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1.  Non-invasive monitoring of BMP-2 retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes 
Non-invasive imaging can provide essential information for the optimization of new drug delivery-based bone regeneration strategies to repair damaged or impaired bone tissue. This study investigates the applicability of nuclear medicine and radiological techniques to monitor growth factor retention profiles and subsequent effects on bone formation. Recombinant human bone morphogenetic protein-2 (BMP-2, 6.5 μg/scaffold) was incorporated into a sustained release vehicle consisting of poly(lactic-co-glycolic acid) microspheres embedded in a poly(propylene fumarate) scaffold surrounded by a gelatin hydrogel and implanted subcutaneously and in 5-mm segmental femoral defects in 9 rats for a period of 56 days. To determine the pharmacokinetic profile, BMP-2 was radiolabeled with 125I and the local retention of 125I-BMP-2 was measured by single photon emission computed tomography (SPECT), scintillation probes and ex vivo scintillation analysis. Bone formation was monitored by micro-computed tomography (μCT). The scaffolds released BMP-2 in a sustained fashion over the 56-day implantation period. A good correlation between the SPECT and scintillation probe measurements was found and there were no significant differences between the non-invasive and ex-vivo counting method after 8 weeks of follow up. SPECT analysis of the total body and thyroid counts showed a limited accumulation of 125I within the body. Ectopic bone formation was induced in the scaffolds and the femur defects healed completely. In vivo μCT imaging detected the first signs of bone formation at days 14 and 28 for the orthotopic and ectopic implants, respectively, and provided a detailed profile of the bone formation rate. Overall, this study clearly demonstrates the benefit of applying non-invasive techniques in drug delivery-based bone regeneration strategies by providing detailed and reliable profiles of the growth factor retention and bone formation at different implantation sites in a limited number of animals.
doi:10.1016/j.jconrel.2008.11.023
PMCID: PMC3974410  PMID: 19105972
Drug delivery; Controlled release; Bone morphogenetic protein-2; Single photon emission computed; tomography; Scintillation probes; Micro-computed tomography
2.  Effect of Autologous Bone Marrow Stromal Cell Seeding and Bone Morphogenetic Protein-2 Delivery on Ectopic Bone Formation in a Microsphere/Poly(Propylene Fumarate) Composite 
Tissue Engineering. Part A  2008;15(3):587-594.
A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8 μg BMP-2 per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2–loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2–impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8 μg/mg BMP-2–loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2–impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.
doi:10.1089/ten.tea.2007.0376
PMCID: PMC2810278  PMID: 18925831
3.  Retention of in vitro and in vivo BMP-2 bioactivity in sustained delivery vehicles for bone tissue engineering 
Biomaterials  2008;29(22):3245-3252.
In this study, we investigated the in vitro and in vivo biologic activity of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated in 1) a gelatin hydrogel, 2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, 3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and 4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with 125I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivity of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (implant 3, p < 0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than in the gelatin and microsphere/gelatin hydrogels (p < 0.001), however there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.
doi:10.1016/j.biomaterials.2008.04.031
PMCID: PMC2577841  PMID: 18472153
4.  A Differential Effect of Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor Release Timing on Osteogenesis at Ectopic and Orthotopic Sites in a Large-Animal Model 
Tissue Engineering. Part A  2012;18(19-20):2052-2062.
In bone tissue engineering, growth factors are widely used. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are the most well-known regulators of osteogenesis and angiogenesis. We investigated whether the timing of dual release of VEGF and BMP-2 influences the amount of bone formation in a large-animal model. Poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) were loaded with BMP-2 or VEGF to create sustained-release profiles, and rapidly degrading gelatin was loaded with either growth factor for fast-release profiles. To study in vivo osteogenicity, the two delivery vehicles were combined with biphasic calcium phosphate (BCP) scaffolds and implanted in 10 Beagle dogs for 9 weeks, at both ectopic (paraspinal muscles) and orthotopic sites (critical-size ulnar defect). The 9 ectopic groups contained combined or single BMP/VEGF dosage, in sustained- or fast-release profiles. In the ulnae of 8 dogs, fast VEGF and sustained BMP-2 were applied to one leg, and the other received the opposite release profiles. The two remaining dogs received bilateral control scaffolds. Bone growth dynamics was analyzed by fluorochrome injection at weeks 3, 5, and 7. Postoperative and posteuthanization X-rays of the ulnar implants were taken. After 9 weeks of implantation, bone quantity and bone growth dynamics were studied by histology, histomorphometry, and fluorescence microscopy. The release of the growth factors resulted in both enhanced orthotopic and ectopic bone formation. Bone formation started before 3 weeks and continued beyond 7 weeks. The ectopic BMP-2 fast groups showed significantly more bone compared to sustained release, independent of the VEGF profile. The ulna implants revealed no significant differences in the amount of bone formed. This study shows that timing of BMP-2 release largely determines speed and amount of ectopic bone formation independent of VEGF release. Furthermore, at the orthotopic site, no significant effect on bone formation was found from a timed release of growth factors, implicating that timed-release effects are location dependent.
