Intermittent Parathyroid Hormone Treatment Research Articles

Abstract 6772: A 3D model of breast cancer-bone metastasis to mimic dysregulation of bone remodeling and drug response

Abstract Introduction: Bone metastases are common among advanced stage breast cancer patients and induce dysregulation of bone remodeling. Crosstalk between cancer cells and the bone niche drives the vicious cycle of bone loss and breast cancer growth. To accelerate discovery of targeted therapies, there is a critical need for experimental models that can mimic breast cancer-induced dysregulation of bone remodeling in patients. Mouse models are the current gold standard, but are low throughput, costly, and cannot fully recapitulate human-human cell interactions. To address these limitations, our goal is to engineer a fully human 3D in vitro model of breast cancer-bone metastasis that can mimic dysregulation of bone remodeling and drug responses in patients, and enable screening of potential new drug candidates. Methods: Two human breast cancer cell lines (MDA-MB-231, MCF-7) were chosen given their well characterized clinical features and tendency to invade bone. Our 3D model comprises an outer ring of tissue engineered (TE) bone and a center of breast cancer cells to mimic invasion through the bone marrow/long bone interface. Human mesenchymal stem cells were encapsulated in 3D gelatin microribbon scaffolds and cultured in osteogenic media for 28 days to form TE bone. Human monocyte-derived osteoclasts were seeded onto the bone, and the center was replaced with GFP-labeled breast cancer cells. Denosumab and zoledronic acid were selected as they are standard treatments for reducing bone loss in patients, and were used to treat the model at 30 µg/mL and 2 µg/mL, respectively. Intermittent parathyroid hormone (iPTH) treatment was selected as it showed efficacy in animal models, and was used to treat the model at 10 µg/mL for 6 hours daily. All groups were treated with drugs for 14 days. Bone volume was assessed using microCT. Cancer cell proliferation and invasion into bone was monitored with fluorescence confocal microscopy. Results: MicroCT imaging showed both breast cancer cell lines induced bone loss in the triculture model, compared to the control with no breast cancer cells in the center. Consistent with known clinical features, the more aggressive cell line (MDA-MB-231) induced a greater amount of bone loss than the less aggressive breast cancer cell line (MCF-7). Consistent with clinical findings, both denosumab and zoledronic acid reduced bone loss in our 3D model, and zoledronic acid further reduced breast cancer cell invasion and proliferation. Finally, the 3D model recapitulated the in vivo drug response of iPTH treatment, reducing bone resorption and breast cancer invasion. Conclusions: Here we report a fully human triculture model of breast cancer-bone metastasis that can mimic key clinical features of breast cancer-induced bone resorption and drug response in patients. Such a 3D model could enable screening of drug candidates with reduced time and cost and enable identification of novel druggable targets. Citation Format: Michelle Tai, Eva C. González Díaz, Callan E. Monette, Joy Wu, Fan Yang. A 3D model of breast cancer-bone metastasis to mimic dysregulation of bone remodeling and drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6772.

Mitochondrial β-oxidation of adipose-derived fatty acids by osteoblasts fuels parathyroid hormone-induced bone formation.

The energetic costs of bone formation require osteoblasts to coordinate their activities with tissues, like adipose, that can supply energy-dense macronutrients. In the case of intermittent parathyroid hormone (PTH) treatment, a strategy used to reduce fracture risk, bone formation is preceded by a change in systemic lipid homeostasis. To investigate the requirement for fatty acid oxidation by osteoblasts during PTH-induced bone formation, we subjected mice with osteoblast-specific deficiency of mitochondrial long-chain β-oxidation as well as mice with adipocyte-specific deficiency for the PTH receptor or adipose triglyceride lipase to an anabolic treatment regimen. PTH increased the release of fatty acids from adipocytes and β-oxidation by osteoblasts, while the genetic mouse models were resistant to the hormone's anabolic effect. Collectively, these data suggest that PTH's anabolic actions require coordinated signaling between bone and adipose, wherein a lipolytic response liberates fatty acids that are oxidized by osteoblasts to fuel bone formation.

