Bone morphogenetic protein 6 (BMP6) antagonises experimental proliferative vitreoretinopathy established by TGF-ß2 stimulation in retinal pigment epithelial cells through modulation of the p38 and JNK MAPK pathways.

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-ß2 (TGF-ß2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-ß2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-ß2. The treatment of RPE cells with TGF-ß2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-ß2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-ß2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-ß2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR.

Recombinant Human BMP6 Applied Within Autologous Blood Coagulum Accelerates Bone Healing: Randomized Controlled Trial in High Tibial Osteotomy Patients.

Bone morphogenetic proteins (BMPs) are potent osteogenic proteins that induce new bone formation in vivo. However, their effect on bone healing in the trabecular bone surfaces remains challenging. We evaluated the safety and efficacy of recombinant human BMP6 (rhBMP6) applied within an autologous blood coagulum (ABC) in a surgically created wedge defect of the proximal tibia in patients undergoing high tibial osteotomy (HTO) for varus deformity and medial osteoarthritis of the knee. We enrolled 20 HTO patients in a randomized, placebo-controlled, double-blinded phase I/II clinical trial. RhBMP6/ABC (1.0 mg/10 mL ABC prepared from peripheral blood) or placebo (10 mL ABC containing excipients) was administered into the tibial wedge defects. Patients were followed for 0 to 24 months by clinical examination (safety) and computed tomography (CT) and serial radiographic analyses (efficacy). The results show that there were no detectable anti-rhBMP6 antibodies in the blood of any of the 20 patients at 14 weeks after implantation. During the 24 months of follow-up, there were no serious adverse reactions recorded. The CT scans from defects of patients treated with rhBMP6/ABC showed an accelerated bone healing compared with placebo at 9 weeks (47.8 ± 24.1 versus 22.2 ± 12.3 mg/cm3 ; p = 0.008) and at 14 weeks (89.7 ± 29.1 versus 53.6 ± 21.9 mg/cm3 ; p = 0.006) follow-up. Radiographic analyses at weeks 6 and 24 and months 12 and 24 suggested the advanced bone formation and remodeling in rhBMP6/ABC-treated patients. In conclusion, we show that rhBMP6/ABC at a dose of 100 µg/mL accelerated bone healing in patients undergoing HTO without serious adverse events and with a good tolerability compared with placebo alone. Overall, for the first time, a BMP-based osteogenic implant was examined against a placebo for bone healing efficacy in the trabecular bone surface, using an objective bone mineral density measurement system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

LIMK1 is involved in the protective effects of bone morphogenetic protein 6 against amyloid-ß-induced neurotoxicity in rat hippocampal neurons.

Bone morphogenetic protein 6 (BMP6) has neuroprotective effects against various neuronal injuries, but its effect on amyloid-ß (Aß)-induced neurotoxicity remains unclear. We exposed rat hippocampal neurons to different concentrations of Aß25-35 to induce neurotoxicity, and then treated cells with BMP6 to assess the neuroprotective effects. In vivo, we bilaterally injected Aß1-40 into basal forebrain to simulate the neuropathological process of Alzheimer's disease (AD), and explored changes in the expression of BMP6 and LIMK1. Our data demonstrated that BMP6 prevented apoptosis induced by a high dose of Aß25-35, and inhibited rod formation induced by low dose of Aß25-35 in hippocampal neurons. Forebrain injection of Aß1-40 led to a significant downregulation of BMP6, and inactivation of LIMK1 pathway in basal forebrain, whereas the opposite changes were observed in hippocampus. Our results suggest that BMP6 has neuroprotective effects against Aß25-35. The BMP6 and LIMK1 pathways may have different expression patterns at different stages of AD, and be self-regulating via a negative feedback mechanism between different brain regions.

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.

Effects of bone morphogenetic protein-6 on periodontal wound healing/regeneration in supraalveolar periodontal defects in dogs.

