Continuous Parathyroid Hormone Research Articles

SAT182 Effects Of An Orally Bioavailable Nonpeptide Parathyroid Hormone Receptor Type 1 (PTH1R) Antagonist In Rodent Models Of Primary Hyperparathyroidism (PHPT)

Abstract Disclosure: A.S. Antwan: Employee; Self; Crinetics Pharmaceuticals. E. Rico-Bautista: Employee; Self; Crinetics Pharmaceuticals. J. Pontillo: Employee; Self; Crinetics Pharmaceuticals. S. Wang: Employee; Self; Crinetics Pharmaceuticals. M. Johns: Employee; Self; Crinetics Pharmaceuticals. B. Ramms: Employee; Self; Crinetics Pharmaceuticals. M.A. Fowler: Employee; Self; Crinetics Pharmaceuticals. J.B. Nguyen: Employee; Self; Crinetics. A.A. Castellanos Gonzalez: Employee; Self; Crinetics. B. Fleck: Employee; Self; Crinetics Pharmaceuticals. K. Retting: Employee; Self; Crinetics Pharmaceuticals. D. Dalvie: Employee; Self; Crinetics Pharmaceuticals. S.F. Betz: Employee; Self; Crinetics Pharmaceuticals. S. Markison: Employee; Self; Crinetics Pharmaceuticals. Primary hyperparathyroidism (PHPT) is a condition resulting from an over-secretion of parathyroid hormone (PTH) from one or more overactive parathyroid glands. PTH is a peptide hormone that regulates blood calcium concentrations through effects on bone, intestines, and kidney. PTH acts on parathyroid hormone receptor type 1 (PTH1R) in the kidney to increase calcium reabsorption and block phosphate reabsorption. The loss of phosphate ions then causes an increase in ionized calcium in the blood and cAMP in the urine. At the bone, continuous PTH infusion stimulates osteoclast activity while inhibiting osteoblast activity, leading to breakdown of bone and release of calcium into the extracellular fluid. Conditions that cause excess PTH, such as PHPT, result in elevated concentrations of plasma calcium, increased bone loss, increased fracture risk, and higher susceptibility to kidney stone development. PHPT affects approximately 100,000 patients per year, but many patients are asymptomatic and remain undiagnosed. While a partial or total parathyroidectomy may be suitable for patients diagnosed with severe cases of PHPT, there are limited treatment options for those who do not meet the criteria or do not wish to undergo surgery. The goals for medicinal treatment are to normalize blood calcium levels and urinary calcium excretion, as well as increase bone mineral density to reduce fracture risk. Blocking PTH action directly via a potent PTH1R antagonist may provide an important new therapeutic mechanism to treat patients with PHPT. Using iterative medicinal chemistry and pharmacology, Crinetics has identified several potent and orally bioavailable PTH1R antagonists with good drug-like properties. Lead molecules were evaluated in vivo in preclinical rodent models for their ability to suppress the effects of excess PTH on serum calcium, bone turnover, and cAMP levels in the kidney. One of these compounds, ANT-5, was characterized as a potent negative allosteric modulator of both human and rat PTH1R. In rats, oral administration of ANT-5 dose-dependently suppressed PTH-induced hypercalcemia. ANT-5 also suppressed PTH-stimulated urinary cAMP in rats, indicating its ability to inhibit the actions of PTH in the kidney. We further evaluated the effect of ANT-5 in a rodent model of continuous PTH infusion-induced bone turnover. Once-daily oral administration of ANT-5 mitigated PTH-induced bone resorption, suggesting that inhibiting PTH1R may have beneficial clinical effects on bone density. The culmination of these studies has led to a subset of candidate molecules that are being evaluated in safety studies to identify the compound(s) suitable for evaluation in human clinical trials. Support: Crinetics Pharmaceuticals. Presentation: Saturday, June 17, 2023

Site specific differences in vBMD and geometry in postmenopausal women with primary hyperparathyroidism.