Intermittent parathyroid hormone increases stability and improves osseointegration of initially unstable implants.

AimsTo develop an early implant instability murine model and explore the use of intermittent parathyroid hormone (iPTH) treatment for initially unstable implants.Methods3D-printed titanium implants were inserted into an oversized drill-hole in the tibiae of C57Bl/6 mice (n = 54). After implantation, the mice were randomly divided into three treatment groups (phosphate buffered saline (PBS)-control, iPTH, and delayed iPTH). Radiological analysis, micro-CT (µCT), and biomechanical pull-out testing were performed to assess implant loosening, bone formation, and osseointegration. Peri-implant tissue formation and cellular composition were evaluated by histology.ResultsiPTH reduced radiological signs of loosening and led to an increase in peri-implant bone formation over the course of four weeks (timepoints: one week, two weeks, and four weeks). Observational histological analysis shows that iPTH prohibits the progression of fibrosis. Delaying iPTH treatment until after onset of peri-implant fibrosis still resulted in enhanced osseointegration and implant stability. Despite initial instability, iPTH increased the mean pull-out strength of the implant from 8.41 N (SD 8.15) in the PBS-control group to 21.49 N (SD 10.45) and 23.68 N (SD 8.99) in the immediate and delayed iPTH groups, respectively. Immediate and delayed iPTH increased mean peri-implant bone volume fraction (BV/TV) to 0.46 (SD 0.07) and 0.34 (SD 0.10), respectively, compared to PBS-control mean BV/TV of 0.23 (SD 0.03) (PBS-control vs immediate iPTH, p < 0.001; PBS-control vs delayed iPTH, p = 0.048; immediate iPTH vs delayed iPTH, p = 0.111).ConclusioniPTH treatment mediated successful osseointegration and increased bone mechanical strength, despite initial implant instability. Clinically, this suggests that initially unstable implants may be osseointegrated with iPTH treatment.Cite this article: Bone Joint Res 2022;11(5):260–269.

Intermittent parathyroid hormone treatment affects the bone structural parameters and mechanical strength of the femoral neck after ovariectomy-induced osteoporosis in rats

BackgroundMenopause-induced decline in estrogen levels in women is a main factor leading to osteoporosis. The objective of this study was to investigate the effect of intermittent parathyroid hormone (PTH) on bone structural parameters of the femoral neck in ovariectomized rats, in addition to correlations of maximum fracture force.MethodsFifteen female Wister rats were divided into three groups: (1) control group; (2) ovariectomized (OVX) group; and (3) OVX + PTH group. All rats were then killed and the femurs extracted for microcomputed tomography scanning to measure volumetric bone mineral density (vBMD) and bone structural parameters of the femoral neck. Furthermore, the fracture forces of femoral neck were measured using a material testing system.ResultsCompared with the control and OVX + PTH groups, the OVX group had significantly lower aBMD, bone parameter, and mechanical strength values. A comparison between OVX and OVX + PTH groups indicated that PTH treatment increased several bone parameters. However, the OVX + PTH groups did not significantly differ with the control group with respect to the bone structural parameters, except for trabecular bone thickness of cancellous bone, which was greater. In addition, among the bone structural parameters, the CSA and BSI of cortical bone were significantly correlated with the maximum fracture force of the femoral neck, with correlations of, respectively, 0.682 (p = 0.005) and 0.700 (p = 0.004).ConclusionIntermittent PTH helped treat ovariectomy-induced osteoporosis of cancellous bone and cortical bone in the femoral necks of rats. The ability of the femoral neck to resist fracture was highly correlated with the two parameters, namely cross-sectional area (CSA) and bone strength index (= vBMD × CSA), of cortical bone in the femoral neck and was less correlated with aBMD or other bone structural parameters.

