Synergistic effect of umbilical cord extracellular vesicles and rhBMP-2 to enhance the regeneration of a metaphyseal femoral defect in osteoporotic rats.

BACKGROUND: The aim of this study was to evaluate potential synergistic effects of a single, local application of human umbilical cord MSC-derived sEVs in combination with a low dose of recombinant human rhBMP-2 to promote the regeneration of a metaphyseal femoral defect in an osteoporotic rat model. METHODS: 6 weeks after induction of osteoporosis by bilateral ventral ovariectomy and administration of a special diet, a total of 64 rats underwent a distal femoral metaphyseal osteotomy using a manual Gigli wire saw. Defects were stabilized with an adapted Y-shaped mini-locking plate and were subsequently treated with alginate only, or alginate loaded with hUC-MSC-sEVs (2 × 109), rhBMP-2 (1.5 µg), or a combination of sEVs and rhBMP-2 (n = 16 for each group). 6 weeks post-surgery, femora were evaluated by µCT, descriptive histology, and biomechanical testing. RESULTS: Native radiographs and µCT analysis confirmed superior bony union with callus formation after treatment with hUC-MSC-sEVs in combination with a low dose of rhBMP-2. This finding was further substantiated by histology, showing robust defect consolidation 6 weeks after treatment. Torsion testing of the explanted femora revealed increased stiffness after application of both, rhBMP-2 alone, or in combination with sEVs, whereas torque was only significantly increased after treatment with rhBMP-2 together with sEVs. CONCLUSION: The present study demonstrates that the co-application of hUC-MSC-sEVs can improve the efficacy of rhBMP-2 to promote the regeneration of osteoporotic bone defects.

rhBMP-2 induces terminal differentiation of human bone marrow mesenchymal stromal cells only by synergizing with other signals.

BACKGROUND: Recombinant human bone morphogenetic protein 2 (rhBMP-2) and human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied for research and translational bone regeneration purposes. rhBMP-2 induces bone formation in vivo, and hBM-MSCs are its target, bone-forming cells. In this article, we studied how rhBMP-2 drives the multilineage differentiation of hBM-MSCs both in vivo and in vitro. METHODS: rhBMP-2 and hBM-MSCs were tested in an in vivo subcutaneous implantation model to assess their ability to form mature bone and undergo multilineage differentiation. Then, the hBM-MSCs were treated in vitro with rhBMP-2 for short-term or long-term cell-culture periods, alone or in combination with osteogenic, adipogenic or chondrogenic media, aiming to determine the role of rhBMP-2 in these differentiation processes. RESULTS: The data indicate that hBM-MSCs respond to rhBMP-2 in the short term but fail to differentiate in long-term culture conditions; these cells overexpress the rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, in combination with other differentiation signals, rhBMP-2 acts as a potentiator of multilineage differentiation, not only of osteogenesis but also of adipogenesis and chondrogenesis, both in vitro and in vivo. CONCLUSIONS: Altogether, our data indicate that rhBMP-2 alone is unable to induce in vitro osteogenic terminal differentiation of hBM-MSCs, but synergizes with other signals to potentiate multiple differentiation phenotypes. Therefore, rhBMP-2 triggers on hBM-MSCs different specific phenotype differentiation depending on the signalling environment.

Litsea glutinosa extract promotes fracture healing and prevents bone loss via BMP2/SMAD1 signaling.

Estrogen deficiency is one of the main causes for postmenopausal osteoporosis. Current osteoporotic therapies are of high cost and associated with serious side effects. So there is an urgent need for cost-effective anti-osteoporotic agents. Anti-osteoporotic activity of Litsea glutinosa extract (LGE) is less explored. Moreover, its role in fracture healing and mechanism of action is still unknown. In the present study we explore the osteoprotective potential of LGE in osteoblast cells and fractured and ovariectomized (Ovx) mice models. Alkaline phosphatase (ALP), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and mineralization assays revealed that LGE treatment increased osteoblast cell differentiation, viability and mineralization. LGE treatment at 0.01 µg increased the expression of BMP2, PSMAD, RUNX2 and type 1 col. LGE also mitigated RANKL-induced osteoclastogenesis. Next, drill hole injury Balb/C mice model was treated with LGE for 12 days. Micro-CT analysis and Calcein labeling at the fracture site showed that LGE (20 mg/kg) enhanced new bone formation and bone regeneration, also increased expression of BMP2/SMAD1 signaling genes at fracture site. Ovx mice were treated with LGE for 1 month. µCT analysis indicated that the treatment of LGE at 20 mg/kg dose prevented the alteration in bone microarchitecture and maintained bone mineral density and bone mineral content. Treatment also increased bone strength and restored the bone turnover markers. Furthermore, in bone samples, LGE increased osteogenesis by enhancing the expression of BMP2/SMAD1 signaling components and decreased osteoclast number and surface. We conclude that LGE promotes osteogenesis via modulating the BMP2/SMAD1 signaling pathway. The study advocates the therapeutic potential of LGE in osteoporosis treatment.