Primary Hyperparathyroidism (PHPT) is associated with catabolic effects at both trabecular and cortical bone. Mechanical loading is one of the most important natural anabolic stimuli for bone at all ages. The present study was designed to assess the impact of PHPT on vBMD and bone geometry using peripheral quantitative computed tomography (pQCT) at the radius and tibia, sites with similar structural characteristics, but subject to different loading conditions. We evaluated the impact of PHPT on bone, by comparing the z-scores of volumetric Bone Mineral Density (vBMD) and indices of bone geometry simultaneously at the tibia and the radius by pQCT, skeletal sites with similar structure, but subject to different loading conditions. Forty-one postmenopausal women with PHPT and 79 controls, comprised the study group. At both trabecular and cortical sites, vBMD and bone geometry indices were significantly lower in patients compared with controls. In patients with PHPT, apart from a lower z-score for total vBMD (p = 0.01) at the radius, there was no other difference between the radius and the tibia at the trabecular sites. On the contrary, at cortical sites, the z-scores of cortical bone mineral content (p = 0.02), cortical vBMD (p = 0.01) and cortical cross-sectional area (p = 0.05) were significantly lower at the radius compared with the tibia, indicating that cortical bone at the weight bearing tibia might be less affected by the catabolic actions of continuous parathyroid hormone (PTH) exposure. PTH levels were positively associated with the difference in z-scores of cort BMD (r = 0.439, p < 0.01) indicating that in more severe cases, as expressed by higher PTH levels, the deleterious effects at the non-weight bearing radius might be accentuated. We found that in postmenopausal women with PHPT, both trabecular and cortical bone are adversely affected. However, at the weight bearing tibia as compared with the radius, the deleterious effects, especially on cortical bone, seem to be attenuated. NCT05426512, 21/06/2022, "retrospectively registered".

β-Arrestin 2 knockout prevents bone loss in response to continuous parathyroid hormone stimulation in male and female mice

ABSTRACT Background β-Arrestin 2 (β-arr2) binds activated parathyroid hormone (PTH) receptors stimulating internalization. PTH stimulates both anabolic and catabolic effect on bone depending on the way it is administered. Intermittent PTH stimulation increases trabecular bone formation in mice, but this is decreased in mice lacking β-arr 2, suggesting a role for β-arr 2 in the anabolic effects of PTH. The role of β-arr 2 in the catabolic effects of continuous PTH (cPTH) treatment is not known. Objective To assess the effects of cPTH administration on bone in mice lacking β-arr 2 compared to wild-type (WT). Methods Groups of male and female WT or β-arr2 knockout (KO) mice were administered either PTH or phosphate-buffered saline by osmotic pumps for 2 weeks. Following treatment, serum calcium and phosphate levels were measured, bone structure and mineral density were measured by microcomputed tomography, and bone cells measured by static and dynamic histomorphometry. Results β-arr2 KO had no effects on skeletal development in mice of either sex. PTH treatment caused hypercalcemia and hypophosphatemia and decreased trabecular and cortical bone only in male WT mice. β-arr2 KO in male mice completely abrogated the effects of PTH on bone, while in female β-arr2 KO mice, PTH treatment increased trabecular bone with no effects on cortical bone. Conclusions These results demonstrate a profound sex effect on skeletal responses to cPTH treatment, suggesting a protective effect of estrogen on bone loss. β-arr2 plays a role in restraining the anabolic effects of PTH in both male and female mice.

Increased osteoblast GαS level determines bone response to hyperparathyroidism in female mice.

GS, the stimulatory heterotrimeric G protein, is an essential regulator of osteogenesis and bone turnover. To determine if increasing GαS in osteoblasts alters bone responses to hyperparathyroidism, we used a transgenic mouse line overexpressing GαS in osteoblasts (GS-Tg mice). Primary osteoblasts from GS-Tg mice showed increased basal and parathyroid hormone (PTH)-stimulated cAMP and greater responses to PTH than cells from WT mice. Skeletal responses to 2-week continuous PTH administration (cPTH) in female mice resulted in trabecular bone loss in WT mice but 74% and 34% increase in trabecular bone mass in long bones and vertebrae, respectively, in GS-Tg mice. Vertebral biomechanical strength was compromised by cPTH treatment in WT mice but not in GS-Tg. Increased peritrabecular fibrosis was greatly increased by cPTH in Gs-Tg compared to WT mice and corresponded with greater increases in Wnt pathway proteins in trabecular bone. Cortical bone responded negatively to cPTH in WT and Gs-Tg mice with large increases in porosity, decreased cortical thickness and compromised biomechanical properties. These results demonstrate that hyperparathyroidism can increase trabecular bone when GS expression and cAMP stimulation in osteoblasts are increased but this is not the case in cortical bone where increased GS expression exacerbates cortical bone loss.