doi:10.1089/ten.tea.2011.0560
PMCID: PMC3463278  PMID: 22563713
5.  PTH Induces Differentiation of Mesenchymal Stem Cells by Enhancing BMP Signaling 
Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with PTH1R and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1+CD45−CD11b− MSCs showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone lead to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMPRII significantly in C2C12 cells and PTH treatment significantly enhanced cell surface binding of 125I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted GFP-labeled Sca-1+CD45−CD11b− MSCs into bone marrow cavity of Rag2−/− immunodeficient mice showed PTH-enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH-enhancement of MSCs differentiation to the osteoblast lineage occurs through a PTH and LRP6 dependent pathway by endocytosis of LRP6/PTH1R complex, allowing enhancement of BMP signaling.
doi:10.1002/jbmr.1663
PMCID: PMC3423493  PMID: 22589223
PARATHYROID HORMONE; BONE MORPHOGENETIC PROTEINS; DIFFERENTIATION; MESENCHYMAL STEM CELLS; LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 6
6.  Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb;128(4):following 713] 
The Journal of Cell Biology  1994;127(6):1755-1766.
The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP- 2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)- positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.
PMCID: PMC2120318  PMID: 7798324
7.  Effect of Intermittent PTH(1–34) on Human Periodontal Ligament Cells Transplanted into Immunocompromised Mice 
Tissue Engineering. Part A  2012;18(17-18):1849-1856.
Residual periodontal ligament (PDL) cells in the damaged tissue are considered a prerequisite for a successful regeneration of the periodontal architecture with all its components, including gingiva, PDL, cementum, and bone. Among other approaches, current concepts in tissue engineering aim at a hormonal support of the regenerative capacity of PDL cells as well as at a supplementation of lost cells for regeneration. Here, we investigated how far an anabolic, intermittent parathyroid hormone (iPTH) administration would enhance the osteoblastic differentiation of PDL cells and the cellular ability to mineralize the extracellular matrix in an in vivo transplantation model. PDL cells were predifferentiated in a standard osteogenic medium for 3 weeks before subcutaneous transplantation into CD-1 nude mice using gelatin sponges as carrier. Daily injections of 40 μg/kg body weight PTH(1–34) or an equivalent dose of vehicle for 4 weeks were followed by explantation of the specimens and an immunohistochemical analysis of the osteoblastic marker proteins alkaline phosphatase (ALP), osteopontin, and osteocalcin. Signs of biomineralization were visualized by means of alizarin red staining. For verification of the systemic effect of iPTH application, blood serum levels of osteocalcin were determined. The osteogenic medium stimulated the expression of ALP and PTH1-receptor mRNA in the cultures. After transplantation, iPTH resulted in an increased cytoplasmic and extracellular immunoreactivity for all markers investigated. In contrast to only sporadic areas of mineralization under control conditions, several foci of mineralization were observed in the iPTH group. Blood serum levels of osteocalcin were elevated significantly with iPTH. These data indicate that the osteoblastic differentiation of human PDL cells and their ability for biomineralization can be positively influenced by iPTH in vivo. These findings hold out a promising prospect for the support of periodontal regeneration.
doi:10.1089/ten.tea.2011.0626
PMCID: PMC3432899  PMID: 22497226
8.  A Single Injection of the Anabolic Bone Agent, Parathyroid Hormone–Collagen Binding Domain (PTH–CBD), Results in Sustained Increases in Bone Mineral Density for up to 12 Months in Normal Female Mice 
Calcified tissue international  2012;91(3):196-203.