Goat mammary epithelial cells provide a better expression system for production of recombinant human bone morphogenetic protein 2 compared to Chinese hamster ovarian cells.

Bone Morphogenetic Protein 2 (BMP2), a member of the Transforming Growth Factor-ß (TGF-ß) super family of proteins and is instrumental in the repair of fractures. The synthesis of BMP2 involves extensive post-translational processing and several studies have demonstrated the abysmally low production of rhBMP2 in eukaryotic systems, which may be due to the short half-life of the bioactive protein. Consequently, production costs of rhBMP2 are quite high, limiting its availability to the general populace. Therefore, there is an urgent need to identify better in-vitro systems for large scale production of rhBMP2. In the present study, we have carried out a comparative analysis of rhBMP2 production by the conventionally used Chinese Hamster ovarian cells (CHO) and goat mammary epithelial cells (GMEC), upon transfection with appropriate construct. Udder gland cells are highly secretory, and we reasoned that such cells may serve as a better in-vitro model for large scale production of rhBMP2. Our results indicated that the synthesis and secretion of bioactive rhBMP2 by goat mammary epithelial cells was significantly higher as compared to that by CHO-K1 cells. Our results provide strong evidence that GMECs may serve as a better alternative to other mammalian cells used for therapeutic protein production.

The bone morphogenetic protein 2 analogue L51P enhances spinal fusion in combination with BMP2 in an in vivo rat tail model.

Bone morphogenic protein 2 (BMP2) is known to induce osteogenesis and is applied clinically to enhance spinal fusion despite adverse effects. BMP2 needs to be used in high doses to be effective due to the presence of BMP2 inhibitors. L51P is a BMP2 analogue that acts by inhibition of BMP2 inhibitors. Here, we hypothesized that mixtures of BMP2 and L51P could achieve better spinal fusion outcomes regarding ossification. To test whether mixtures of both cytokines are sufficient to improve ossification, 45 elderly Wistar rats (of which 21 were males) were assigned to seven experimental groups, all which received spinal fusion surgery, including discectomy at the caudal 4-5 level using an external fixator and a porous ß-tricalcium phosphate (ßTCP) carrier. These ßTCP carriers were coated with varying concentrations of BMP2 and L51P. X-rays were taken immediately after surgery and again six and twelve weeks post-operatively. Histological sections and µCT were analyzed after twelve weeks. Spinal fusion was assessed using X-ray, µCT and histology according to the Bridwell scale by voxel-based quantification and a semi-quantitative histological score, respectively. The results were congruent across modalities and revealed high ossification for high-dose BMP2 (10 µg), while PBS induced no ossification. Low-dose BMP2 (1 µg) or 10 µg L51P alone did not induce relevant bone formation. However, all combinations of low-dose BMP2 with L51P (1 µg + 1/5/10 µg) were able to induce similar ossificationas high-dose BMP2. These results are of high clinical relevance, as they indicate L51P is sufficient to increase the efficacy of BMP2 and thus lower the required dose for spinal fusion. STATEMENT OF SIGNIFICANCE: Spinal fusion surgery is frequently applied to treat spinal pathologies. Bone Morphogenic Protein-2 (BMP2) has been approved by the U .S. Food and Drug Administration (FDA-) and by the "Conformité Européenne" (CE)-label. However, its application is expensive and high concentrations cause side-effects. This research targets the improvement of the efficacy of BMP2 in spinal fusion surgery.

Enhanced bone regeneration in rat calvarial defects through BMP2 release from engineered poly(ethylene glycol) hydrogels.