Continuous and intermittent parathyroid hormone administration promotes osteogenic differentiation and activity of programmable cells of monocytic origin

ABSTRACT Bone healing deficiencies are challenging for orthopedic practice. The use of stem cells with scaffolds to treat bone tissue losses currently is popular for promoting regeneration of tissue. Programmable cells of monocytic origin (PCMO) may differentiate into three germ layers and may be a promising alternative treatment due to their stem cell-like properties. Parathyroid hormone (PTH) participates in bone metabolism. Intermittent administration of PTH promotes osteogenic activity of mesenchymal stem cdells (MSC). We investigated the osteogenic effects of continuous and intermittent administration of PTH on PCMO. Mononuclear cells were harvested from the peripheral blood of healthy donors. Isolated cells were cultured for six days in a de-differentiation medium. Indirect immunocytochemistry using anti-CD14, anti-CD45 and anti-CD90 primary antibodies, as well as electron microscopy were used to detect PCMO. PCMO then were cultured in an osteogenic differentiation medium supplemented with continuous or intermittent 50 ng/ml PTH. The PTH-free control group (CG), intermittent PTH treated group (IPG) and continuous PTH treated group (CPG) were cultured and assessed for their differentiation into osteogenic lineage cells by indirect immunocytochemistry using anti-collagen I, anti-osteonectin and anti-osteocalcin primary antibodies. Osteoblast-like cells obtained by continuous or intermittent PTH administration exhibited increased levels of collagen I, osteonectin and osteocalcin immunoreactivity. We found that continuous and intermittent PTH administration to PCMO enhanced their differentiation to osteogenic lineage cells and increased osteoblastic activity.

Gut microbiome, parathyroid hormone, and bone.

Microorganisms in the gut (the 'microbiome') and the metabolites they produce (the 'metabolome') regulate bone mass through interactions between parathyroid hormone (PTH), the immune system, and bone. This review summarizes these data and details how this physiology may relate to CKD-mediated bone disease. The actions of PTH on bone require microbial metabolite activation of immune cells. Butyrate is necessary for CD4+ T-cell differentiation, T-reg cell expansion and CD8+ T-cell secretion of the bone-forming factor Wnt10b ligand. By contrast, mice colonized with segmented filamentous bacteria exhibit an expansion of gut Th17 cells and continuous PTH infusion increases the migration of Th17 cells to the bone marrow, contributing to bone resorption. In the context of CKD, a modified diet, frequent antibiotic therapy, altered intestinal mobility, and exposure to multiple medications together contribute to dysbiosis; the implications for an altered microbiome and metabolome on the pathogenesis of renal osteodystrophy and its treatment have not been explored. As dysregulated interactions between PTH and bone ('skeletal resistance') characterize CKD, the time is ripe for detailed, mechanistic studies into the role that gut metabolites may play in the pathogenesis of CKD-mediated bone disease.

Recent advances in understanding and managing hypoparathyroidism

Hypoparathyroidism is a rare endocrine disorder which leads to hypocalcemia, hypercalciuria, and hyperphosphatemia. Complications include nephrocalcinosis with renal dysfunction, reduced quality of life, and abnormal skeletal properties. Conventional therapy with calcium and vitamin D analogs addresses hypocalcemia but has important limitations. Parathyroid hormone (PTH) therapy is a fundamental advance, although the effects of PTH on long-term complications require additional testing. Continuous PTH therapy is likely to be particularly advantageous for addressing renal, quality of life, and skeletal complications. Overall, much progress has been made, yet more information is needed to improve our understanding and management of hypoparathyroidism.

