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Hypothesis: Dental bleaching with H2O2 is a common daily practice in dentistry to correct discoloration of anterior teeth. The aim of this study has been to determine whether this treatment of human teeth affects growth, differentiation and activity of osteoclast-like cells, as well as the putative modulatory action of osteostatin and fibroblast growth factor 2 (FGF-2)., Experiments: Previously to the in vitro assays, structural, physical-chemical and morphological features of teeth after bleaching were studied. Osteoclast-like cells were cultured on human dentin disks, pre-treated or not with 38% H2O2 bleaching gel, in the presence or absence of osteostatin (100 nM) or FGF-2 (1 ng/ml). Cell proliferation and viability, intracellular content of reactive oxygen species (ROS), pro-inflammatory cytokine (IL-6 and TNFα) secretion and resorption activity were evaluated., Findings: Bleaching treatment failed to affect either the structural or the chemical features of both enamel and dentin, except for slight morphological changes, increased porosity in the most superficial parts (enamel), and a moderate increase in the wettability degree. In this scenario, bleaching produced an increased osteoclast-like cell proliferation but decreased cell viability and cytokine secretion, while it augmented resorption activity on dentin. The presence of either osteostatin or FGF-2 reduced the osteoclast-like cell proliferation induced by bleaching. FGF-2 enhanced ROS content, whereas osteostatin decreased ROS but increased TNFα secretion. The bleaching effect on resorption activity was increased by osteostatin, but this effect was less evident with FGF-2., Conclusions: These findings further confirm the deleterious effects of tooth bleaching by affecting osteoclast growth and function as well as different modulatory actions of osteostatin and FGF-2., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A promising bone graft substitute is porous titanium. Porous titanium, produced by selective laser melting (SLM), can be made as a completely open porous and load-bearing scaffold that facilitates bone regeneration through osteoconduction. In this study, the bone regenerative capacity of porous titanium is improved with a coating of osteostatin, an osteoinductive peptide that consists of the 107-111 domain of the parathyroid hormone (PTH)-related protein (PTHrP), and the effects of this osteostatin coating on bone regeneration were evaluated in vitro and in vivo. SLM-produced porous titanium received an alkali-acid-heat treatment and was coated with osteostatin through soaking in a 100 nM solution for 24 h or left uncoated. Osteostatin-coated scaffolds contained ∼0.1 μg peptide/g titanium, and in vitro 81% was released within 24 h. Human periosteum-derived osteoprogenitor cells cultured on osteostatin-coated scaffolds did not induce significant changes in osteogenic (alkaline phosphatase [ALP], collagen type 1 [Col1], osteocalcin [OCN], runt-related transcription factor 2 [Runx2]), or angiogenic (vascular endothelial growth factor [VEGF]) gene expression; however, it resulted in an upregulation of osteoprotegerin (OPG) gene expression after 24 h and a lower receptor activator of nuclear factor kappa-B ligand (RankL):OPG mRNA ratio. In vivo, osteostatin-coated, porous titanium implants increased bone regeneration in critical-sized cortical bone defects (p=0.005). Bone regeneration proceeded until 12 weeks, and femurs grafted with osteostatin-coated implants and uncoated implants recovered, respectively, 66% and 53% of the original femur torque strength (97±31 and 77±53 N·mm, not significant). In conclusion, the osteostatin coating improved bone regeneration of porous titanium. This effect was initiated after a short burst release and might be related to the observed in vitro upregulation of OPG gene expression by osteostatin in osteoprogenitor cells. Long-term beneficial effects of osteostatin-coated, porous titanium implants on bone regeneration or mechanical strength were not established here and may require optimization of the pace and dose of osteostatin release.

