The Role Of BMPs in the Regulation of Osteoclasts Resorption and Bone Remodeling: From Experimental Models to Clinical Applications.

In response to mechanical forces and the aging process, bone in the adult skeleton is continuously remodeled by a process in which old and damaged bone is removed by bone-resorbing osteoclasts and subsequently is replaced by new bone by bone-forming cells, osteoblasts. During this essential process of bone remodeling, osteoclastic resorption is tightly coupled to osteoblastic bone formation. Bone-resorbing cells, multinuclear giant osteoclasts, derive from the monocyte/macrophage hematopoietic lineage and their differentiation is driven by distinct signaling molecules and transcription factors. Critical factors for this process are Macrophage Colony Stimulating Factor (M-CSF) and Receptor Activator Nuclear Factor-κB Ligand (RANKL). Besides their resorption activity, osteoclasts secrete coupling factors which promote recruitment of osteoblast precursors to the bone surface, regulating thus the whole process of bone remodeling. Bone morphogenetic proteins (BMPs), a family of multi-functional growth factors involved in numerous molecular and signaling pathways, have significant role in osteoblast-osteoclast communication and significantly impact bone remodeling. It is well known that BMPs help to maintain healthy bone by stimulating osteoblast mineralization, differentiation and survival. Recently, increasing evidence indicates that BMPs not only help in the anabolic part of bone remodeling process but also significantly influence bone catabolism. The deletion of the BMP receptor type 1A (BMPRIA) in osteoclasts increased osteoblastic bone formation, suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone-formation activity during bone remodeling. The dual effect of BMPs on bone mineralization and resorption highlights the essential role of BMP signaling in bone homeostasis and they also appear to be involved in pathological processes in inflammatory disorders affecting bones and joints. Certain BMPs (BMP2 and -7) were approved for clinical use; however, increased bone resorption rather than formation were observed in clinical applications, suggesting the role BMPs have in osteoclast activation and subsequent osteolysis. Here, we summarize the current knowledge of BMP signaling in osteoclasts, its role in osteoclast resorption, bone remodeling, and osteoblast-osteoclast coupling. Furthermore, discussion of clinical application of recombinant BMP therapy is based on recent preclinical and clinical studies.

Iron overload in aging Bmp6­/­ mice induces exocrine pancreatic injury and fibrosis due to acinar cell loss.

The relationship between hemochromatosis and diabetes has been well established, as excessive iron deposition has been reported to result in impaired function of the endocrine and exocrine pancreas. Therefore, the objective of the present study was to analyze the effects of iron accumulation on the pancreata and glucose homeostasis in a bone morphogenetic protein 6­knockout (Bmp6­/­) mouse model of hemochromatosis. The sera and pancreatic tissues of wild­type (WT) and Bmp6­/­ mice (age, 3 and 10 months) were subjected to biochemical and histological analyses. In addition, 18F­fluorodeoxyglucose biodistribution was evaluated in the liver, muscle, heart, kidney and adipose tissue of both animal groups. The results demonstrated that 3­month­old Bmp6­/­ mice exhibited iron accumulation preferentially in the exocrine pancreas, with no signs of pancreatic injury or fibrosis. No changes were observed in the glucose metabolism, as pancreatic islet diameter, insulin and glucagon secretion, blood glucose levels and glucose uptake in the liver, muscle and adipose tissue remained comparable with those in the WT mice. Aging Bmp6­/­ mice presented with progressive iron deposits in the exocrine pancreas, leading to pancreatic degeneration and injury that was characterized by acinar atrophy, fibrosis and the infiltration of inflammatory cells. However, the aging mice exhibited unaltered blood glucose levels and islet structure, normal insulin secretion and moderately increased α­cell mass compared with those in the age­matched WT mice. Additionally, iron overload and pancreatic damage were not observed in the aging WT mice. These results supported a pathogenic role of iron overload in aging Bmp6­/­ mice leading to iron­induced exocrine pancreatic deficiency, whereas the endocrine pancreas retained normal function.

A novel autologous bone graft substitute comprised of rhBMP6 blood coagulum as carrier tested in a randomized and controlled Phase I trial in patients with distal radial fractures.