Continuous rhPTH (1-34) treatment in chronic hypoparathyroidism.

SummaryStandard treatment of hypoparathyroidism consists of supplementation of calcium and vitamin D analogues, which does not fully restore calcium homeostasis. In some patients, hypoparathyroidism is refractory to standard treatment with persistent low serum calcium levels and associated clinical complications. Here, we report on three patients (58-year-old male, 52-year-old female, and 48-year-old female) suffering from severe treatment-refractory postsurgical hypoparathyroidism. Two patients had persistent hypocalcemia despite oral treatment with up to 4 µg calcitriol and up to 4 g calcium per day necessitating additional i.v. administration of calcium gluconate 2–3 times per week, whereas the third patient presented with high frequencies of hypocalcemic and treatment-associated hypercalcemic episodes. S.c. administration of rhPTH (1–34) twice daily (40 µg/day) or rhPTH (1–84) (100 µg/day) only temporarily increased serum calcium levels but did not lead to long-term stabilization. In all three cases, treatment with rhPTH (1–34) as continuous s.c. infusion via insulin pump was initiated. Normalization of serum calcium and serum phosphate levels was observed within 1 week at daily 1–34 parathyroid hormone doses of 15 µg to 29.4 µg. Oral vitamin D and calcium treatment could be stopped or reduced and regular i.v. calcium administration was no more necessary. Ongoing efficacy of this treatment has been documented for up to 7 years so far. Therefore, we conclude that hypoparathyroidism that is refractory to both conventional treatment and s.c. parathyroid hormone (single or twice daily) may be successfully treated with continuous parathyroid hormone administration via insulin pump.Learning points:Standard treatment of hypoparathyroidism still consists of administration of calcium and active vitamin D.Very few patients with hypoparathyroidism also do not respond sufficiently to standard treatment or administration of s.c. parathyroid hormone once or twice daily.In those cases, continuous s.c. administration of parathyroid hormone via insulin pump may represent a successful treatment alternative.

The effects of intro-oral parathyroid hormone on the healing of tooth extraction socket: an experimental study on hyperglycemic rats.

Objective To investigate the effects of intro-oral injection of parathyroid hormone (PTH) on tooth extraction wound healing in hyperglycemic rats.Methodology 60 male Sprague-Dawley rats were randomly divided into the normal group (n=30) and DM group (n=30). Type 1 diabetes mellitus (DM) was induced by streptozotocin. After extracting the left first molar of all rats, each group was further divided into 3 subgroups (n=10 per subgroup), receiving the administration of intermittent PTH, continuous PTH and saline (control), respectively. The intermittent-PTH group received intra-oral injection of PTH three times per week for two weeks. A thermosensitive controlled-release hydrogel was synthesized for continuous-PTH administration. The serum chemistry was determined to evaluate the systemic condition. All animals were sacrificed after 14 days. Micro-computed tomography (Micro-CT) and histological analyses were used to evaluate the healing of extraction sockets.Results The level of serum glucose in the DM groups was significantly higher than that in the non-DM groups (p<0.05); the level of serum calcium was similar in all groups (p>0.05). Micro-CT analysis showed that the DM group had a significantly lower alveolar bone trabecular number (Tb.N) and higher trabecular separation (Tb.Sp) than the normal group (p<0.05). The histological analyses showed that no significant difference in the amount of new bone (hard tissue) formation was found between the PTH and non-PTH groups (p>0.05).Conclusions Bone formation in the extraction socket of the type 1 diabetic rats was reduced. PTH did not improve the healing of hard and soft tissues. The different PTH administration regimes (continuous vs. intermittent) had similar effect on tissue healing. These results demonstrated that the metabolic characteristics of the hyperglycemic rats produced a condition that was unable to respond to PTH treatment.