Parathyroid hormone (PTH) is the most effective osteoporosis treatment, but it is only effective if administered by daily injections. We fused PTH(1–33) to a collagen binding domain (PTH–CBD) to extend its activity, and have shown an anabolic bone effect with monthly dosing. We tested the duration of action of this compound with different routes of administration. Normal young C57BL/6J mice received a single intraperitoneal injection of PTH–CBD (320 μg/kg). PTH–CBD treated mice showed a 22.2 % increase in bone mineral density (BMD) at 6 months and 12.8 % increase at 12 months. When administered by subcutaneous injection, PTH–CBD again caused increases in BMD, 15.2 % at 6 months and 14.3 % at 12 months. Radiolabeled PTH–CBD was concentrated in bone and skin after either route of administration. We further investigated skin effects of PTH–CBD, and histological analysis revealed an apparent increase in anagen VI hair follicles. A single dose of PTH–CBD caused sustained increases in BMD by >10 % for 1 year in normal mice, regardless of the route of administration, thus showing promise as a potential osteoporosis therapy.
doi:10.1007/s00223-012-9626-1
PMCID: PMC3693552  PMID: 22806683
Animal models; Bone density technology; DXA; Osteoporosis therapy
9.  Biodegradable Composite Scaffolds Incorporating an Intramedullary Rod and Delivering Bone Morphogenetic Protein-2 for Stabilization and Bone Regeneration in Segmental Long Bone Defects 
Acta biomaterialia  2011;7(10):3627-3637.
In this study, a two part bone tissue engineering scaffold was investigated consisting of a solid poly(propylene fumarate) (PPF) intramedullary rod for mechanical support surrounded by a porous PPF sleeve for osseointegration and delivery of poly(DL-lactic-co-glycolic acid) (PLGA) microspheres with adsorbed recombinant human bone morphogenetic protein-2 (rhBMP-2). Scaffolds were implanted into critical size rat segmental femoral defects with internal fixation for 12 weeks. Bone formation was assessed throughout the study via radiography, and following euthanasia via micro-CT and histology. Mechanical stabilization was evaluated further via torsional testing. Experimental implant groups included the PPF rod alone and the rod with a porous PPF sleeve containing PLGA microspheres with 0, 2, or 8 μg of rhBMP-2 adsorbed onto their surface. Results showed that presence of the scaffold increased mechanical stabilization of the defect, as evidenced by the increased torsional stiffness of the femurs by the presence of a rod compared to the empty defect. Although the presence of a rod decreased bone formation, the presence of a sleeve combined with a low or high dose of rhBMP-2 increased the torsional stiffness to 2.06 ± 0.63 N·mm and 1.68 ± 0.56 N·mm, respectively, from 0.56 ± 0.24 N·mm for the rod alone. The results indicate that, while scaffolds may provide structural support to regenerating tissues and increase their mechanical properties, the presence of scaffolds within defects may hinder overall bone formation if they interfere with cellular processes.
doi:10.1016/j.actbio.2011.06.043
PMCID: PMC3163689  PMID: 21757034
Bone Tissue Engineering; Bone Morphogenetic Protein; Bone Regeneration; Intramedullary Rod; Composite Scaffold
10.  Systemic intermittent parathyroid hormone treatment improves osseointegration of press-fit inserted implants in cancellous bone 
Acta Orthopaedica  2012;83(4):411-419.
Background and purpose
Intermittent administration of parathyroid hormone (PTH) has an anabolic effect on bone, as confirmed in human osteoporosis studies, distraction osteogenesis, and fracture healing. PTH in rat models leads to improved fixation of implants in low-density bone or screw insertion transcortically.
Material and methods
We examined the effect of human PTH (1–34) on the cancellous osseointegration of unloaded implants inserted press-fit in intact bone of higher animal species. 20 dogs were randomized to treatment with human PTH (1–34), 5 μg/kg/day subcutaneously, or placebo for 4 weeks starting on the day after insertion of a cylindrical porous coated plasma-sprayed titanium alloy implant in the proximal metaphyseal cancellous bone of tibia. Osseointegration was evaluated by histomorphometry and fixation by push-out test to failure.
Results
Surface fraction of woven bone at the implant interface was statistically significantly higher in the PTH group by 1.4 fold with (median (interquartile range) 15% (13–18)) in the PTH group and 11% (7–13) in control. The fraction of lamellar bone was unaltered. No significant difference in bone or fibrous tissue was observed in the circumferential regions of 0–500, 500–1,000, and 1,000–2,000 μm around the implant. Mechanically, the implants treated with PTH showed no significant differences in total energy absorption, maximum shear stiffness, or maximum shear strength.