Procalcitonin is expressed in osteoblasts and limits bone resorption through inhibition of macrophage migration during intermittent PTH treatment

Intermittent injections of parathyroid hormone (iPTH) are applied clinically to stimulate bone formation by osteoblasts, although continuous elevation of parathyroid hormone (PTH) primarily results in increased bone resorption. Here, we identified Calca, encoding the sepsis biomarker procalcitonin (ProCT), as a novel target gene of PTH in murine osteoblasts that inhibits osteoclast formation. During iPTH treatment, mice lacking ProCT develop increased bone resorption with excessive osteoclast formation in both the long bones and axial skeleton. Mechanistically, ProCT inhibits the expression of key mediators involved in the recruitment of macrophages, representing osteoclast precursors. Accordingly, ProCT arrests macrophage migration and causes inhibition of early but not late osteoclastogenesis. In conclusion, our results reveal a potential role of osteoblast-derived ProCT in the bone microenvironment that is required to limit bone resorption during iPTH.

Short Cyclic Regimen With Parathyroid Hormone (PTH) Results in Prolonged Anabolic Effect Relative to Continuous Treatment Followed by Discontinuation in Ovariectomized Rats.

Despite the potent effect of intermittent parathyroid hormone (PTH) treatment on promoting new bone formation, bone mineral density (BMD) rapidly decreases upon discontinuation of PTH administration. To uncover the mechanisms behind this adverse phenomenon, we investigated the immediate responses in bone microstructure and bone cell activities to PTH treatment withdrawal and the associated long-term consequences. Unexpectedly, intact female and estrogen-deficient female rats had distinct responses to the discontinuation of PTH treatment. Significant tibial bone loss and bone microarchitecture deterioration occurred in estrogen-deficient rats, with the treatment benefits of PTH completely lost 9 weeks after discontinuation. In contrast, no adverse effect was observed in intact rats, with sustained treatment benefit 9 weeks after discontinuation. Intriguingly, there is an extended anabolic period during the first week of treatment withdrawal in estrogen-deficient rats, during which no significant change occurred in the number of osteoclasts, whereas the number of osteoblasts remained elevated compared with vehicle-treated rats. However, increases in number of osteoclasts and decreases in number of osteoblasts occurred 2 weeks after discontinuation of PTH treatment, leading to significant reduction in bone mass and bone microarchitecture. To leverage the extended anabolic period upon early withdrawal from PTH, a cyclic administration regimen with repeated cycles of on and off PTH treatment was explored. We demonstrated that the cyclic treatment regimen efficiently alleviated the PTH withdrawal-induced bone loss, improved bone mass, bone microarchitecture, and whole-bone mechanical properties, and extended the treatment duration. © 2021 American Society for Bone and Mineral Research (ASBMR).

Parathyroid hormone increases alveolar bone homoeostasis during orthodontic tooth movement in rats with periodontitis via crosstalk between STAT3 and \u03b2-catenin

Periodontitis patients are at risk of alveolar bone loss during orthodontic treatment. The aim of this study was to investigate whether intermittent parathyroid hormone (1–34) treatment (iPTH) could reduce alveolar bone loss during orthodontic tooth movement (OTM) in individuals with periodontitis and the underlying mechanism. A rat model of OTM in the context of periodontitis was established and alveolar bone loss was observed. The control, iPTH and iPTH + stattic groups received injections of vehicle, PTH and vehicle, or PTH and the signal transducer and activator of transcription 3 (STAT3) inhibitor stattic, respectively. iPTH prevented alveolar bone loss by enhancing osteogenesis and suppressing bone resorption in the alveolar bone during OTM in rats with periodontitis. This effect of iPTH was along with STAT3 activation and reduced by a local injection of stattic. iPTH promoted osteoblastic differentiation and might further regulate the Wnt/β-catenin pathway in a STAT3-dependent manner. The findings of this study suggest that iPTH might reduce alveolar bone loss during OTM in rats with periodontitis through STAT3/β-catenin crosstalk.

Distinct Responses of Modeling- and Remodeling-Based Bone Formation to the Discontinuation of Intermittent Parathyroid Hormone Treatment in Ovariectomized Rats.