SUN-512 Denosumab Improves Trabecular but Not Cortical Bone Osteoporosis in a Patient with Refractory Primary Hyperparathyroidism

Background: Primary hyperparathyroidism is a disorder of parathyroid glands in which one or more of these glands is/are overactive. As a result, the overactive gland releases too much parathyroid hormone (PTH) which increases serum calcium levels from thinning of the bones. Osteoporosis occurs in patients with hyperparathyroidism, but it is usually reversible post parathyroidectomy. In the United States, about 100,000 people develop primary hyperparathyroidism each year. The disorder is diagnosed most often in people between age 50 and 60, and women are affected about three times as often as men. Case: This is the case of a 71-year-old lady with history of primary hyperparathyroidism treated with left parathyroidectomy for severe hypercalcemia and osteoporosis. However, although quiescent for few months, the hyperparathyroidism recurred and the osteoporosis worsened. She was referred to our Osteoporosis clinic for evaluation and management. Patient had multiple Sestamibi scans for re-localization of the abnormal parathyroid which were all unsuccessful. Also, she went through a second neck exploration which failed to find the abnormal parathyroid gland. She even underwent fine needle aspiration of intrathyroidal cysts with PTH washouts for identification of possible parathyroid adenoma with negative results. Patient was then treated with bisphosphonates for 5 years and her DEXA scan T score of (-) 2.9 at the radius never improved. Bisphosphonates were discontinued, and patient was treated with Denosumab infusions every 6 months for the last two years in an attempt to help with both osteoporosis and hypercalcemia. Repeated DEXA scan revealed significant improvement of bone mineral density at the level of the lumbar spine, with worsening of osteoporosis at the femurs and radius. Hypercalcemia normalized while PTH remained inappropriately high normal. Discussion: Osteoporosis from hyperparathyroidism affects predominantly long bones. Aside from parathyroidectomy of parathyroid adenomas, no other therapy has been shown to improve cortical bone osteoporosis due to hyperparathyroidism. Denosumab is a human monoclonal antibody which is used for the treatment of osteoporosis in postmenopausal women who have high risk of bone fracture. It has been used lately also for treatment of hypercalcemia of malignancy. Although our patient’s hypercalcemia and trabecular bone osteoporosis improved with Denosumab, cortical bone osteoporosis worsened. This case illustrates the power of continuous PTH secretion on cortical bone resorption which is able to counteract the beneficial effect of Rank ligand blockade at the trabecular bone level. It also demonstrates the need for more research on potential therapies to address this PTH specific action.

Effects of continuous and intermittent parathyroid hormone administration on midpalatal suture expansion in rats

ObjectivesThe aim of this study was to investigate the effects of continuous parathyroid hormone (cPTH) and intermittent parathyroid hormone (iPTH) on bone formation and bone resorption in midpalatal suture during maxillary expansion. MethodsForty-eight male SD rats were randomly divided into four groups (n = 12 each), including the control, the expansion (E), the E + cPTH, and the E + iPTH. A thermosensitive controlled-release hydrogel was synthesized for cPTH administration. All animals were sacrificed after seven days. Microcomputed tomography, histochemical staining and real-time PCR were used to investigate the bone remodeling of midpalatal suture. Serum chemistry was adopted to evaluate the systemic condition of experimental animals. ResultsThe suture width was increased by the expansion, and further elevated by cPTH and iPTH administration. Both regimes improved bone volume fraction and trabecular thickness of suture bone region. Moreover, both cPTH and iPTH decreased SOST expression and enhanced the expression of β-catenin and Col-I. cPTH increased RANKL expression, inhibited OPG expression, and resulted in an increment of osteoclasts, while iPTH had no influence on osteoclastogenesis. The serum calcium concentration was enhanced by PTH administration. ConclusionBoth cPTH and iPTH promote midpalatal suture expansion by enhancing bone formation, probably via SOST downregulation and the resulting β-catenin activation. Our results demonstrated that PTH administration may have potential to be an adjunctive approach for maxillary expansion treatment.