Bone morphogenetic proteins (BMPs) are known to induce new bone formation in vivo but treating trabecular bone defects with a BMP based therapeutic remains controversial. Here, we evaluated the safety and efficacy of a novel Autologous Bone Graft Substitute (ABGS) comprised of recombinant human BMP6 (rhBMP6) dispersed within an autologous blood coagulum (ABC) as a physiological natural carrier in patients with a closed distal radial fracture (DRF). We enrolled 32 patients in a randomized, standard of care (SoC) and placebo (PBO) controlled, double-blinded Phase I First in Human (FiH) clinical trial. ABGS was prepared from peripheral blood as 250 µg rhBMP6/mL ABC or PBO (1 mL ABC containing excipients only) and was administered dorsally via a syringe injection into the fracture site following closed fracture fixation with 3 Kirschner wires. Patients carried an immobilization for 5 weeks and were followed-up for 0 to 26 weeks by clinical examination, safety, serial radiographic analyses and CT. During the 13 weeks follow-up and at 26 weeks post study there were no serious adverse reactions recorded. The results showed that there were no detectable anti-rhBMP6 antibodies in the blood of any of the 32 patients at 13- and 26-weeks following treatment. Pharmacokinetic analyses of plasma from patients treated with ABGS showed no detectable rhBMP6 at any time point within the first 24 h following administration. The CT image and radiographic analyses score from patients treated with AGBS showed significantly accelerated bone healing as compared to PBO and SoC at 5 and 9 weeks (with high effect sizes and P = 0.027), while at week 13 all patients had similar healing outcomes. In conclusion, we show that intraosseous administration of ABGS (250 µg rhBMP6/mL ABC) into the distal radial fracture site demonstrated a good tolerability with no serious adverse reactions as well as early accelerated trabecular bone healing as compared to control PBO and SoC patients.

Autologous blood coagulum containing rhBMP6 induces new bone formation to promote anterior lumbar interbody fusion (ALIF) and posterolateral lumbar fusion (PLF) of spine in sheep.

In the present study, we evaluated an autologous bone graft substitute (ABGS) composed of recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) used as a physiological carrier for new bone formation in spine fusion sheep models. The application of ABGS included cervical cage for use in the anterior lumbar interbody fusion (ALIF), while for the posterolateral lumbar fusion (PLF) sheep model allograft devitalized bone particles (ALLO) were applied with and without use of instrumentation. In the ALIF model, ABGS (rhBMP6/ABC/cage) implants fused significantly when placed in between the L4-L5 vertebrae as compared to control (ABC/cage) which appears to have a fibrocartilaginous gap, as examined by histology and micro CT analysis at 16 weeks following surgery. In the PLF model, ABGS implants with or without ALLO showed a complete fusion when placed ectopically in the gutter bilaterally between two decorticated L4-L5 transverse processes at a success rate of 88% without instrumentation and at 80% with instrumentation; however the bone volume was 50% lower in the instrumentation group than without, as examined by histology, radiographs, micro CT analyses and biomechanical testing at 27 weeks following surgery. The newly formed bone was uniform within ABGS implants resulting in a biomechanically competent and histologically qualified fusion with an optimum dose in the range of 100 µg rhBMP6 per mL ABC, while in the implants that contained ALLO, the mineralized bone particles were substituted by the newly formed remodeling bone via creeping substitution. These findings demonstrate for the first time that ABGS (rhBMP6/ABC) without and with ALLO particles induced a robust bone formation with a successful fusion in sheep models of ALIF and PLF, and that autologous blood coagulum (ABC) can serve as a preferred physiological native carrier to induce new bone at low doses of rhBMP6 and to achieve a successful spinal fusion.

Recombinant Human Bone Morphogenetic Protein 6 Delivered Within Autologous Blood Coagulum Restores Critical Size Segmental Defects of Ulna in Rabbits.

BMP2 and BMP7, which use bovine Achilles tendon-derived absorbable collagen sponge and bovine bone collagen as scaffold, respectively, have been approved as bone graft substitutes for orthopedic and dental indications. Here, we describe an osteoinductive autologous bone graft substitute (ABGS) that contains recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) scaffold. The ABGS is created as an injectable or implantable coagulum gel with rhBMP6 binding tightly to plasma proteins within fibrin meshwork, as examined by dot-blot assays, and is released slowly as an intact protein over 6 to 8 days, as assessed by ELISA. The biological activity of ABGS was examined in vivo in rats (Rattus norvegicus) and rabbits (Oryctolagus cuniculus). In a rat subcutaneous implant assay, ABGS induced endochondral bone formation, as observed by histology and micro-CT analyses. In the rabbit ulna segmental defect model, a reproducible and robust bone formation with complete bridging and restoration of the defect was observed, which is dose dependent, as determined by radiographs, micro-CT, and histological analyses. In ABGS, ABC scaffold provides a permissive environment for bone induction and contributes to the use of lower doses of rhBMP6 compared with BMP7 in bovine bone collagen as scaffold. The newly formed bone undergoes remodeling and establishes cortices uniformly that is restricted to implant site by bridging with host bone. In summary, ABC carrier containing rhBMP6 may serve as an osteoinductive autologous bone graft substitute for several orthopedic applications that include delayed and nonunion fractures, anterior and posterior lumbar interbody fusion, trauma, and nonunions associated with neurofibromatosis type I.