Anabolic agents, such as intermittent parathyroid hormone (PTH), exert their treatment efficacy through activation of two distinct bone formation processes, namely, remodeling-based bone formation (RBF, bone formation coupled with prior bone resorption) and modeling-based bone formation (MBF, bone formation without prior activation of bone resorption). However, if not followed by an antiresorptive agent, treatment benefit was quickly lost upon withdrawal from anabolic agents. By using in vivo micro-computed tomography imaging and multiplex cryohistology with sequential immunofluorescence staining, we investigated the temporal response of newly formed bone tissue from MBF and RBF and the preexisting bone tissue to withdrawal from PTH treatment and the associated cellular activity in an ovariectomized (OVX) rat model. We first demonstrated continued mineral apposition at both RBF and MBF sites following PTH discontinuation, resulting in an extended anabolic effect after 1-week withdrawal from PTH. It was further discovered that MBF sites had a greater contribution than RBF sites to the extended anabolic effect upon early withdrawal from PTH, evidenced by a higher percentage of alkaline phosphatase-positive (ALP+) surfaces and far greater bone formation activity at MBF versus RBF sites. Furthermore, significant bone loss occurred after 3 weeks of discontinuation from PTH, resulting from marked loss of newly formed bone tissue from RBF and preexisting bone tissue prior to treatment. In contrast, MBF surfaces had a delayed increase of tartrate-resistant acid phosphatase activity following PTH discontinuation. As a result, newly formed bone tissue from MBF had greater resistance to PTH discontinuation-induced bone loss than those from RBF and preexisting bone. Understanding various responses of two distinct bone formation types and preexisting bone to anabolic treatment discontinuation is critical to inform the design of follow-up treatment or cyclic treatment strategies to maximize treatment benefit of anabolic agents. © 2022 American Society for Bone and Mineral Research (ASBMR).

CCN1/Cyr61 Is Required in Osteoblasts for Responsiveness to the Anabolic Activity of PTH

CCN1/Cyr61 is a dynamically expressed matricellular protein that serves regulatory functions in multiple tissues. Previous studies from our laboratory demonstrated that CCN1 regulates bone maintenance. Using an osteoblast and osteocyte conditional knockout mouse model (Ccn1OCN ), we found a significant decrease in trabecular and cortical bone mass in vivo, in part through suppression of Wnt signaling since the expression of the Wnt antagonist sclerostin (SOST) is increased in osteoblasts lacking CCN1. It has been established that parathyroid hormone (PTH) signaling also suppresses SOST expression in bone. We therefore investigated the interaction between CCN1 and PTH-mediated responses in this study. We find that loss of Ccn1 in osteoblasts leads to impaired responsiveness to anabolic intermittent PTH treatment in Ccn1OCN mice in vivo and in osteoblasts from these mice in vitro. Analysis of Ccn1OCN mice demonstrated a significant decrease in parathyroid hormone receptor-1 (PTH1R) expression in osteoblasts in vivo and in vitro. We investigated the regulatory role of a non-canonical integrin-binding domain of CCN1 because several studies indicate that specific integrins are critical to mechanotransduction, a PTH-dependent response, in bone. These data suggest that CCN1 regulates the expression of PTH1R through interaction with the αvβ3 and/or αvβ5 integrin complexes. Osteoblasts that express a mutant form of CCN1 that cannot interact with αvβ3/β5 integrin demonstrate a significant decrease in mRNA and protein expression of both PTH1R and αv integrin. Overall, these data suggest that the αvβ3/β5-binding domain of CCN1 is required to endow PTH signaling with anabolic activity in bone cells. © 2020 American Society for Bone and Mineral Research (ASBMR).

Effects of intermittent treatment with parathyroid hormone (PTH) on osteoblastic differentiation and mineralization of mouse induced pluripotent stem cells in a 3D culture model.