Anabolic action of teriparatide to osteoporotic patients

Intermittent administration of human parathyroid hormone (PTH) [1-34], or teriparatide, has been used for osteoporotic treatment, which is available for daily and weekly administration for the osteoporotic patients in Japan, increasing bone mass and reduce bone fracture risk. In general, continuous PTH infusion shows catabolic effects in bone, while the intermittent administration of PTH results in anabolic action in osteoporotic patients. Intermittent PTH administration promotes preosteoblastic proliferation, as well as stimulates osteoblastic bone formation dependent on cell coupling with osteoclasts. Dosing frequency of PTH administration may affect resultant bone mass, and therefore, we have examined the anabolic effects of the high and low frequency of PTH administration using a mouse model. As a consequence, the high frequency of PTH administration accelerated the preosteoblastic proliferation with forming thick preosteoblastic network, osteoclastogenesis inside the preosteoblastic network, as well as osteoblastic bone formation. In contrast, the low frequency of PTH administration promoted osteoblastic bone formation, but, did not stimulate preosteoblastic proliferation and osteoclastogenesis. In addition, the high or low frequency of PTH administration demonstrated bone formation by manners of accelerated bone remodeling or bone remodeling/mini-modeling, respectively. Thus, the different dosing frequency of PTH administration may induce the different cellular mechanism of anabolic effects in bone.

Different duration of parathyroid hormone exposure distinctively regulates primary response genes Nurr1 and RANKL in osteoblasts.

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis.

Continuous infusion of PTH1\u201334 delayed fracture healing in mice

Hyperparathyroidism, which is increased parathyroid hormone (PTH) levels in the blood, could cause delayed or non-union of bone fractures. But, no study has yet demonstrated the effects of excess continuous PTH exposure, such as that seen in hyperparathyroidism, for fracture healing. Continuous human PTH1–34 (teriparatide) infusion using an osmotic pump was performed for stabilized tibial fractures in eight-week-old male mice to determine the relative bone healing process compared with saline treatment. Radiographs and micro-computed tomography showed delayed but increased calcified callus formation in the continuous PTH1–34 infusion group compared with the controls. Histology and quantitative histomorphometry confirmed that continuous PTH1–34 treatment significantly increased the bone callus area at a later time point after fracture, since delayed endochondral ossification occurred. Gene expression analyses showed that PTH1–34 resulted in sustained Col2a1 and reduced Col10a1 expression, consistent with delayed maturation of the cartilage tissue during fracture healing. In contrast, continuous PTH1–34 infusion stimulated the expression of both Bglap and Acp5 through the healing process, in accordance with bone callus formation and remodeling. Mechanical testing showed that continuously administered PTH1–34 increased the maximum load on Day 21 compared with control mice. We concluded that continuous PTH1–34 infusion resulted in a delayed fracture healing process due to delayed callus cell maturation but ultimately increased biomechanical properties.

Differential effects on bone and mesenchymal stem cells caused by intermittent and continuous PTH administration

Objective: To investigate the distinct effects of intermittent and continuous administration of parathyroid hormone (PTH) on bone and mesenchymal stem cell (MSC). Methods: Six weeks old mice with C57/BL6J background and SOX9-creERT/Td-tomato/Osteocalcin-GFP genotype were divided into 6 groups: intermittent administration and withdraw group (subcutaneous injection with PTH 500 μg·kg-1·d-1), continuous administration and withdraw group (subcutaneous implantation of PTH pump, 80 μg·kg-1·d-1, with a rate of 0.25 μl/h), control administration and withdraw group, with 8 mice in each group. Serum calcium level and bone mineral density (BMD) were measured after two weeks' treatment and two weeks after drug withdraw. Histopathology and immunofluorescence analyses were performed to assess the effects of PTH on bone and mesenchymal stem cell. Results: Serum calcium level increased transiently in intermittent group[(1.36±0.03) mmol/L]and increased gradually in continuous group[up to (2.33±0.03) mmol/L], but reduced to normal level (1.12-1.27 mmol/L) 14 days after drug withdraw. BMD of both intermittent[(0.047±0.002) g/cm2]and continuous[(0.046±0.001) g/cm2]PTH administration groups increased compared with control group[(0.044±0.001) g/cm2], but there was no significant difference among three groups 2 weeks after drug withdraw. Femoral histopathology showed that bone mass, trabecular number and little fibrous tissue hyperplasia in intermittent PTH group increased. Osteoblasts number increased, but lining cells decreased. There was no significant difference in osteocyte and osteoclast numbers. After withdrawing of intermittent PTH, osteocyte and osteoblast number declined significantly, but there was an increased number of lining cells. Continuous PTH caused very high amount of fibrosis, and osteoclast number increased significantly, while osteoblast and osteocyte number increased slightly. After withdrawing of continuous PTH, fibrosis disappeared significantly, and lining cell number increased. Immunofluorescence staining in the epiphyseal-metaphyseal regions in fibula showed intermittent PTH increased undifferentiated Td-Tomato MSC, but declined significantly after withdrawing. Undifferentiated Td-Tomato MSC in continuous PTH increased slightly and decreased after drug withdraw. Conclusions: Intermittent PTH increased undifferentiated Td-Tomato MSC and osteoblast number, and might transform lining cell into osteocytes and osteoblasts, and thus lead to bone formation. Continuous PTH increased undifferentiated Td/Tomato MSC, osteoblast and osteocyte number slightly, but high amount of fibrosis and osteoclasts can be seen, leading to metabolic bone disease. However, lining cell ascended after drug withdraw, which may be beneficial to bone remodeling.