Biopolymer-coated nanocrystalline hydroxyapatite (HA) made as macroporous foams which are degradable and flexible are promising candidates as orthopaedic implants. The C-terminal (107-111) epitope of parathyroid hormone-related protein (PTHrP) exhibits osteogenic properties. The main aim of this study was to evaluate whether PTHrP (107-111) loading into gelatin-glutaraldehyde biopolymer-coated HA (HAGlu) scaffolds would produce an optimal biomaterial for tissue engineering applications. HAGlu scaffolds with and without PTHrP (107-111) were implanted into a cavitary defect performed in both distal tibial metaphysis of adult rats. Animals were sacrificed after 4 weeks for histological, microcomputerized tomography and gene expression analysis of the callus. At this time, bone healing occurred only in the presence of PTHrP (107-111)-containing HAGlu implant, related to an increase in bone volume/tissue volume and trabecular thickness, cortical thickness and gene expression of osteocalcin and vascular cell adhesion molecule 1, but a decreased gene expression of Wnt inhibitors, SOST and dickkopf homolog 1. The autonomous osteogenic effect of the PTHrP (107-111)-loaded HAGlu scaffolds was confirmed in mouse and human osteoblastic cell cultures. Our findings demonstrate the advantage of loading PTHrP (107-111) into degradable HAGlu scaffolds for achieving an optimal biomaterial that is promising for low load bearing clinical applications., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Insulin-like growth factor-I (IGF-I) deficiency causes growth delay, and IGF-I has been shown to partially mediate bone anabolism by parathyroid hormone (PTH). PTH-related protein (PTHrP) is abundant in bone, and has osteogenic features by poorly defined mechanisms. We here examined the capacity of PTHrP (1-36) and PTHrP (107-111) (osteostatin) to reverse the skeletal alterations associated with IGF-I deficiency. Igf1-null mice and their wild type littermates were treated with each PTHrP peptide (80 µg/Kg/every other day/2 weeks; 2 males and 4 females for each genotype) or saline vehicle (3 males and 3 females for each genotype). We found that treatment with either PTHrP peptide ameliorated trabecular structure in the femur in both genotypes. However, these peptides were ineffective in normalizing the altered cortical structure at this bone site in Igf1-null mice. An aberrant gene expression of factors associated with osteoblast differentiation and function, namely runx2, osteoprotegerin/receptor activator of NF-κB ligand ratio, Wnt3a , cyclin D1, connexin 43, catalase and Gadd45, as well as in osteocyte sclerostin, was found in the long bones of Igf1-null mice. These mice also displayed a lower amount of trabecular osteoblasts and osteoclasts in the tibial metaphysis than those in wild type mice. These alterations in Igf1-null mice were only partially corrected by each PTHrP peptide treatment. The skeletal expression of Igf2, Igf1 receptor and Irs2 was increased in Igf1-null mice, and this compensatory profile was further improved by treatment with each PTHrP peptide related to ERK1/2 and FoxM1 activation. In vitro, PTHrP (1-36) and osteostatin were effective in promoting bone marrow stromal cell mineralization in normal mice but not in IGF-I-deficient mice. Collectively, these findings indicate that PTHrP (1-36) and osteostatin can exert several osteogenic actions even in the absence of IGF-I in the mouse bone.

Parathyroid hormone-related protein (PTHrP) stimulates osteoblastic function through its N- and C-terminal domains. Since the osteogenic action of the latter domain appears to depend at least in part on its interaction with the vascular endothelial growth factor (VEGF) system, we aimed to explore the putative mechanism underlying this interaction in osteoblasts. Using native conditions for protein extraction and immunoblotting, we found that both PTHrP (107-139) and the shorter PTHrP (107-111) peptide (known as osteostatin), at 100 nM, promoted the appearance of a VEGF receptor (VEGFR) 2 protein band of apparent Mr. wt. 230 kDa, which likely represents its activation by dimer formation, in mouse osteoblastic MC3T3-E1 cells. Moreover, osteostatin (100 nM) maximally increased VEGFR2 phosphorylation at Tyr-1059 within 5-10 min in both MC3T3-E1 and rat osteoblastic osteosarcoma UMR-106 cells. This phosphorylation elicited by osteostatin appears to be VEGF-independent, but prevented by the VEGFR2 activation inhibitor SU1498 and also by the Src kinase inhibitors SU6656 and PP1. Furthermore, osteostatin induced phosphorylation of Src, extracellular signal-regulated kinase (ERK) and Akt with a similar time course to that observed for VEGFR2 activation in these osteoblastic cells. This osteostatin-dependent induction of ERK and Akt activation was abrogated by SU6656. Up-regulation of VEGF and osteoprotegerin gene expression as well as the pro-survival effect induced by osteostatin treatment were all prevented by both SU1498 and SU6656 in these osteoblastic cells. Collectively, these findings demonstrate that the osteostatin domain of C-terminal PTHrP phosphorylates VEGFR2 through Src activation, which represents a mechanism for modulating osteoblastic function., (Copyright © 2012 Wiley Periodicals, Inc.)