PTH plays an important role in bone remodeling, and different actions have been reported depending on its administration method. iPSCs are promising as a cell source for regeneration of periodontal tissue due to their ability of proliferation and pluripotency. However, the effects of PTH on iPSCs remain mostly unknown. The purpose of this study was to investigate in vitro effects of parathyroid hormone (PTH) on osteoblastic differentiation of induced pluripotent stem cells (iPSCs) in a 3D culture model. Following embryoid body (EB) induction from mouse iPSCs (miPSCs), dissociated cells (miPS-EB-derived cells) were seeded onto atelocollagen sponge (ACS) in osteoblast differentiation medium (OBM). Cell-ACS constructs were divided into three groups: continuous treatment with human recombinant PTH (1-34) (PTH-C), intermittent PTH treatment (PTH-I) or OBM control. To confirm the expression of PTH receptor-1(PTH1R), the expression of Pth1r and cAMP production over time were assessed. Real-time PCR was used to assess the expression of genes encoding osterix (Sp7), runt-related transcription factor 2 (Runx2), collagen type 1 (Col1a1), and osteocalcin (Bglap) at different time points. Mineralization was assessed by von Kossa staining. Histochemical staining was used to analyze alkaline phosphatase (ALP) activity, and immunolocalization of SP7 and BGLAP was analyzed by confocal laser scanning microscopy (CLSM). On days 7 and 14, expression of the Pth1r in miPS-EB-derived cells was increased in all groups. Production of cAMP, the second messenger of the PTH1R, tended to increase in the PTH-I group compared with PTH-C group on day 14. Expression of Col1a1 in the PTH-I group on day 14 was significantly higher than other groups. There was a time-dependent increase in the expression of Sp7 in all groups. On day 14, the expression level of Sp7 in the PTH-I group was significantly higher than other groups. In von Kossa staining, the PTH-I group showed higher level of staining compared with other groups on day 14, whereas the level was slightly attenuated in the PTH-C group. In histochemical staining, ALP-positive cells were significantly increased in the PTH-I group compared with other groups on day 14. In CLSM analysis, the numbers of SP7- and BGLAP-positive cells showed a gradual increase over time, and on day 14, a significantly greater SP7 expression was observed in the PTH-I group than other groups. These results suggested that the intermittent PTH treatment promotes osteoblastic differentiation and mineralization of miPSCs in the ACS scaffold.

Alcohol-induced Wnt signaling inhibition during bone fracture healing is normalized by intermittent parathyroid hormone treatment.

Nearly half of orthopaedic trauma patients are intoxicated at the time of injury, and excess alcohol consumption increases the risk for fracture nonunion. Previous studies show alcohol disrupts fracture associated Wnt signaling required for normal bone fracture repair. Intermittent parathyroid hormone (PTH) promotes bone growth through canonical Wnt signaling, however, no studies have investigated the effect of PTH on alcohol‐inhibited bone fracture repair. Male C57BL/6 mice received two‐3 day alcohol binges separated by 4 days before receiving a mid‐shaft tibia fracture. Postoperatively, mice received PTH daily until euthanasia. Wnt/β‐catenin signaling was analyzed at 9 days post‐fracture. As previously observed, acute alcohol exposure resulted in a >2‐fold decrease in total and the active form of β‐catenin and a 2‐fold increase in inactive β‐catenin within the fracture callus. Intermittent PTH abrogated the effect of alcohol on β‐catenin within the fracture callus. Upstream of β‐catenin, alcohol‐treated animals had a 2‐fold decrease in total LRP6, the Wnt co‐receptor, which was restored with PTH treatment. Alcohol nor PTH had any significant effect on GSK‐3β. These data show that intermittent PTH following a tibia fracture restores normal expression of Wnt signaling proteins within the fracture callus of alcohol‐treated mice.

The Transcription Factor Foxc1 Promotes Osteogenesis by Directly Regulating Runx2 in Response of Intermittent Parathyroid Hormone (1-34) Treatment.