OBJECTIVE: Application of a synthetic BMP-6 polypeptide in a rat periodontal fenestration defect model enhanced periodontal wound healing/regeneration including new bone and cementum formation. The purpose of this study was to translate the relevance of these initial observations into a discriminating large animal model. METHODS: Critical-size (4-5 mm) supraalveolar periodontal defects were created at the 2(nd) and 3(rd) mandibular premolar teeth in 11 Beagle dogs. Experimental sites received BMP-6 at 0.25, 1.0 and 2.0 mg/ml soak-loaded onto an absorbable collagen sponge (ACS) carrier or ACS alone (control) each condition repeated in four jaw quadrants. The animals were euthanized at 8 weeks when block biopsies were collected and processed for histologic/histometric analysis. RESULTS: BMP-6 at 0.25, 1.0 and 2.0 mg/ml soak-loaded onto the ACS yielded significantly enhanced new bone (0.99 ± 0.07 versus 0.23 ± 0.13 mm/BMP-6 at 0.25 mg/ml) and cementum (2.45 ± 0.54 versus 0.73 ± 0.15 mm/BMP-6 at 0.25 mg/ml) formation including a functionally oriented periodontal ligament compared with control (p < 0.05). A significant inverse linear association between BMP-6 dose and new bone (ß = -0.21 ± 0.09 mm, p = 0.016) and cementum height (ß = -0.34 ± 0.15 mm, p = 0.023) was observed. Minimal root resorption was observed without significant differences between groups. Ankylosis was not observed for any of the experimental groups. CONCLUSIONS: Surgical application of BMP-6/ACS onto critical-size supraalveolar defects enhanced periodontal wound healing/regeneration, in particular cementogenesis including a functionally oriented periodontal ligament; the low BMP-6 0.25 mg/ml concentration apparently providing the most effective dose.

Gene-modified adult stem cells regenerate vertebral bone defect in a rat model.

Vertebral compression fractures (VCFs), the most common fragility fractures, account for approximately 700,000 injuries per year. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, a new minimally invasive biological solution to vertebral bone repair is needed. Previously, we showed that adipose-derived stem cells (ASCs) overexpressing a BMP gene are capable of inducing spinal fusion in vivo. We hypothesized that a direct injection of ASCs, designed to transiently overexpress rhBMP6, into a vertebral bone void defect would accelerate bone regeneration. Porcine ASCs were isolated and labeled with lentiviral vectors that encode for the reporter gene luciferase (Luc) under constitutive (ubiquitin) or inductive (osteocalcin) promoters. The ASCs were first labeled with reporter genes and then nucleofected with an rhBMP6-encoding plasmid. Twenty-four hours later, bone void defects were created in the coccygeal vertebrae of nude rats. The ASC-BMP6 cells were suspended in fibrin gel (FG) and injected into the bone void. A control group was injected with FG alone. The regenerative process was monitored in vivo using microCT, and cell survival and differentiation were monitored using tissue specific reporter genes and bioluminescence imaging (BLI). The surgically treated vertebrae were harvested after 12 weeks and subjected to histological and immunohistochemical (against porcine vimentin) analyses. In vivo BLI detected Luc-expressing cells at the implantation site over a 12-week period. Beginning 2 weeks postoperatively, considerable defect repair was observed in the group treated with ASC-BMP6 cells. The rate of bone formation in the stem cell-treated group was two times faster than that in the FG-treated group, and bone volume at the end point was 2-fold compared to the control group. Twelve weeks after cell injection the bone volume within the void reached the volume measured in native vertebrae. Immunostaining against porcine vimentin indicated that the ASC-BMP6 cells contributed to new bone formation. Here we show the potential of injections of BMP-modified ASCs to repair vertebral bone defects in a rat model. Our results could pave the way to a novel approach for the biological treatment of traumatic and osteoporosis-related vertebral bone injuries.

Direct delayed human adenoviral BMP-2 or BMP-6 gene therapy for bone and cartilage regeneration in a pony osteochondral model.