Preprogrammed Long-Term Systemic Pulsatile Delivery of Parathyroid Hormone to Strengthen Bone.

Parathyroid hormone (PTH) is the only US Food and Drug Administration (FDA)-approved anabolic agent for the treatment of osteoporosis. The anabolic action of PTH depends on the mode of PTH administration. Pulsatile administration promotes bone formation, however continuous PTH exposure results in bone resorption. In addition, the therapeutic effect of PTH is optimal when the dose and duration fit the therapeutic window. Current PTH treatment requires daily injection, which is neither a convenient nor a favorable choice of patients. Here, an implantable and biodegradable device capable of long-term pulsatile delivery of PTH is developed as a patient-friendly alternative. The advanced materials and fabrication techniques developed in this work enable us to preprogram a pulsatile delivery device to systemically deliver 21 daily pulses of PTH that build bone in vivo. In addition, the device is biodegradable and absorbable in vivo so that no retraction procedure is needed. Therefore, this implantable and biodegradable pulsatile device holds promise to promote bone growth and treat various conditions of bone loss without the burden of daily injections or secondary surgeries.

Effect of parathyroid hormone on prechondroblast differentiation

Parathyroid hormone (PTH) exerts an anabolic effect that is manifested upon periodic exposure to the hormone and a catabolic effect that is observed in case of a prolonged uninterrupted exposure to high doses of the hormone. The latter effect is characteristic of a range of pathologies, such as the hyperfunction of the parathyroid glands. The analysis of the effects of periodic and continuous administration of PTH fragment 1–34 on the differentiation of bovine prechondroblasts maintained in chondrogenic and osteogenic media was the aim of the present study. Alcian blue staining of monolayers of cultured cells maintained in a chondrogenic medium revealed the stimulation of chondrogenesis (manifested as higher intensity of matrix staining) in the case of continuous PTH administration. Alizarin red staining of monolayers of cultured cells maintained in an osteogenic medium revealed more intensive mineralization of the matrix in plates with cells that were exposed to PTH in a periodical manner. The results of the histochemical study were confirmed using electrophoretic detection of osteocrin and type II collagen.

The Expression of PHOSPHO1, nSMase2 and TNAP is Coordinately Regulated by Continuous PTH Exposure in Mineralising Osteoblast Cultures.