Parathyroid hormone (PTH) is crucial for bone remodeling. Intermittent PTH (1–34) administration stimulates osteogenesis and promotes bone formation; however, the possible targets and underlying mechanisms still remain unclear. In this study, functional links between PTH and Foxc1, a transcription factor reported to be predominant in skeletal development and formation, were indicated. We determined the impacts of Foxc1 on in vitro osteogenic differentiation and in vivo bone regeneration under intermittent PTH induction, and further explored its possible targets. We found that the expression level of Foxc1 was upregulated during osteogenic induction by intermittent PTH treatment, and the elevated expression of Foxc1 induced by PTH was inhibited by PTH1R silencing, while rescued by intermittent PTH supplement. By gain- and loss-of-function strategies targeting Foxc1 in MC3T3-E1 cells, we demonstrated that Foxc1 could promote in vitro osteogenic differentiation by intermittent PTH induction. Moreover, immunofluorescence analysis indicated the nuclear co-localization of Foxc1 with Runx2. Luciferase-reporter and chromatin immunoprecipitation analysis further confirmed that Foxc1 could bind to the P1 promoter region of Runx2 directly, which plays an indispensable part in osteogenic differentiation and bone mineralization. Meanwhile, we also revealed that Foxc1 could promote bone regeneration induced by intermittent PTH treatment in vivo. Taken together, this study revealed the role and mechanism of Foxc1 on in vitro osteogenic differentiation and in vivo bone regeneration in response of intermittent PTH treatment.

Preactivation of β-catenin in osteoblasts improves the osteoanabolic effect of PTH in type 1 diabetic mice.

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1-3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.

Parathyroid Hormone Shifts Cell Fate of a Leptin Receptor-Marked Stromal Population from Adipogenic to Osteoblastic Lineage.

Intermittent parathyroid hormone (iPTH) treatment induces bone anabolic effects that result in the recovery of osteoporotic bone loss. Human PTH is usually given to osteoporotic patients because it induces osteoblastogenesis. However, the mechanism by which PTH stimulates the expansion of stromal cell populations and their maturation toward the osteoblastic cell lineage has not be elucidated. Mouse genetic lineage tracing revealed that iPTH treatment induced osteoblastic differentiation of bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs), which carried the leptin receptor (LepR)-Cre. Although these findings suggested that part of the PTH-induced bone anabolic action is exerted because of osteoblastic commitment of MSPCs, little is known about the in vivo mechanistic details of these processes. Here, we showed that LepR+ MSPCs differentiated into type I collagen (Col1)+ mature osteoblasts in response to iPTH treatment. Along with osteoblastogenesis, the number of Col1+ mature osteoblasts increased around the bone surface, although most of them were characterized as quiescent cells. However, the number of LepR-Cre-marked lineage cells in a proliferative state also increased in the vicinity of bone tissue after iPTH treatment. The expression levels of SP7/osterix (Osx) and Col1, which are markers for osteoblasts, were also increased in the LepR+ MSPCs population in response to iPTH treatment. In contrast, the expression levels of Cebpb, Pparg, and Zfp467, which are adipocyte markers, decreased in this population. Consistent with these results, iPTH treatment inhibited 5-fluorouracil- or ovariectomy (OVX)-induced LepR+ MSPC-derived adipogenesis in BM and increased LepR+ MSPC-derived osteoblasts, even under the adipocyte-induced conditions. Treatment of OVX rats with iPTH significantly affected the osteoporotic bone tissue and expansion of the BM adipose tissue. These results indicated that iPTH treatment induced transient proliferation of the LepR+ MSPCs and skewed their lineage differentiation from adipocytes toward osteoblasts, resulting in an expanded, quiescent, and mature osteoblast population. © 2019 American Society for Bone and Mineral Research.

Examining the influence of PTH(1-34) on tissue strength and composition.

