BV/TV to the same extent. Cortical thickness was improved by DP and further enhanced by PTH. DP tended to decrease urinary deoxypyridinoline and calcium, but did not alter alkaline phosphatase or osteocalcin. We conclude that though the degree of improvement was not equivalent to PTH with regard to all parameters, DP reverses bone loss due to ORX and the mechanisms should be further investigated.

Effects of cyclic vs. daily treatment with human parathyroid hormone (1–34) on murine bone structure and cellular activity

Previously, we demonstrated that the human parathyroid hormone (1–34) fragment (hPTH(1–34)) increased bone strength in proportion to its effects on BMD and cortical bone structure in the murine femur by comparing cyclic vs. daily administration of hPTH(1–34). Both cyclic and daily regimens increased vertebral BMD similarly at 7 weeks. Here, we have examined the effects of daily and cyclic PTH regimens on bone structure and cellular activity by static and dynamic histomorphometry. Twenty-week-old, intact female C57BL/J6 mice were treated with the following regimens ( n = 7 for each group): daily injection with vehicle for 7 weeks [control]; daily injection with hPTH(1–34) (40 μg/kg/day) for 7 weeks [daily PTH]; and daily injection with hPTH(1–34) (40 μg/kg/day) and vehicle alternating weekly for 7 weeks [cyclic PTH]. At days 9 and 10, and 2 and 3 prior to euthanasia, calcein (10 mg/kg) was injected subcutaneously. At the end of study, the lumbar vertebrae 1–3 and the left femora were excised, cleaned, and processed for histomorphometry. In the lumbar vertebrae, daily and cyclic PTH regimens significantly increased cancellous bone volume (BV/TV), trabecular number, trabecular osteoclast and osteoblast perimeters, trabecular mineral apposition rate (MAR) and bone formation rate (BFR), and periosteal MAR and BFR compared to control, with no significant difference between the two PTH-treated groups. Increased trabecular tunneling was observed in both PTH-treated groups. Both regimens tended to increase vertebral cortical bone formation parameters with the effects at the periosteum site being more marked than those at the endosteum site, resulting in a significant increase in cortical width. In the femur, the effects of cyclic PTH on BV/TV, trabecular width and number, trabecular and endocortical osteoblast and osteoclast perimeters, cortical width, and trabecular and periosteal BFR were less marked than those of daily PTH. A cyclic PTH regimen was as effective as a daily regimen in improving cancellous and cortical bone microarchitecture and cellular activity in the murine vertebra.

Serum proteome profiles identifies parathyroid hormone physiologic response

Parathyroid hormone (amino acids 1-34) (PTH) regulates bone and calcium homeostasis. The magnitude of the effects of PTH on bone varies in osteoporosis patients. We employed ProteinChip technology to generate protein profiles from sera of mice treated once daily with PTH or vehicle for 3 or 11 days. Data analyses on selected arrays indicated significant increases in serum proteins or peptides in PTH-treated groups, compared to vehicle-controls. The magnitude of change increased with duration of treatment. Anion-exchange fractionation of sera prior to profiling on array surfaces increased the number of proteins detected that were regulated by PTH. The optimized purification conditions developed "on-chip" for subsets of proteins, reflected corresponding behavior with process-compatible chromatographic resins under elution chromatography. We have identified and evaluated subsets of serum proteins regulated by PTH treatment, using a combination of ProteinChip technology, column chromatography, PAGE and LC-MS/MS. Our data demonstrate the feasibility of using a panel of serum proteins to detect PTH responsiveness in humans.