A novel role of bone morphogenetic protein 6 (BMP6) in glucose homeostasis.

AIMS: Bone morphogenetic proteins (BMPs) are involved in the development and homeostasis of multiple organs and tissues. There has been a significant focus on understanding the role of BMPs in pancreatic ß-cell dysfunction associated with type 2 diabetes (T2D). Our objective was to investigate the relationship between BMP6 and glucose homeostasis. METHODS: Ob/ob mice were treated with BMP6 for 6 days and analyzed for insulin release, body weight, lipid parameters and glucose tolerance. Quantitative real-time PCR, chromatin immunoprecipitation and glucose output assays were used to assess BMP6 effect on gluconeogenesis in rat hepatoma H4IIE cells. Specificity of BMP6 receptors was characterized by the utilization of various receptor Fc fusion proteins in luciferase reporter gene and glucose output assays in INS1 and H4IIE cells. RESULTS: Treatment of ob/ob mice with BMP6 for 6 days resulted in a reduction of circulating glucose and lipid levels, followed by a significantly elevated plasma insulin level in a dose-dependent manner. In addition, BMP6 improved the glucose excursion during an oral glucose tolerance test, lowering the total glycemic response by 21%. In rat H4IIE hepatoma cells, BMP6 inhibited gluconeogenesis and glucose output via downregulation the PepCK expression. Moreover, BMP6 inhibited glucose production regardless of the presence of cAMP, antagonizing its glycogenolytic effect. BMP6 acted on pancreatic and liver cells utilizing Alk3, Alk6 and ActRIIA serine/threonine kinase receptors. CONCLUSIONS: Collectively, we demonstrate that BMP6 improves glycaemia in T2D mice and regulates glucose metabolism in hepatocytes representing an exciting prospect for future treatments of diabetes.

Exogenous BMP7 corrects plasma iron overload and bone loss in Bmp6-/- mice.

PURPOSE: Iron overload accelerates bone loss in mice lacking the bone morphogenetic protein 6 (Bmp6) gene, which is the key endogenous regulator of hepcidin, iron homeostasis gene. We investigated involvement of other BMPs in preventing haemochromatosis and subsequent osteopenia in Bmp6-/- mice. METHODS: Iron-treated wild-type (WT) and Bmp6-/- mice were analysed for hepcidin messenger RNA (mRNA) and tissue and blood BMP levels by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry, Western blot, enzyme-linked immunosorbent assay (ELISA) and proximity extension assay. BMPs labeled with technetium-99m were used in pharmacokinetic studies. RESULTS: In WT mice, 4 h following iron challenge, liver Bmp6 and hepcidin expression were increased, while expression of other Bmps was not affected. In parallel, we provided the first evidence that BMP6 circulates in WT mice and that iron increased the BMP6 serum level and the specific liver uptake of (99m)Tc-BMP6. In Bmp6-/- mice, iron challenge led to blunted activation of liver Smad signaling and hepcidin expression with a delay of 24 h, associated with increased Bmp5 and Bmp7 expression and increased Bmp2, 4, 5 and 9 expression in the duodenum. Liver Bmp7 expression and increased circulating BMP9 eventually contributed to the late hepcidin response. This was further supported by exogenous BMP7 therapy resulting in an effective hepcidin expression followed by a rapid normalisation of plasma iron values and restored osteopenia in Bmp6-/- mice. CONCLUSION: In Bmp6-/- mice, iron activated endogenous compensatory mechanisms of other BMPs that were not sufficient for preventing hemochromatosis and bone loss. Administration of exogenous BMP7 was effective in correcting the plasma iron level and bone loss, indicating that BMP6 is an essential but not exclusive in vivo regulator of iron homeostasis.

Systemically available bone morphogenetic protein two and seven affect bone metabolism.