The lacunar-canaliculi system is a network of channels that is created and maintained by osteocytes as they are embedded throughout cortical bone. As osteocytes modify their lacuna space, the local tissue composition and tissue strength are subject to change. Although continual exposure to parathyroid hormone (PTH) can induce adaptation at the lacunar wall, the impact of intermittent PTH treatment on perilacunar adaptation remains unclear. Therefore, the primary objective of this study was to establish how intermittent PTH(1-34) treatment influences perilacunar adaptation with respect to changes in tissue composition. We hypothesized that local changes in tissue composition following PTH(1-34) are associated with corresponding gains in tissue strength and resistance to microdamage at the whole bone level. Adult male C57BL/6J mice were treated daily with PTH(1-34) or vehicle for 3 weeks. In response to PTH(1-34), Raman spectroscopy revealed a significant decrease in the carbonate-to-phosphate ratio and crystallinity across the entire tissue, while the mineral-to-matrix ratio demonstrated a significant decrease in just the perilacunar region. The shift in perilacunar composition largely explained the corresponding increase in tissue strength, while the degree of new tissue added at the endosteum and periosteum did not produce any significant changes in cortical area or moment of inertia that would explain the increase in tissue strength. Furthermore, fatigue testing revealed a greater resistance to crack formation within the existing tissue following PTH(1-34) treatment. As a result, the shift in perilacunar composition presents a unique mechanism by which PTH(1-34) produces localized differences in tissue quality that allow more energy to be dissipated under loading, thereby increasing tissue strength and resistance to microdamage. In addition, our findings demonstrate the potential for PTH(1-34) to amplify osteocytes' mechanotransduction by producing a more compliant tissue. Overall, the present study demonstrates that changes in tissue composition localized at the lacuna wall contribute to the strength and fatigue resistance of cortical bone gained in response to intermittent PTH(1-34) treatment.

Enhancement of Bone Augmentation in Osteoporotic Conditions by the Intermittent Parathyroid Hormone: An Animal Study in the Calvarium of Ovariectomized Rat.

Intermittent parathyroid hormone (PTH) is the commonly used therapeutic approach for patients with severe osteoporosis. The goal of this study was to elucidate the effect of the intermittent PTH treatment on guided bone augmentation (GBA) in the calvarium of ovariectomized (OVX) rats. Surgical ovariectomy on 14 rats and sham surgery on 7 rats were conducted on all rats as the first surgery. GBA surgery was conducted 8 weeks following the first surgery in the rat calvarium by placing 5-mm-diameter cylindrical plastic caps. Following surgery, rats were treated with 40 μg/kg PTH (OVX-PTH) or saline (Sham-Saline, OVX-Saline) via intraperitoneal injection three times per week during the all-observational period. Longitudinal microcomputed tomography (micro-CT) imaging was performed every 2 weeks following the GBA surgery without euthanasia, and the amount of newly generated bone volume (BV) was calculated. All rats were euthanized 12 weeks after GBA surgery, and histology was obtained. Sections stained with hematoxylin and eosin were used for the quantitative analysis of newly generated tissue, and immunohistology was used to visualize Runx2-positive cells and TRAP-positive cells. Throughout the monitoring period, the BVs of OVX rats without PTH treatment (OVX-Saline) were significantly lower than that of the other two groups at weeks 8 and 12 in micro-CT analysis. During all experimental periods, the BV was highest in the OVX rats that were treated with PTH (OVX-PTH). Histologic analysis confirmed the result of micro-CT, and determined that the OVX-PTH presented a greater number of Runx2-positive cells. The number of TRAP-positive multinucleated osteoclasts was highest in OVX-PTH rats; there were no significant differences between the other two groups. The results of this study suggest that treatment with intermittent PTH was associated with increased newly regenerated bone volume in ovariectomized rat calvarial bone augmentation, which may have important clinical implications.

Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss.

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis byinversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis byinducing osteoprotegerin through newly discovered p38-GSK3β-β-catenin and WNT5A-LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serumS1P levels were positively associated with serumcalcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and thebone formation markerPINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.

Regulation of Bone Remodeling by Parathyroid Hormone.