OBJECTIVE: To evaluate healing of surgically created large osteochondral defects in a weight-bearing femoral condyle in response to delayed percutaneous direct injection of adenoviral (Ad) vectors containing coding regions for either human bone morphogenetic proteins 2 (BMP-2) or -6. METHODS: Four 13mm diameter and 7mm depth circular osteochondral defects were drilled, 1/femoral condyle (n=20 defects in five ponies). At 2 weeks, Ad-BMP-2, Ad-BMP-6, Ad-green fluorescent protein (GFP), or saline was percutaneously injected into the central drill hole of the defect. Quantitative magnetic resonance imaging (qMRI) and computed tomography (CT) were serially performed at 12, 24, and 52 weeks. At 12 (one pony) or 52 weeks, histomorphometry and microtomographic analyses were performed to assess subchondral bone and cartilage repair tissue quality. RESULTS: Direct delivery of Ad-BMP-6 demonstrated delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and histologic evidence of greater Glycosaminoglycan (GAG) content in repair tissue at 12 weeks, while Ad-BMP-2 had greater non-mineral cartilage at the surface at 52 weeks (p<0.04). Ad-BMP-2 demonstrated greater CT subchondral bone mineral density (BMD) by 12 weeks and both Ad-BMP-2 and -6 had greater subchondral BMD at 52 weeks (p<0.05). Despite earlier (Ad-BMP-6) and more persistent (Ad-BMP-2) chondral tissue and greater subchondral bone density (Ad-BMP-2 and -6), the tissue within the large weight-bearing defects at 52 weeks was suboptimal in all groups due to poor quality repair cartilage, central fibrocartilage retention, and central bone cavitation. Delivery of either BMP by this method had greater frequency of subchondral bone cystic formation (p<0.05). CONCLUSIONS: Delivery of Ad-BMP-2 or Ad-BMP-6 via direct injection supported cartilage and subchondral bone regeneration but was insufficient to provide long-term quality osteochondral repair.

BMP6 treatment compensates for the molecular defect and ameliorates hemochromatosis in Hfe knockout mice.

BACKGROUND & AIMS: Abnormal hepcidin regulation is central to the pathogenesis of HFE hemochromatosis. Hepatic bone morphogenetic protein 6 (BMP6)-SMAD signaling is a main regulatory mechanism controlling hepcidin expression, and this pathway was recently shown to be impaired in Hfe knockout (Hfe(-/-)) mice. To more definitively determine whether HFE regulates hepcidin expression through an interaction with the BMP6-SMAD signaling pathway, we investigated whether hepatic Hfe overexpression activates the BMP6-SMAD pathway to induce hepcidin expression. We then investigated whether excess exogenous BMP6 administration overcomes the BMP6-SMAD signaling impairment and ameliorates hemochromatosis in Hfe(-/-) mice. METHODS: The BMP6-SMAD pathway and the effects of neutralizing BMP6 antibody were examined in Hfe transgenic mice (Hfe Tg) compared with wild-type (WT) mice. Hfe(-/-) and WT mice were treated with exogenous BMP6 and analyzed for hepcidin expression and iron parameters. RESULTS: Hfe Tg mice exhibited hepcidin excess and iron deficiency anemia. Hfe Tg mice also exhibited increased hepatic BMP6-SMAD target gene expression compared with WT mice, whereas anti-BMP6 antibody administration to Hfe Tg mice improved the hepcidin excess and iron deficiency. In Hfe(-/-) mice, supraphysiologic doses of exogenous BMP6 improved hepcidin deficiency, reduced serum iron, and redistributed tissue iron to appropriate storage sites. CONCLUSIONS: HFE interacts with the BMP6-SMAD signaling pathway to regulate hepcidin expression, but HFE is not necessary for hepcidin induction by BMP6. Exogenous BMP6 treatment in mice compensates for the molecular defect underlying Hfe hemochromatosis, and BMP6-like agonists may have a role as an alternative therapeutic strategy for this disease.