Effects of low-dose, intermittent treatment with recombinant human parathyroid hormone (1–34) on chondrogenesis in a model of experimental fracture healing

Recent studies have demonstrated that intermittent administration of parathyroid hormone (PTH) enhances osteogenesis (hard callus formation) and increases mechanical strength in experimental fracture healing. Thus far, however, effects of PTH on chondrogenesis (soft callus formation) during fracture healing have not been fully elucidated. In the present study, we analyzed the underlying molecular mechanism by which exogenous PTH would affect chondrogenesis in a model of experimental fracture healing. Unilateral femoral fractures were produced in 2-month-old Sprague–Dawley rats. Daily subcutaneous injections of 10 μg/kg of recombinant human PTH(1–34) [rhPTH(1–34)] were administered over a 28-day period of fracture healing. Control animals were injected with vehicle solution (normal saline) alone. The results showed that, on day 14 after fracture, cartilage area in the PTH-treated group was significantly increased (1.4-fold) compared with the controls, but this increase was not observed at days 21 and 28. In the early stage of chondrogenesis (days 4–7), cell proliferation, expressed as the rate of proliferating cell nuclear antigen-positive cells, was increased in mesenchymal (chondroprogenitor) cells but not chondrocytes in the PTH-treated group compared with controls. In addition, gene expression of SOX-9 was up-regulated in the PTH-treated group on day 4 (1.4-fold), and this was accompanied by enhanced expression of pro-α1 (II) collagen (1.8-fold). After 14 days, there were no significant differences between groups in either cell proliferation or the expression levels of cartilage differentiation-related genes (SOX-9, pro-α1 (II) collagen, pro-α1 (X) collagen and osteopontin). These results suggest that intermittent treatment with low-dose rhPTH(1–34) induces a larger cartilaginous callus but does not delay chondrocyte differentiation during fracture healing.

Combined treatment with PTH (1–34) and OPG increases bone volume and uniformity of mineralization in aged ovariectomized rats

The combination of PTH with OPG has been proposed as a potential therapy in patients with severe osteoporosis. In the present study, we examined the bone material of aged ovariectomized (OVX) rats treated either with PTH (1–34) or OPG alone or in combination of both. The micro- and nanostructural characteristics of the mineralized bone were evaluated using quantitative backscattered electron imaging (qBEI) and small-angle X-ray scattering (SAXS). Rats ( n = 68) were either sham-operated or ovariectomized (OVX) at the age of 3 months, and 15 months later, OVX animals were treated either with vehicle, OPG (10 mg/kg), PTH (80 μg/kg) or a combination of both during 5.5 months. All treatments were by subcutaneous injection, 3 days per week. Secondary metaphyseal spongiosa from distal femora was assessed for mineralized bone volume (BV/TV), for the mean Ca-concentration (Ca mean), the width of the bone mineralization density distribution (Ca width), as well as the average mineral particle thickness parameter ( T) and the degree of alignment of the mineral particles ( ρ). A remarkable increase of BV/TV up to 139% ( P < 0.001) was observed in the PTH-treated groups independently of OPG. Ca mean was slightly increased (+1.7%, P < 0.05) in the OPG-treated group. Ca width was reduced (−6.4%, P < 0.01, and −8.9%, P < 0.001) in animals treated with OPG and PTH + OPG, respectively. In contrast, Ca width in sham-operated rats was 16.0% ( P < 0.001) higher than in OVX. The T parameter was not altered in the trabecular bone within the group of treated and untreated OVX rats. However, the non-ovariectomized animals exhibited a significantly lower T value (−7.1%, P < 0.01) with respect to OVX. In conclusion, qBEI and SAXS data of OVX rats suggest that PTH alone was responsible for increase of bone volume, whereas OPG positively influenced the homogeneity and density of mineralization without affecting the nanostructure of the bone material.

Enhancement of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced new bone formation by concurrent treatment with parathyroid hormone and a phosphodiesterase inhibitor, pentoxifylline.