The clinical standard therapy for large bone defects, typically addressed through autograft or allograft donor tissue, faces significant limitations. Tissue engineering offers a promising alternative strategy for the regeneration of substantial bone lesions. In this study, we harnessed poly(ethylene glycol) (PEG)-based hydrogels, optimizing critical parameters including stiffness, incorporation of arginine-glycine-aspartic acid (RGD) cell adhesion motifs, degradability, and the release of BMP2 to promote bone formation. In vitro we demonstrated that human bone marrow derived stromal cell (hBMSC) proliferation and spreading strongly correlates with hydrogel stiffness and adhesion to RGD peptide motifs. Moreover, the incorporation of the osteogenic growth factor BMP2 into the hydrogels enabled sustained release, effectively inducing bone regeneration in encapsulated progenitor cells. When used in vivo to treat calvarial defects in rats, we showed that hydrogels of low and intermediate stiffness optimally facilitated cell migration, proliferation, and differentiation promoting the efficient repair of bone defects. Our comprehensive in vitro and in vivo findings collectively suggest that the developed hydrogels hold significant promise for clinical translation for bone repair and regeneration by delivering sustained and controlled stimuli from active signaling molecules.

A Combined Treatment of BMP2 and Soluble VEGFR1 for the Enhancement of Tendon-Bone Healing by Regulating Injury-Activated Skeletal Stem Cell Lineage.

BACKGROUND: Bone morphogenetic protein 2 (BMP2) is an appealing osteogenic and chondrogenic growth factor for promoting tendon-bone healing. Recently, it has been reported that soluble vascular endothelial growth factor (VEGF) receptor 1 (sVEGFR1) (a VEGF receptor antagonist) could enhance BMP2-induced bone repair and cartilage regeneration; thus, their combined application may represent a promising treatment to improve tendon-bone healing. Moreover, BMP2 could stimulate skeletal stem cell (SSC) expansion and formation, which is responsible for wounded tendon-bone interface repair. However, whether the codelivery of BMP2 and sVEGFR1 increases tendon enthesis injury-activated SSCs better than does BMP2 alone needs further research. PURPOSE: To study the effect of BMP2 combined with sVEGFR1 on tendon-bone healing and injury-activated SSC lineage. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 128 C57BL/6 mice that underwent unilateral supraspinatus tendon detachment and repair were randomly assigned to 4 groups: (1) untreated control group; (2) hydrogel group, which received a local injection of the blank hydrogel at the injured site; (3) BMP2 group, which received an injection of hydrogel with BMP2; and (4) BMP2 with sVEGFR1 group, which received an injection of hydrogel with BMP2 and sVEGFR1. Histology, micro-computed tomography, and biomechanical tests were conducted to evaluate tendon-bone healing at 4 and 8 weeks after surgery. In addition, flow cytometry was performed to detect the proportion of SSCs and their downstream differentiated subtypes, including bone, cartilage, and stromal progenitors; osteoprogenitors; and pro-chondrogenic progenitors within supraspinatus tendon enthesis at 1 week postoperatively. RESULTS: The repaired interface in BMP2 with sVEGFR1 group showed a significantly improved collagen fiber continuity, increased fibrocartilage, greater newly formed bone, and elevated mechanical properties compared with the other 3 groups. There were more SSCs; bone, cartilage, and stromal progenitors; osteoprogenitors; and pro-chondrogenic progenitors in the BMP2 with sVEGFR1 group than that in the other groups. CONCLUSION: Our study suggests that the combined delivery of BMP2 and sVEGFR1 could promote tendon-bone healing and stimulate the expansion of SSCs and their downstream progeny within the injured tendon-bone interface. CLINICAL RELEVANCE: Combining BMP2 with sVEGFR1 may be a good clinical treatment for wounded tendon enthesis healing.

Mitigation of BMP-induced inflammation in craniofacial bone regeneration and improvement of bone parameters by dietary hesperidin.