Interpretation
Intermittent treatment with PTH (1–34) improved xhistological osseointegration of a prosthesis inserted press-fit at surgery in cancellous bone, with no additional improvement of the initial mechanical fixation at this time point.
doi:10.3109/17453674.2012.702388
PMCID: PMC3427634  PMID: 22880714
11.  Segmental Bone Regeneration Using a Load Bearing Biodegradable Carrier of Bone Morphogenetic Protein-2 
Biomaterials  2006;28(3):459-467.
Segmental defect regeneration has been a clinical challenge. Current tissue engineering approach using porous biodegradable scaffolds to delivery osteogenic cells and growth factors demonstrated success in facilitating bone regeneration in these cases. However, due to the lack of mechanical property, the porous scaffolds were evaluated in non-load bearing area or were stabilized with stress-shielding devices (bone plate or external fixation). In this paper, we tested a scaffold that does not require a bone plate because it has sufficient biomechanical strength. The tube-shaped scaffolds were manufactured from poly(propylene) fumarate/tricalcium phosphate (PPF/TCP) composites. Dicalcium phosphate dehydrate (DCPD) were used as bone morphogenetic protein -2 (BMP-2) carrier. Twenty two scaffolds were implanted in 5 mm segmental defects in rat femurs stabilized with k-wire for 6 and 15 weeks with and without 10 μg of rhBMP-2. Bridging of the segmental defect was evaluated first radiographically and was confirmed by histology and micro- computer tomography (μ-CT) imaging. The scaffolds in the BMP group maintained the bone length throughout the duration of the study and allow for bridging. The scaffolds in the control group failed to induce bridging and collapsed at 15 weeks. Peripheral computed tomography (pQCT) showed that BMP-2 does not increase the bone mineral density in the callus. Finally, the scaffold in BMP group was found to restore the mechanical property of the rat femur after 15 weeks. Our results demonstrated that the load-bearing BMP-2 scaffold can maintain bone length and allow successfully regeneration in segmental defects.
doi:10.1016/j.biomaterials.2006.09.004
PMCID: PMC1986795  PMID: 16996588
12.  Anabolic Therapies 
Current Osteoporosis Reports  2010;8(1):23-27.
The striking clinical benefits of intermittent parathyroid hormone in osteoporosis have begun a new era of skeletal anabolic agents. Recombinant human parathyroid hormone (rhPTH) (1–34) is the first US Food and Drug Administration–approved anabolic therapy. Its use has been limited by the need for subcutaneous injection. Newer delivery systems include transdermal and oral preparations. Newer anabolic therapies include monoclonal antibody to sclerostin, a potent inhibitor of osteoblastogenesis; and use of bone morphogenetic proteins and parathyroid hormone–related protein PTHrP, a calcium-regulating hormone similar to PTH.
doi:10.1007/s11914-010-0005-4
PMCID: PMC2841260  PMID: 20425087
Wnt; Sclerostin; BMP; PTH; BMD; Osteoporosis
13.  Alendronate Inhibits PTH (1–34)-induced Bone Morphogenetic Protein Expression in MC3T3-E1 Preosteoblastic Cells 
HSS Journal  2007;3(2):169-172.
The bisphosphonate class of antiresorptive drugs and active forms of parathyroid hormone (PTH (1–34)) have been used clinically to enhance bone mass and density in patients with osteoporosis. Abundant evidence suggests that the mechanism by which PTH (1–34) increases bone density is stimulation of osteoblast differentiation. Although bisphosphonates have been classically thought to increase bone density by inhibiting osteoclasts, there is increasing evidence to suggest that bisphosphonates have direct stimulatory effects on osteoblast differentiation. Interestingly, in patients with osteoporosis, combination therapy with bisphosphonates and PTH (1–34) is not synergistic in increasing bone density; bisphosphonates appear to blunt the effect of PTH (1–34). To begin to understand the mechanism governing the effects of these agents on osteoblasts and a possible explanation for their apparent antagonism, we examined the expression of several bone morphogenetic proteins (BMPs) in MC3T3-E1 preosteoblastic cells either untreated, or treated with alendronate, parathyroid hormone, or a combination of the two agents. We find by reverse transcriptase-polymerase chain reaction (RT-PCR) that while alendronate fails to induce the expression of any of the BMPs tested, several BMPs are induced by PTH (1–34). The induction of the PTH (1–34)-inducible BMPs is blocked with simultaneous alendronate treatment. These data suggest that alendronate interferes with PTH (1–34)-induced BMP gene transcription and provides a possible basis for the antagonism observed between the two agents in increasing bone density.
doi:10.1007/s11420-007-9042-7
PMCID: PMC2504255  PMID: 18751789
osteoblasts; bisphosphonates; parathyroid hormone; BMPs
14.  Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats 
Journal of Clinical Investigation  2000;105(11):1595-1604.
Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a “calcilytic”) of the parathyroid cell Ca2+ receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17β-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca2+ receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.
PMCID: PMC300853  PMID: 10841518
15.  Atherogenic Phospholipids Attenuate Osteogenic Signaling by BMP-2 and Parathyroid Hormone in Osteoblasts* 
The Journal of biological chemistry  2007;282(29):21237-21243.
Cardiovascular disease, such as atherosclerosis, has been associated with reduced bone mineral density and fracture risk. A major etiologic factor in atherogenesis is believed to be oxidized phospholipids. We previously found that these phospholipids inhibit spontaneous osteogenic differentiation of marrow stromal cells, suggesting that they may account for the clinical link between atherosclerosis and osteoporosis. Currently, anabolic agents that promote bone formation are increasingly used as a new treatment for osteoporosis. It is not known, however, whether atherogenic phospholipids alter the effects of bone anabolic agents, such as bone morphogenetic protein (BMP)-2 and parathyroid hormone (PTH). Therefore we investigated the effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on osteogenic signaling induced by BMP-2 and PTH in MC3T3-E1 cells. Results showed that ox-PAPC attenuated BMP-2 induction of osteogenic markers alkaline phosphatase and osteocalcin. Ox-PAPC also inhibited both spontaneous and BMP-induced expression of PTH receptor. Consistently, pretreatment of cells with ox-PAPC inhibited PTH-induced cAMP production and expression of immediate early genes Nurr1 and IL-6. Results from immunofluorescence and Western blot analyses showed that inhibitory effects of ox-PAPC on BMP-2 signaling were associated with inhibition of SMAD 1/5/8 but not p38-MAPK activation. These effects appear to be due to ox-PAPC activation of the ERK pathway, as the ERK inhibitor PD98059 reversed ox-PAPC inhibitory effects on BMP-2-induced alkaline phosphatase activity, osteocalcin expression, and SMAD activation. These results suggest that atherogenic lipids inhibit osteogenic signaling induced by BMP-2 and PTH, raising the possibility that hyperlipidemia and atherogenic phospholipids may interfere with anabolic therapy.
doi:10.1074/jbc.M701341200
PMCID: PMC3001330  PMID: 17522049
16.  Anatomically Shaped Tooth and Periodontal Regeneration by Cell Homing 
Journal of Dental Research  2010;89(8):842-847.
Tooth regeneration by cell delivery encounters translational hurdles. We hypothesized that anatomically correct teeth can regenerate in scaffolds without cell transplantation. Novel, anatomically shaped human molar scaffolds and rat incisor scaffolds were fabricated by 3D bioprinting from a hybrid of poly-ε-caprolactone and hydroxyapatite with 200-µm-diameter interconnecting microchannels. In each of 22 rats, an incisor scaffold was implanted orthotopically following mandibular incisor extraction, whereas a human molar scaffold was implanted ectopically into the dorsum. Stromal-derived factor-1 (SDF1) and bone morphogenetic protein-7 (BMP7) were delivered in scaffold microchannels. After 9 weeks, a putative periodontal ligament and new bone regenerated at the interface of rat incisor scaffold with native alveolar bone. SDF1 and BMP7 delivery not only recruited significantly more endogenous cells, but also elaborated greater angiogenesis than growth-factor-free control scaffolds. Regeneration of tooth-like structures and periodontal integration by cell homing provide an alternative to cell delivery, and may accelerate clinical applications.
doi:10.1177/0022034510370803
PMCID: PMC3318060  PMID: 20448245
tooth regeneration; cell homing; stem cells; bioprinting; periodontal
17.  Daily Parathyroid Hormone 1-34 Replacement Therapy for Hypoparathyroidism Induces Marked Changes in Bone Turnover and Structure 
Journal of Bone and Mineral Research  2012;27(8):1811-1820.