We investigated the enhancement of new bone |formation elicited ectopically by recombinant human bone morphogenetic protein-2 (rhBMP-2), using parathyroid hormone (PTH) and a phosphodiesterase inhibitor (PDEi), pentoxifylline (PTX), in an animal model. Collagen sponge sheet discs containing rhBMP were implanted onto the back muscles of mice. PTX alone (200 mg/kg body weight [BW]), PTH(1-34) (10 microg/kg BW), PTX plus PTH (200 mg/kg BW and 10 microg/kg BW, respectively), or vehicle (control) were injected subcutaneously daily for 3 weeks after implantation. At the end of this period, rhBMP-2-induced ectopic ossicles were harvested from each group of animals. Ossicles from the PTX-treated group were significantly larger in size, with unchanged bone mineral density (BMD), as compared with the ossicles from the controls. In contrast, the ossicles from the PTH-treated group had significantly higher BMD, but showed no difference in size when compared with those from the control animals. The ossicles of the PTX + PTH treatment group were significantly larger than those of the control and PTH treatment groups. In addition, the BMD of the harvested tissues from the PTX + PTH treatment group was signifi-cantly higher than that of tissues from the control and PTX treatment groups. Although the calcium content of ossicles was significantly higher in the PTX-, PTH-, and PTX + PTH-treated groups than in the control group, the Ca content of ossicles from the PTH + PTX-treated group was highest (two times that of controls), followed by the PTH- and PTX-treated groups.

Efficacy and Safety of Human Parathyroid Hormone-(1–84) in Increasing Bone Mineral Density in Postmenopausal Osteoporosis

Many osteoporotic patients continue to have subnormal bone mineral density (BMD) despite treatment. Anabolic agents, including parathyroid hormone (PTH) and N-terminal fragment analogs of PTH, are able to induce large increases in bone mass and bone strength. This prospective, double-blind, placebo-controlled study evaluated 1 year of treatment with full-length recombinant human PTH (PTH-[1–84]) in postmenopausal women with osteoporosis. All participants were 50 to 75 years of age and had been postmenopausal for 5 years or longer. BMD in the lumbar spine was more than 2.5 standard deviations below the young adult mean. A total of 206 women were assigned to self-inject 50, 75, or 100 μg daily of human PTH-(1–84) or a placebo subcutaneously. All patients received calcium and vitamin D supplements. PTH treatment led to dose-related increases in BMD in the lumbar spine (Fig. 1). With the highest dose (100 μg daily), there was no tendency toward plateauing of the response. Average increases in BMD after 12 months were 3.0%, 5.1%, and 7.8%, respectively, for the 50-, 75-, and 100-μg doses. The 0.9% increase in BMD in placebo patients was not significantly greater than baseline BMD. There also was a treatment-related increase in bone area in the lumbar spine that was significant at the highest dose level. PTH treatment had a greater effect on bone mineral content than on BMD. The more severely osteoporotic women had the greatest responses to PTH treatment. Changes in BMD at the total hip and femoral neck were relatively small and inconsistent. Markers of bone remodeling such as osteocalcin were significantly increased in all PTH-treated groups after only 30 days of treatment. Serum calcium levels increased by a small but statistically significant degree in PTH-treated women. Compliance with self-injection exceeded 94% in all groups. Treatment was, in general, well tolerated. The dropout rate was 14%, noncompliance being the major cause. Only 1 patient, in the highest-dose group, withdrew because of repeated hypercalcemia. The most common adverse effect was a reaction at the injection site; it was not dose-related. Creatinine clearance tended to increase in all the PTH groups. Daily treatment with full-length PTH over 1 year led to a significant dose-related increase in lumbar spinal BMD in these postmenopausal women with osteoporosis. The treatment appears to be safe.Fig. 1: Changes in bone mineral density, bone area, and bone mineral content in the lumbar spine (L1–L4) of postmenopausal osteoporotic women treated with full-length parathyroid hormone or placebo for 1 year. Results are the mean ± standard error (n = 43–51/time point). *P <0.05 versus placebo. Reproduced with permission. J Clin Endocrinol Metab 2003;88:5212–5220. Copyright © 2003, The Endocrine Society. All rights reserved.