Based on anti-inflammatory and osteogenic properties of hesperidin (HE), we hypothesized its systemic administration could be a cost-effective method of improving BMP-induced bone regeneration. Sprague-Dawley rats were allocated into 4 groups (n = 10/group): a 5-mm critical-sized mandible defect + collagen scaffold or, scaffold + 1 µg of BMP2 with and without dietary HE at 100 mg/kg. HE was administered by oral gavage 4 weeks prior to surgeries until euthanasia at day 7 or 14 post-surgery. The healing tissue within the defect collected at day 7 was subjected to gene expression analysis. Mandibles harvested at day 14 were subjected to microcomputed tomography and histology. HE + BMP2-treated rats had a statistically significant decrease in expression of inflammatory genes compared to BMP2 alone. The high-dose BMP2 alone caused cystic-like regeneration with incomplete defect closure. HE + BMP2 showed virtually complete bone fusion. Collagen fibril birefringence pattern (red color) under polarized light indicated high organization in BMP2-induced newly formed bone (NFB) in HE-supplemented group (p < 0.05). Clear changes in osteocyte lacunae as well as a statistically significant increase in osteoclasts were found around NFB in HE-treated rats. A significant increase in trabecular volume and thickness, and trabecular and cortical density was found in femurs of HE-supplemented rats (p < 0.05). Our findings show, for the first time, that dietary HE has a remarkable modulatory role in the function of locally delivered high-dose BMP2 in bone regeneration possibly via control of inflammation, osteogenesis, changes in osteocyte and osteoclast function and collagen maturation in regenerated and native bone. In conclusion, HE had a significant skeletal bone sparing effect and the ability to provide a more effective BMP-induced craniofacial regeneration.

Polydioxanone Enhances Bone Regeneration After Resection and Reconstruction of Rat Femur with rhBMP2.

The aim of this study was to assess the bone regeneration potential of a polydioxanone (PDO) scaffold together with recombinant human bone morphogenetic protein-2 (rhBMP-2) for the reconstruction of large bone defect. In total, 24 male rats (6 months old) were subjected to bilateral femoral stabilization using titanium plates to create a 2 mm gap, and reconstruction using rhBMP-2 (Infuse®; 3.25 µg). The bone defects were covered with PDO (PDO group), or with titanium mesh (Ti group). Animals were euthanized on days 14 and 60. Simultaneously, 16 rats received PDO and Ti in their dorsum for the purpose of biocompatibility analysis at 3, 5, 7, and 10 days postoperatively. X-ray densitometry showed a higher density in the PDO group on day 14. On day 60, coverage of the bone defect with PDO showed a larger quantity of newly formed bone than that found for the Ti group, a lower inflammatory infiltrate value, and a more significant number of blood vessels on day 14. By immunohistochemical assessment, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) showed higher labeling on day 14 in the PDO group. On day 60, bone morphogenetic protein-2 (BMP-2) showed higher labeling in the PDO group, whereas Ti showed higher labeling for osteoprotegerin, nuclear factor kappa B ligand-activating receptor, RUNX2, and OCN. Furthermore, biocompatibility analysis showed a higher inflammatory response in the Ti group. The PDO scaffold enhanced bone regeneration when associated with rhBMP-2 in rat femur reconstruction. Impact statement Regeneration of segmental bone defects is a difficult task, and several techniques and materials have been used. Recent advances in the production of synthetic polymers, such as polydioxanone (PDO), produced by three-dimensional printing, have shown distinct characteristics that could improve tissue regeneration even in an important bone defect. The present preclinical study showed that PDO membranes used as scaffolds to carry recombinant human bone morphogenetic protein-2 (rhBMP-2) improved bone tissue regeneration by more than 8-fold when compared with titanium mesh, suggesting that PDO membranes could be a feasible and useful material for use in guided bone regeneration. (In English, viable is only used for living creatures capable of sustaining life.

Stiffness and BMP-2 Mimetic Peptide Jointly Regulate the Osteogenic Differentiation of Rat Bone Marrow Stromal Cells in a Gelatin Cryogel.

Both biochemical and mechanical cues could regulate the function of stem cells, but the interaction mechanism of their signaling pathway remains unclear, especially in the three-dimensional (3D) culture mode. Higher matrix stiffness promotes osteogenic differentiation of stem cells, and bone morphogenic protein-2 (BMP-2) has been clinically applied to promote bone regeneration. Here, the crosstalk of extracellular mechanical signals on BMP-2 signaling was investigated in rat bone marrow stromal cells (rMSCs) cultured inside cryogels with interconnective pores. Stiff cryogel independently promoted osteogenic differentiation and enhanced the autocrine secretion of BMP-2, thus stimulating increased phosphorylation levels of the Smad1/5/8 complex. BMP-2 mimetic peptide (BMMP) and high cryogel stiffness jointly guided the osteogenic differentiation of rMSCs. Inhibition of rho-associated kinase (ROCK) by Y-27632 or inhibition of nonmuscle myosin II (NM II) by blebbistatin showed that osteogenesis induction by BMP-2 signaling, as well as autocrine secretion of BMP-2 and phosphorylation of the Smad complex, requires the involvement of cytoskeletal tension and ROCK pathway signaling. An interconnective microporous cryogel scaffold promoted rMSC osteogenic differentiation by combining matrix stiffness and BMMP, and it accelerated critical cranial defect repair in the rat model.