PURPOSE: Bone morphogenetic protein (BMP)-2 and -7 are used in patients with long-bone fractures, nonunions and spinal fusions. It is unknown whether their potential systemic bioavailability following local bone administration might affect skeletal metabolism. To answer this question, we examined effects of systemically administered BMP-2 and -7 on bone in a newly developed rat model with a low level of calciotropic hormones. METHODS: Removal of thyroid and parathyroid glands (TPTx) in rats resulted in a decreased level of calciotropic hormones and subsequent bone loss assessed by micro computed tomography (micro-CT) and measurement of serum bone formation and resorption markers, including osteocalcin, C-telopeptide, osteoprotegerin and receptor activator of nuclear factor kappa-B ligand. Results were complemented with in vitro studies on osteoblast and osteoclast activity by both BMP-2 and -7. The doses used were calculated from published pharmacodynamic studies and bioavailability results from preclinical BMP-2 and -7 studies. RESULTS: TPTx resulted in bone loss, which was restored by systemic administration of 10-70 µg/kg of BMP-2 and 10-250 µg/kg of BMP-7. BMP-2 showed a higher capacity for enhancing trabecular microarchitecture, whereas BMP-7 augmented trabecular thickness. In vitro experiments revealed that BMP-2 and -7 when uncoupled increased the number and activity of both osteoblasts and osteoclasts. CONCLUSIONS: Surprisingly, both BMP-2 and -7 showed an increased bone volume in an in vivo environment of low calciotropic hormones. Locally administered BMP-2 and -7 from bone devices might become partially available in circulation but will not mediate systemic bone loss.

The clinical use of bone morphogenetic proteins revisited: a novel biocompatible carrier device OSTEOGROW for bone healing.

PURPOSE: The purpose of this study was to revise the clinical use of commercial BMP2 (Infuse) and BMP7 (Osigraft) based bone devices and explore the mechanism of action and efficacy of low BMP6 doses in a novel whole blood biocompatible device OSTEOGROW. METHODS: Complications from the clinical use of BMP2 and BMP7 have been systemically reviewed in light of their role in bone remodeling. BMP6 function has been assessed in Bmp6-/- mice by µCT and skeletal histology, and has also been examined in mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) and osteoclasts. Safety and efficacy of OSTEOGROW have been assessed in rats and rabbits. RESULTS: Clinical use issues of BMP2 and BMP7 have been ascribed to the limited understanding of their role in bone remodeling at the time of device development for clinical trials. BMP2 and BMP7 in bone devices significantly promote bone resorption leading to osteolysis at the endosteal surfaces, while in parallel stimulating exuberant bone formation in surrounding tissues. Unbound BMP2 and BMP7 in bone devices precipitate on the bovine collagen and cause inflammation and swelling. OSTEOGROW required small amounts of BMP6, applied in a biocompatible blood coagulum carrier, for stimulating differentiation of MSCs and accelerated healing of critical size bone defects in animals, without bone resorption and inflammation. BMP6 decreased the number of osteoclasts derived from HSC, while BMP2 and BMP7 increased their number. CONCLUSIONS: Current issues and challenges with commercial bone devices may be resolved by using novel BMP6 biocompatible device OSTEOGROW, which will be clinically tested in metaphyseal bone fractures, compartments where BMP2 and BMP7 have not been effective.

Hyperserotonemia in autism: activity of 5HT-associated platelet proteins.

Disturbances in serotonin (5HT) neurotransmission have been indicated as biological substrates in several neuropsychiatric disorders including autism. Blood 5HT concentrations, elevated in about one-third of autistic subjects, are regulated through the action of peripheral 5HT-associated proteins. We have measured the activity of two platelet 5HT-associated proteins: 5HT transporter (5HTT) and monoamine oxidase B (MAOB), and indirectly studied the activity of 5HT(2A) receptor (5HT(2A)r) in 15 hyperserotonemic (HS) and 17 normoserotonemic (NS) autistic subjects, and 15 healthy controls (C). While mean velocities of 5HTT kinetics did not significantly differ among the groups, significant elevation in the mean velocity of MAOB kinetics was observed in NS subjects and was even more pronounced in HS subjects in comparison to controls. Also, a decrease in adenosine 5'-diphosphate-induced platelet aggregation of borderline significance was observed in NS subjects, compared to C subjects. The results suggest a possibility of upregulation of monoaminergic synthesis/degradation and, probably consequential, downregulation of 5HT(2A)r in autistic subjects.