Sustained exposure to high levels of parathyroid hormone (PTH), as observed in hyperparathyroidism, is catabolic to bone. The increase in the RANKL/OPG ratio in response to continuous PTH, resulting in increased osteoclastogenesis, is well established. However, the effects of prolonged PTH exposure on key regulators of skeletal mineralisation have yet to be investigated. This study sought to examine the temporal expression of PHOSPHO1, TNAP and nSMase2 in mineralising osteoblast-like cell cultures and to investigate the effects of continuous PTH exposure on the expression of these enzymes in vitro. PHOSPHO1, nSMase2 and TNAP expression in cultured MC3T3-C14 cells significantly increased from day 0 to day 10. PTH induced a rapid downregulation of Phospho1 and Smpd3 gene expression in MC3T3-C14 cells and cultured hemi-calvariae. Alpl was differentially regulated by PTH, displaying upregulation in cultured MC3T3-C14 cells and downregulation in hemi-calvariae. PTH was also able to abolish the stimulatory effects of bone morphogenic protein 2 (BMP-2) on Smpd3 and Phospho1 expression. The effects of PTH on Phospho1 expression were mimicked with the cAMP agonist forskolin and blocked by the PKA inhibitor PKI (5-24), highlighting a role for the cAMP/PKA pathway in this regulation. The potent down-regulation of Phospho1 and Smpd3 in osteoblasts in response to continuous PTH may provide a novel explanation for the catabolic effects on the skeleton of such an exposure. Furthermore, our findings support the hypothesis that PHOSPHO1, nSMase2 and TNAP function cooperatively in the initiation of skeletal mineralisation.

Serum Amyloid A3 Secreted by Preosteoclasts Inhibits Parathyroid Hormone-stimulated cAMP Signaling in Murine Osteoblasts

Continuous parathyroid hormone (PTH) blocks its own osteogenic actions in marrow stromal cell cultures by inducing Cox2 and receptor activator of nuclear factor κB ligand (RANKL) in the osteoblastic lineage cells, which then cause the hematopoietic lineage cells to secrete an inhibitor of PTH-stimulated osteoblast differentiation. To identify this inhibitor, we used bone marrow macrophages (BMMs) and primary osteoblasts (POBs) from WT and Cox2 knock-out (KO) mice. Conditioned medium (CM) from RANKL-treated WT, but not KO, BMMs blocked PTH-stimulated cAMP production in POBs. Inhibition was reversed by pertussis toxin (PTX), which blocks Gαi/o activation. Saa3 was the most highly differentially expressed gene in a microarray comparison of RANKL-treated WT versus Cox2 KO BMMs, and RANKL induced Saa3 protein secretion only from WT BMMs. CM from RANKL-stimulated BMMs with Saa3 knockdown did not inhibit PTH-stimulated responses in POBs. SAA added to POBs inhibited PTH-stimulated cAMP responses, which was reversed by PTX. Selective agonists and antagonists of formyl peptide receptor 2 (Fpr2) suggested that Fpr2 mediated the inhibitory actions of Saa3 on osteoblasts. In BMMs committed to become osteoclasts by RANKL treatment, Saa3 expression peaked prior to appearance of multinucleated cells. Flow sorting of WT marrow revealed that Saa3 was secreted only from the RANKL-stimulated B220(-) CD3(-)CD11b(-/low) CD115(+) preosteoclast population. We conclude that Saa3 secretion from preosteoclasts, induced by RANKL in a Cox2-dependent manner, inhibits PTH-stimulated cAMP signaling and osteoblast differentiation via Gαi/o signaling. The induction of Saa3 by PTH may explain the suppression of bone formation when PTH is applied continuously and may be a new therapeutic target for osteoporosis.

T cells, osteoblasts, and osteocytes: interacting lineages key for the bone anabolic and catabolic activities of parathyroid hormone.

Osteoimmunology is a field of research dedicated to the study of the interactions between the immune system and bone. Among the cells of the immune system that regulate bone turnover and the responsiveness of bone cells to calciothropic hormones are bone marrow T lymphocytes. T cells secrete osteoclastogenic cytokines such as RANKL and TNF-α, as well as factors that stimulate bone formation, one of which is Wnt10b. In addition, T cells regulate the differentiation and life span of stromal cells (SCs) and their responsiveness to parathyroid hormone (PTH) via costimulatory molecules expressed on their surface. The conditioning effect of T cells on SCs is inherited by the osteoblastic and osteocytic progeny of SCs. As a result, osteoblastic cells of T cell-deficient mice have functional characteristics different from corresponding cells of T cell-replete mice. These differences include the ratio of RANKL/OPG produced in response to continuous PTH treatment, and the osteoblastogenic response to intermittent PTH treatment. This article reviews the evidence indicating that the effects of PTH are mediated not only by osteoblasts and osteocytes but also by T cells.