BMP-9 Improves the Osteogenic Differentiation Ability over BMP-2 through p53 Signaling In Vitro in Human Periosteum-Derived Cells.

Bone morphogenetic proteins (BMPs) have tremendous therapeutic potential regarding the treatment of bone and musculoskeletal disorders due to their osteo-inductive ability. More than twenty BMPs have been identified in the human body with various functions, such as embryonic development, skeleton genesis, hematopoiesis, and neurogenesis. BMPs can induce the differentiation of MSCs into the osteoblast lineage and promote the proliferation of osteoblasts and chondrocytes. BMP signaling is also involved in tissue remodeling and regeneration processes to maintain homeostasis in adults. In particular, growth factors, such as BMP-2 and BMP-7, have already been approved and are being used as treatments, but it is unclear as to whether they are the most potent BMPs that induce bone formation. According to recent studies, BMP-9 is known to be the most potent inducer of the osteogenic differentiation of mesenchymal stem cells, both in vitro and in vivo. However, its exact role in the skeletal system is still unclear. In addition, research results suggest that the molecular mechanism of BMP-9-mediated bone formation is also different from the previously known BMP family, suggesting that research on signaling pathways related to BMP-9-mediated bone formation is actively being conducted. In this study, we performed a phosphorylation array to investigate the signaling mechanism of BMP-9 compared with BMP-2, another influential bone-forming growth factor, and we compared the downstream signaling system. We present a mechanism for the signal transduction of BMP-9, focusing on the previously known pathway and the p53 factor, which is relatively upregulated compared with BMP-2.

Complementary modulation of BMP signaling improves bone healing efficiency.

The bone morphogenetic protein (BMP) signaling pathway plays a crucial role in bone development and regeneration. While BMP-2 is widely used as an alternative to autograft, its clinical application has raised concerns about adverse side effects and deteriorated bone quality. Therefore, there is a need to develop more sophisticated approaches to regulate BMP signaling and promote bone regeneration. Here, we present a novel complementary strategy that targets both BMP antagonist noggin and agonist Trb3 to enhance bone defect repair without the application of exogenous BMP-2. In vitro studies showed that overexpression of Trb3 with simultaneous noggin suppression significantly promotes osteogenic differentiation of mesenchymal stem cells. This was accompanied by increased BMP/Smad signaling. We also developed sterosome nanocarriers, a non-phospholipid liposomal system, to achieve non-viral mediated noggin suppression and Trb3 overexpression. The gene-loaded sterosomes were integrated onto an apatite-coated polymer scaffold for in vivo calvarial defect implantation, resulting in robust bone healing compared to BMP-2 treatments. Our work provides a promising alternative for high-quality bone formation by regulating expression of BMP agonists and antagonists.

[Advantages and disadvantages of bone graft materials activated by BMP-2 and constructs carrying its gene]. / Preimushchestva i nedostatki kostno-plasticheskikh materialov, aktivirovannykh BMP-2 i nesushchimi ego gen konstruktsiyami.

In the review gene constructs and proteins used to impart osteoinductive properties to bone graft materials are compared. On the basis of clinical and experimental data the experience and prospects of their application in maxillofacial surgery and dentistry are described. Information about complications associated with the use of bone morphogenetic protein-2 (BMP-2) and vectors carrying its gene is provided.

Pilot Study on Percutaneous Delivery of Recombinant Human Bone Morphongenetic Protein-2 Augments Fusion in a Nicotine-impaired Rabbit Fusion Model.