Runx2 is a key transcription factor in bone development regulating several processes, including osteoblast apoptosis. The antiapoptotic effects of parathyroid hormone (PTH) in osteoblasts depend on Runx2-mediated transcription of prosurvival genes. In the kidney, PTH-related protein (PTHrP) promotes tubulointerstitial cell survival by activating the PTH/PTHrP type 1 receptor. We found that Runx2 is expressed in renal tubuloepithelial MCT and HK2 cell lines in vitro and in the mouse kidney tubuloepithelium in vivo. The 1-36 amino-acid fragment of PTHrP was found to increase the expression and nuclear translocation of Runx2 in both cell lines in a dose- and time-dependent manner. PTHrP(1-36) protected renal tubuloepithelial cells from folic acid toxicity and serum deprivation, an effect inhibited by a dominant-negative Runx2 construct or a Runx2 siRNA. Furthermore, PTHrP(1-36) upregulated the antiapoptotic proteins Bcl-2 and osteopontin, and these effects were abolished by Runx2 siRNA. Runx2, osteopontin, and Bcl-2 were increased in tubuloepithelial cells from transgenic mice with PTHrP overexpression and in wild-type mice with acute or chronic renal failure. Thus, PTHrP regulates renal tubuloepithelial cell survival via Runx2 in the mammalian kidney.

Parathyroid hormone-related protein (PTHrP) is an important modulator of bone formation. Recently, we reported that PTHrP (107-111) (osteostatin) coating onto mesoporous ceramics confers osteogenic activity to these materials. Bone repair is dramatically compromised in osteopenia/osteoporosis. Thus, we examined the efficacy of unmodified and organically modified SBA15 ceramics loaded with osteostatin in promoting bone repair in an osteoporotic rabbit model. Osteoporosis was induced in New Zealand rabbits by methylprednisolone administration, and healthy rabbits were used as controls. Tested materials were implanted into a femoral cavitary defect, and animals were sacrificed at 2 weeks post-implantation. At this time, implants were encapsulated by a variable layer of fibrotic tissue with no evidence of inflammation. Similarly to observations in normal rabbits, both types of osteostatin-loaded bioceramics induced tissue regeneration associated with increased staining for PCNA, Runx2, osteopontin, and/or vascular endothelial growth factor in osteoporotic rabbits. Our present findings demonstrate that these osteostatin-bearing bioceramics increase the early repair response not only in normal bone but also in osteoporotic bone after a local injury., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

We here compared the changes induced by subcutaneous injection of PTHrP (1-36) or PTHrP (107-139) (80 µg/kg/day, 5 days/week for 4 or 8 weeks) in bone histology and bone remodeling factors, and in bone marrow cells (BMCs) ex vivo, in ovariectomized (OVX) mice. We also examined the osteogenic effects of these peptides in mouse mesenchymal C3H10T1/2 cells under oxidative stress condition in vitro, which recapitulates the effects of OVX. We confirmed that PTHrP (1-36) exerts bone anabolic actions, as assessed by bone histology and osteoblast differentiation markers in the long bones and plasma from OVX mice. PTHrP (107-139) was also efficient in stimulating several bone formation parameters, and it dramatically decreased bone resorption markers. Moreover, both PTHrP peptides modulate DKK-1 and Sost/sclerostin in osteoblast-like UMR-106 cells highly expressing these Wnt pathway inhibitors, related to their osteogenic action in this in vivo scenario. Administration of either PTHrP peptide improved osteogenic differentiation in BMCs from OVX mice ex vivo and in mouse mesenchymal C3H10T1/2 cells under oxidative stress condition in vitro. These data demonstrate that PTHrP (1-36) and PTHrP (107-139) can exert similar osteogenic effects in the appendicular skeleton of OVX mice. Our results suggest that these effects might occur in part by modulating the Wnt pathway. These findings lend credence to the notion that the osteogenic action of PTHrP (107-139) is likely a consequence of its anti-resorptive and anabolic features, and further support the usefulness of PTHrP (1-36) as a bone anabolic peptide in the setting of estrogen-depletion., (Copyright © 2011 Wiley Periodicals, Inc.)