Parathyroid hormone (PTH) has variable actions on bone. Chronically increased PTH is catabolic leading to osteoporosis, yet intermittent administration is anabolic and increases bone mass. PTH deficiency is associated with decreased bone remodeling and increased bone mass. However, the effects of PTH replacement therapy on bone in hypoparathyroidism are not well known. We discontinued calcitriol therapy and treated five hypoparathyroid subjects (2 adults and 3 adolescents) with synthetic human PTH 1-34 (hPTH 1-34), injected 2-3 times daily for 18 months, with doses individualized to maintain serum calcium at 1.9-2.25 mmol/L. Biochemical markers and bone density (BMD) were assessed every 6 months; iliac-crest biopsies were performed before and after 1 year of treatment. hPTH 1-34 therapy significantly increased bone markers to supranormal levels. Histomorphometry revealed that treatment dramatically increased cancellous bone volume and trabecular number and decreased trabecular separation. Changes in trabecular width were variable, suggesting that the increase in trabecular number was due to the observed intratrabecular tunneling. Cortical width remained unchanged, however, hPTH 1-34 treatment increased cortical porosity. Cancellous bone remodeling was also stimulated, inducing significant changes in osteoid, mineralizing surface, and bone formation rate. Similar changes were seen in endocortical and intracortical remodeling. BMD Z-scores were unchanged at the spine and femoral neck. Total hip Z-scores increased, however, total body BMD Z-scores decreased during the first 6 months of treatment and then stabilized, remaining significantly decreased compared to baseline. Radial Z-scores also decreased with treatment; this was most pronounced in the growing adolescent. Daily hPTH 1-34 therapy for hypoparathyroidism stimulated bone turnover, increased bone volume, and altered bone structure in the iliac crest. These findings suggest that treatment with hPTH 1-34 in hypoparathyroid adults and adolescents has varying effects in the different skeletal compartments, leading to an increase in trabecular bone and an apparent trabecularization of cortical bone.
doi:10.1002/jbmr.1627
PMCID: PMC3399961  PMID: 22492501
18.  Pulsatile Release of Parathyroid Hormone from an Implantable Delivery System 
Biomaterials  2007;28(28):4124-4131.
Intermittent (pulsatile) administration of parathyroid hormone (PTH) is known to improve bone micro-architecture, mineral density and strength. Therefore, daily injection of PTH has been clinically used for the treatment of osteoporosis. However, this regimen of administration is not convenient and is not a favorable choice of patients. In this study, an implantable delivery system has been developed to achieve pulsatile release of PTH. A well-defined cylindrical device was first fabricated with a biodegradable polymer, poly(lactic acid) (PLLA), using a reverse solid free form fabrication technique. Three-component polyanhydrides composed of sebacic acid, 1,3-bis(p-carboxyphenoxy) propane and poly(ethylene glycol) were synthesized and used as isolation layers. The polyanhydride isolation layers and PTH-loaded alginate layers were then stacked alternately within the delivery device. The gap between the stacked PTH-releasing core and the device frame was filled with PLLA to seal. Multi-pulse PTH release was achieved using the implantable device. The lag time between two adjacent pulses were modulated by the composition and the film thickness of the polyanhydride. The released PTH was demonstrated to be biologically active using an in vitro assay. Timed sequential release of multiple drugs has also been demonstrated. The implantable device holds promise for both systemic and local therapies.
doi:10.1016/j.biomaterials.2007.05.034
PMCID: PMC2048537  PMID: 17576005
19.  Parathyroid Hormone and Physical Exercise: a Brief Review 
Parathyroid hormone (PTH) is the major hormone regulating calcium metabolism and is involved in both catabolic and anabolic actions on bone. Intermittent PTH exposure can stimulate bone formation and bone mass when PTH has been injected. In contrast, continuous infusion of PTH stimulates bone resorption. PTH concentration may be affected by physical exercise and our review was designed to investigate this relationship. The variation in PTH concentration appears to be influenced by both exercise duration and intensity. There probably exists a stimulation threshold of exercise to alter PTH. PTH regulation is also influenced by the initial bone mineral content, age, gender, training state, and other hormonal and metabolic factors (catecholamines, lactic acid and calcium concentrations).
Key PointsPhysical exercise can improve PTH secretion.Parathyroid hormone has both anabolic and catabolic effects on bone: intermittent treatment of PTH is anabolic whereas continuous treatment is catabolic.
PMCID: PMC3842136  PMID: 24353453
Parathyroid hormone; physical exercise; calcium; catabolic/anabolic effects
20.  Bone Regeneration Mediated by BMP4-Expressing Muscle-Derived Stem Cells Is Affected by Delivery System 
Tissue Engineering. Part A  2008;15(2):285-293.