Parathyroid hormone (PTH) exerts profound effects on skeletal homeostasis through multiple cellular and molecular mechanisms. Continuous hyperparathyroidism causes net loss of bone mass, despite accelerating bone formation by osteoblasts. Intermittent treatment with PTH analogs represents the only Food and Drug Administration (FDA)-approved bone anabolic osteoporosis treatment strategy. Functional PTH receptors are present on cells of the osteoblast lineage, ranging from early skeletal stem cells to matrix-embedded osteocytes. In addition, bone remodeling by osteoclasts liberates latent growth factors present within bone matrix. Here, we will provide an overview of the multiple cellular and molecular mechanisms through which PTH influences bone homeostasis. Notably, net skeletal effects of continuous versus intermittent can differ significantly. Where possible, we will highlight mechanisms through which continuous hyperparathyroidism leads to bone loss, and through which intermittent hyperparathyroidism boosts bone mass. Given the therapeutic usage of intermittent PTH (iPTH) treatment for osteoporosis, particular attention will be paid toward mechanisms underlying the bone anabolic effects of once daily PTH administration.

Bisphosphonate's and Intermittent Parathyroid Hormone's Effect on Human Spinal Fusion: A Systematic Review of the Literature.

There has been a conscious effort to address osteoporosis in the aging population. As bisphosphonate and intermittent parathyroid hormone (PTH) therapy become more widely prescribed to treat osteoporosis, it is important to understand their effects on other physiologic processes, particularly the impact on spinal fusion. Despite early animal model studies and more recent clinical studies, the impact of these medications on spinal fusion is not fully understood. Previous animal studies suggest that bisphosphonate therapy resulted in inhibition of fusion mass with impeded maturity and an unknown effect on biomechanical strength. Prior animal studies demonstrate an improved fusion rate and fusion mass microstructure with the use of intermittent PTH. The purpose of this study was to determine if bisphosphonates and intermittent PTH treatment have impact on human spinal fusion. A systematic review of the literature published between 1980 and 2015 was conducted using major electronic databases. Studies reporting outcomes of human subjects undergoing 1, 2, or 3-level spinal fusion while receiving bisphosphonates and/or intermittent PTH treatment were included. The results of relevant human studies were analyzed for consensus on the effects of these medications in regards to spinal fusion. There were nine human studies evaluating the impact of these medications on spinal fusion. Improved fusion rates were noted in patients receiving bisphosphonates compared to control groups, and greater fusion rates in patients receiving PTH compared to control groups. Prior studies involving animal models found an improved fusion rate and fusion mass microstructure with the use of intermittent PTH. No significant complications were demonstrated in any study included in the analysis. Bisphosphonate use in humans may not be a deterrent to spinal fusion. Intermittent parathyroid use has shown early promise to increase fusion mass in both animal and human studies but further studies are needed to support routine use.

PTH [1\u201334] induced differentiation and mineralization of mandibular condylar cartilage

Intermittent Parathyroid Hormone (I-PTH) is the only FDA approved anabolic drug therapy available for the treatment of osteoporosis in males and postmenopausal females. The effects of I-PTH on the chondrogenic lineage of the mandibular condylar cartilage (MCC) are not well understood. To investigate the role of I-PTH on the MCC and subchondral bone, we carried out our studies using 4 to 5 week old triple transgenic mice (Col1a1XCol2a1XCol10a1). The experimental group was injected with PTH (80 μg/kg) daily for 2 weeks, while control group was injected with saline. Our histology showed that the I-PTH treatment led to an increased number of cells expressing Col1a1, Col2a1 and Col10a1. Additionally, there was an increase in cellular proliferation, increased proteoglycan distribution, increased cartilage thickness, increased TRAP activity, and mineralization. Immunohistochemical staining showed increased expression of pSMAD158 and VEGF in the MCC and subchondral bone. Furthermore our microCT data showed that I-PTH treatment led to an increased bone volume fraction, tissue density and trabecular thickness, with a decrease in trabecular spacing. Morphometric measurements showed increased mandibular length and condyle head length following I-PTH treatment. In conclusion, our study suggests that I-PTH plays a critical role in cellular proliferation, proteoglycan distribution, and mineralization of the MCC.