Parathyroid hormone-related protein (107-111) (osteostatin) induces osteogenic effects in osteoblasts in vitro and in regenerating bone in mice and rabbits. In this study we used osteoblastic MC3T3-E1 cell cultures to evaluate and compare the bioactivity of this peptide either adsorbed or covalently bound (by its C-terminus) to Si-doped hydroxyapatite (Si-HA) scaffolds after organic (-NH(2)) functionalization. By these means osteostatin can be locally released or kept anchored to the scaffold surface. This was confirmed by chemical analysis and by testing the efficiency of osteostatin-loaded Si-HA scaffolds (placed in Transwell chambers) in healing a scratch wound in mouse pluripotent mesenchymal C3H10T1/2 cells. Our results show that exposure of MC3T3-E1 cell monolayers to Si-HA scaffolds with both types of osteostatin coating (deliverable or immobilized), in contrast to those without peptide, similarly stimulated cell growth and matrix mineralization. These findings demonstrate that osteostatin release from Si-HA scaffolds is not essential to promote osteoblastic growth and function in vitro, and lend credence to considering osteostatin a bone regenerating factor., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Parathyroid hormone-related protein (PTHrP) is an important regulator of bone formation and remodeling. Our recent findings demonstrate that PTHrP (107-111) (osteostatin) loaded onto silica-based ordered mesoporous SBA15 materials exhibit osteogenic features in osteoblastic cell cultures. We aimed here to elucidate whether these peptide-coated materials might be suitable for promoting bone repair following a cavitary defect in the rabbit femur. Histological examination revealed the absence of significant inflammation or bone resorption within the time of study (4 and 8 weeks) after implantation. At 8 weeks, the peptide-unloaded materials were still separated from the bone marrow by a fibrous cap, which was greatly diminished by the presence of the PTHrP peptide. By using μCT analysis, new bone formation was evident at different distances from the implants, mainly for the latter peptide-loaded biomaterials. This was confirmed by performing immunostaining for different osteoblast markers. Our findings demonstrate that these PTHrP (107-111)-loaded bioceramics significantly improve local bone induction, as compared to that observed with the unloaded material., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The parathyroid-hormone-related protein (PTHrP) is an important modulator of bone formation and bone remodeling. High and/or prolonged glucocorticoid (GC) treatments inhibit PTHrP expression in osteoblastic cells and bone formation and repair. We assessed the ability of the N- and C-terminal PTHrP fragments to restore the GC-altered bone regeneration after bone marrow ablation in mice. Animals were administered 3-methylprednisolone or vehicle and PTHrP (1-36) or PTHrP (107-139) every other day, beginning 4 days before marrow ablation in the tibia, and euthanized 12 days later. GC-treated mice showed in the ablated tibia a decrease in bone formation and in osteoblast and sclerostin-positive osteocyte numbers, reduced expression of osteoblastic factors, decreased osteogenesis of bone-marrow-derived cells, an increase in the numbers of multinucleated osteoclasts and adipocytes, and decreased cortical vascularization, as well as altered bone structure (measured by microcomputerized tomography) in the intact femur. These effects were reversed at least in part by either PTHrP peptide. The present novel findings support the use of both PTHrP peptides tested as putative bone regenerative therapies in GC-related bone diseases.