This study investigated the delivery of bone morphogenetic protein (BMP)4-secreting muscle-derived stem cells (MDSC-B4) capable of inducing bone formation in mice using collagen gel (CG), fibrin sealant (FS), and gelatin sponge carriers. After implanting these various cell-loaded scaffolds intramuscularly or into critical-size skull defects, we measured the extent of heterotopic ossification and calvarial defect healing over a 6-week period via radiographic, radiomorphometric, histological, and micro-computed tomography analyses. As expected, in the absence of MDSC-B4, there was no ectopic ossification and only minimal calvarial regeneration using each type of scaffold. Although CG and gelatin sponges loaded with BMP4-secreting cells produced the most ectopic bone, FS constructs produced bone with comparably less mineralization. In the mouse calvaria, we observed MDSC-B4-loaded scaffolds able to promote bone defect healing to a variable degree, but there were differences between these implants in the volume, shape, and morphology of regenerated bone. MDSC-B4 delivery in a gelatin sponge produced hypertrophic bone, whereas delivery in a CG and FS healed the defect with bone that closely resembled the quantity and configuration of native calvarium. In summary, hydrogels are suitable carriers for osteocompetent MDSCs in promoting bone regeneration, especially at craniofacial injury sites.
doi:10.1089/ten.tea.2008.0130
PMCID: PMC2810214  PMID: 19061430
21.  Hormones and growth factors in the pathogenesis of spinal ligament ossification 
European Spine Journal  2007;16(8):1075-1084.
Ossification of the spinal ligaments (OSL) is a pathologic condition that causes ectopic bone formation and subsequently results in various degrees of neurological deficit, but the etiology of OSL remains almost unknown. Some systemic hormones, such as 1,25-dihydroxyvitamin D, parathyroid hormone (PTH), insulin and leptin, and local growth factors, such as transforming growth factor-β (TGF-β), and bone morphogenetic protein (BMP), have been studied and are thought to be involved in the initiation and development of OSL. This review article summarizes these studies, delineates the possible mechanisms, and puts forward doubts and new questions. The related findings from studies of genes and target cells in the ligament of OSL are also discussed. Although these findings may be helpful in understanding the pathogenesis of OSL, much more research needs to be conducted in order to investigate the nature of OSL.
doi:10.1007/s00586-007-0356-4
PMCID: PMC2200765  PMID: 17426989
Spine; Ligaments; Ossification; Hormones; Growth factors
22.  Parathyroid hormone PTH(1–34) increases the volume, mineral content, and mechanical properties of regenerated mineralizing tissue after distraction osteogenesis in rabbits 
Acta Orthopaedica  2009;80(6):716-723.
Background and purpose Parathyroid hormone (PTH) has attracted considerable interest as a bone anabolic agent. Recently, it has been suggested that PTH can also enhance bone repair after fracture and distraction osteogenesis. We analyzed bone density and strength of the newly regenerated mineralized tissue after intermittent treatment with PTH in rabbits, which undergo Haversian bone remodeling similar to that in humans.
Methods 72 New Zealand White rabbits underwent tibial mid-diaphyseal osteotomy and the callus was distracted 1 mm/day for 10 days. The rabbits were divided into 3 groups, which received injections of PTH 25 µg/kg/day for 30 days, saline for 10 days and PTH 25 µg/kg/day for 20 days, or saline for 30 days. At the end of the study, the rabbits were killed and the bone density was evaluated with DEXA. The mechanical bone strength was determined by use of a 3-point bending test.
Results In the 2 PTH-treated groups the regenerate callus ultimate load was 33% and 30% higher, absorbed energy was 100% and 65% higher, BMC was 61% and 60% higher, and callus tissue volume was 179% and 197% higher than for the control group.
Interpretation We found that treatment with PTH during distraction osteogenesis resulted in substantially higher mineralized tissue volume, mineral content, and bending strength. This suggests that treatment with PTH may benefit new bone formation during distraction osteogenesis and could form a basis for clinical application of this therapy in humans.
doi:10.3109/17453670903350032
PMCID: PMC2823317  PMID: 19995322
23.  Dual Delivery of an Angiogenic and an Osteogenic Growth Factor for Bone Regeneration in a Critical Size Defect Model 
Bone  2008;43(5):931-940.
This study investigated the effects of dual delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) for bone regeneration in a rat cranial critical size defect. Four groups of scaffolds were generated with VEGF (12 μg), BMP-2 (2 μg), both VEGF (12 μg) and BMP-2 (2 μg), or no growth factor released from gelatin microparticles incorporated within the scaffold pores. These scaffolds were implanted within an 8 mm rat cranial critical size defect (n = 8–9 for each group). At 4 and 12 weeks, implants were retrieved and evaluated by microcomputed tomography (microCT) and histological scoring analysis. Additionally, 4 week animals were perfused with a radiopaque material to visualize and quantify blood vessel formation. Histological analysis revealed that for all groups at 4 weeks, a majority of the porous scaffold volume was filled with vascularized fibrous tissue; however, bone formation appeared most abundant in the dual release group at this time. At 12 weeks, both dual release and BMP-2 groups showed large amounts of bone formation within the scaffold pores and along the outer surfaces of the scaffold; osteoid secretion and mineralization were apparent, and new bone was often in close or direct contact with the scaffold interface. MicroCT results showed no significant difference among groups for blood vessel formation at 4 weeks (<4% blood vessel volume); however, the dual release group showed significantly higher bone formation (16.1±9.2% bone volume) than other groups at this time. At 12 weeks, dual release and BMP-2 groups exhibited significantly higher bone formation (39.7 ± 14.1% and 37.4 ± 18.8% bone volume, respectively) than either the VEGF group or blank scaffolds (6.3 ± 4.8% and 7.8 ± 7.1% bone volume, respectively). This work indicates a synergistic effect of the dual delivery of VEGF and BMP-2 on bone formation at 4 weeks and suggests an interplay between these growth factors for early bone regeneration. For the doses investigated, the results show that the addition of VEGF does not affect the amount of bone formation achieved by BMP-2 at 12 weeks; however, they also indicate that delivery of both growth factors may enhance bone bridging and union of the critical size defect compared to delivery of BMP-2 alone.
doi:10.1016/j.bone.2008.06.019
PMCID: PMC3014108  PMID: 18675385
Controlled release; Gelatin microparticles; Vascular endothelial growth factor; Bone morphogenetic protein-2; Bone tissue engineering
24.  Modulation of Stromal Cell-Derived Factor-1/CXC Chemokine Receptor 4 Axis Enhances rhBMP-2-Induced Ectopic Bone Formation 
Tissue Engineering. Part A  2012;18(7-8):860-869.
Enhancement of in vivo mobilization and homing of endogenous mesenchymal stem cells (MSCs) to an injury site is an innovative strategy for improvement of bone tissue engineering and repair. The present study was designed to determine whether mobilization by AMD3100 and/or local homing by delivery of stromal cell-derived factor-1 (SDF-1) enhances recombinant human bone morphogenetic protein-2 (rhBMP-2) induced ectopic bone formation in an established rat model. Rats received an injection of either saline or AMD3100 treatment 1 h before harvesting of bone marrow for in vitro colony-forming unit-fibroblasts (CFU-F) culture or the in vivo subcutaneous implantation of absorbable collagen sponges (ACSs) loaded with saline, recombinant human bone morphogenetic protein-2 (rhBMP-2), SDF-1, or the combination of SDF-1 and rhBMP-2. AMD3100 treatment resulted in a significant decrease in CFU-F number, compared with saline, which confirmed that a single systemic AMD3100 treatment rapidly mobilized MSCs from the bone marrow. At 28 and 56 days, bone formation in the explanted ACS was assessed by microcomputed tomography (μCT) and histology. At 28 days, AMD3100 and/or SDF-1 had no statistically significant effect on bone volume (BV) or bone mineral content (BMC), but histology revealed more active bone formation with treatment of AMD3100, loading of SDF-1, or the combination of both AMD3100 and SDF-1, compared with saline-treated rhBMP-2 loaded ACS. At 56 days, the addition of AMD3100 treatment, loading of SDF-1, or the combination of both resulted in a statistically significant stimulatory effect on BV and BMC, compared with the saline-treated rhBMP-2 loaded ACS. Histology of the 56-day ACS were consistent with the μCT analysis, exhibiting more mature and mineralized bone formation with AMD3100 treatment, SDF-1 loading, or the combination of both, compared with the saline-treated rhBMP-2 loaded ACS. The present study is the first that provides evidence of the efficacy of AMD3100 and SDF-1 treatment to stimulate trafficking of MSCs to an ectopic implant site, in order to ultimately enhance rhBMP-2 induced long-term bone formation.
doi:10.1089/ten.tea.2011.0187
PMCID: PMC3313617  PMID: 22035136
25.  Transcriptional Regulation of BMP2 Expression by the PTH-CREB Signaling Pathway in Osteoblasts 
PLoS ONE  2011;6(6):e20780.
Intermittent application of parathyroid hormone (PTH) has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs) in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA), we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.
doi:10.1371/journal.pone.0020780
PMCID: PMC3111437  PMID: 21695256

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