It has been suggested that hormones released after nutrient absorption, such as glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 2 (GLP-2), could be responsible for changes in bone resorption. However, information about the role of GLP-1 in this regard is scanty. Diabetes-related bone loss occurs as a consequence of poor control of glucose homeostasis, but the relationship between osteoporosis and type 2 diabetes remains unclear. Since GLP-1 is decreased in the latter condition, we evaluated some bone characteristics in streptozotocin-induced type 2 diabetic (T2D) and fructose-induced insulin-resistant (IR) rat models compared to normal (N) and the effect of GLP-1 or saline (control) treatment (3 days by osmotic pump). Blood was taken before and after treatment for plasma measurements; tibiae and femora were collected for gene expression of bone markers (RT-PCR) and structure (microCT) analysis. Compared to N, plasma glucose and insulin were, respectively, higher and lower in T2D; osteocalcin (OC) and tartrate-resistant alkaline phosphatase 5b were lower; phosphate in IR showed a tendency to be higher; PTH was not different in T2D and IR; all parameters were unchanged after GLP-1 infusion. Bone OC, osteoprotegerin (OPG) and RANKL mRNA were lower in T2D and IR; GLP-1 increased OC and OPG in all groups and RANKL in T2D. Compared to N, trabecular bone parameters showed an increased degree of anisotropy in T2D and IR, which was reduced after GLP-1. These findings show an insulin-independent anabolic effect of GLP-1 and suggest that GLP-1 could be a useful therapeutic agent for improving the deficient bone formation and bone structure associated with glucose intolerance.

Parathyroid hormone-related protein (PTHrP) (107-139), in contrast to the N-terminal fragment PTHrP (1-36), has been shown to interact with the vascular endothelial growth factor (VEGF) system to modulate human osteoblast differentiation. In this study, we evaluated whether this interaction might affect human osteoblastic cell survival. Pre-incubation with PTHrP (107-139) for 1-24 h dose-dependently (0.1-100 nM) inhibited dexamethasone- or etoposide-induced cell death in human osteoblastic MG-63 cells and human osteoblast-like cells from trabecular bone. This effect, but not that elicited by PTHrP (1-36), was abolished by the VEGF receptor (VEGFR)-2 inhibitors SU5614 and SU1498 or VEGFR-2 siRNA transfection in these cells. PTHrP (107-139), but not PTHrP (1-36), at 100 nM, rapidly (within 2 min) increased VEGFR-2 tyrosine-phosphorylation in MG-63 cells; an effect unaffected by several inhibitors of metalloproteinases, neutralizing VEGF(165) or VEGFR-2 antibodies, or the VEGF binding inhibitor CBO-PP1. The latter two antagonists also failed to affect (125)I-[Tyr(116)] PTHrP (107-115) binding to these cells. Consistent with its effect on VEGFR-2 activation, PTHrP (107-139) rapidly induced extracellular signal-regulated kinase (ERK) 1/2 and Akt activaton, and both ERK and phosphatidylinsositol-3 kinase (PI3K) inhibitors abolished its pro-survival effect in human osteoblastic cells. In addition, SU5614 and the latter two types of inhibitors abrogated Runx2 activation by this peptide in MG-63 cells. Transfection with a dominant-negative Runx2 construct abolished the pro-survival effect of PTHrP (107-139), associated with a decrease in Bcl-2/Bax protein ratio. Our findings demonstrate that PTHrP (107-139) interacts with VEGFR-2 to promote human osteoblastic cell survival by a mechanism involving Runx2 activation.

Parathyroid hormone (PTH)-related protein (PTHrP) seems to affect bone resorption by interaction with bone cytokines, among them interleukin-6 (IL-6). Recent studies suggest that nuclear factor (NF)-kappaB activation has an important role in bone resorption. We assessed whether the N-terminal fragment of PTHrP, and its C-terminal region, unrelated to PTH, can activate NF-kappaB, and its relationship with IL-6 gene induction in different rat and human osteoblastic cell preparations. Here we present molecular data demonstrating that both PTHrP (1-36) and PTHrP (107-139) activate NF-kappaB, leading to an increase in IL-6 mRNA, in these cells. Using anti-p65 and anti-p50 antibodies, we detected the presence of both proteins in the activated NF-kappaB complex. This effect induced by either the N- or C-terminal PTHrP domain in osteoblastic cells appears to occur by different intracellular mechanisms, involving protein kinase A or intracellular Ca(2+)/protein kinase C activation, respectively. However, the effect of each peptide alone did not increase further when added together. Our findings lend support to the hypothesis that the C-terminal domain of PTHrP, in a manner similar to its N-terminal fragment, might stimulate bone resorption. These studies also provide further insights into the putative role of PTHrP as a modulator of bone remodeling.