STUDY DESIGN: A nicotine-impaired spinal fusion rabbit model. OBJECTIVE: To examine whether controlled delivery of morselized absorbable collagen sponge recombinant human bone morphogenetic protein-2 (rhBMP2) in a delayed manner postsurgery would allow for improved bone healing. SUMMARY OF BACKGROUND DATA: The current delivery method of rhBMP-2 during surgery causes a burst of rhBMP-2, which is not sustained. Given that bone morphogenetic protein 2 (BMP-2) expression peaks later in the fusion process, there may be the benefit of delivery of rhBMP-2 later in the healing process. METHODS: Sixteen male 1-year-old rabbits underwent a posterolateral spinal fusion with iliac crest bone graft at L5-L6 while being given nicotine to prevent spinal fusion as previously published. Eight were controls, whereas 8 had morselized rhBMP-2 (4.2 mg) injected at the fusion site at 4 weeks postoperatively. Histologic, radiologic, and palpation examinations were performed at 12 weeks to determine fusion status and the volume of bone formed. Hematoxylin and eosin stains were used for histology. A Student t test was used to compare the computed tomography scan measured volume of bone created between the control cohort (CC) and rhBMP-2 delayed delivery cohort (BMP-DDC). RESULTS: Of the total, 7/8 rabbits in the BMP-DDC and 5/8 rabbits in the CC formed definitive fusion with a positive palpation examination, bridging bone between transverse processes on computed tomography scan, and an x-ray showing fusion. Histologic analysis revealed newly remodeled bone within the BMP-DDC. There was an increased average volume of bone formed within the BMP-DDC versus the CC (22.6 ± 13.1 vs 11.1 ± 3.6 cm 3 , P = 0.04). CONCLUSION: Our study shows that injectable morselized absorbable collagen sponge/rhBMP-2 can create twice as much bone within a nicotine-impaired rabbit spine fusion model when delivered 4 weeks out from the time of surgery.

Micellized protein transduction domain-bone morphogenetic protein-2 accelerates bone healing in a rat tibial distraction osteogenesis model.

The clinical application of growth factors such as recombinant human bone morphogenetic protein-2 (rh-BMP-2), for functional bone regeneration remains challenging due to limited in vivo efficacy and adverse effects of previous modalities. To overcome the instability and short half-life of rh-BMP-2 in vivo, we developed a novel osteogenic supplement by fusing a protein transduction domain (PTD) with BMP-2, effectively creating a prodrug of BMP-2. In this study, we first created an improved PTD-BMP-2 formulation using lipid nanoparticle (LNP) micellization, resulting in downsizing from micrometer to nanometer scale and achieving a more even distribution. The micellized PTD-BMP-2 (mPTD-BMP-2) demonstrated improved distribution and aggregation profiles. As a prodrug of BMP-2, mPTD-BMP-2 successfully activated Smad1/5/8 and induced mineralization with osteogenic gene induction in vitro. In vivo pharmacokinetic analysis revealed that mPTD-BMP-2 had a much more stable pharmacokinetic profile than rh-BMP-2, with a 7.5-fold longer half-life. The in vivo BMP-responsive element (BRE) reporter system was also successfully activated by mPTD-BMP-2. In the in vivo rat tibia distraction osteogenesis (DO) model, micro-computed tomography (micro-CT) scan findings indicated that mPTD-BMP-2 significantly increased bone volume, bone surface, axis moment of inertia (MOI), and polar MOI. Furthermore, it increased the expression of osteogenesis-related genes, and induced bone maturation histologically. Based on these findings, mPTD-BMP-2 could be a promising candidate for the next-generation osteogenesis drug to promote new bone formation in DO surgery. STATEMENT OF SIGNIFICANCE: This study introduces micellized bone morphogenetic protein-2 (mPTD-BMP-2), a next-generation osteogenic supplement that combines protein transduction domain (PTD) and nano-sized micelle formulation technique to improve transduction efficiency and stability. The use of PTD represents a novel approach, and our results demonstrate the superiority of mPTD-BMP-2 over rh-BMP-2 in terms of in vivo pharmacokinetic profile and osteogenic potential, particularly in a rat tibial model of distraction osteogenesis. These findings have significant scientific impact and potential clinical applications in the treatment of bone defects that require distraction osteogenesis. By advancing the field of osteogenic supplements, our study has the potential to contribute to the development of more effective treatments for musculoskeletal disorders.

Dual roles of cellular communication network factor 6 (CCN6) in the invasion and metastasis of oral cancer cells to bone via binding to BMP2 and RANKL.

The acquisition of motility via epithelial-mesenchymal transition (EMT) and osteoclast induction are essential for the invasion and metastasis of oral squamous cell carcinoma (OSCC) to bone. However, the molecule suppressing both EMT and osteoclastogenesis is still unknown. In this study, we found that cellular communication network factor 6 (CCN6) was less produced in a human OSCC cell line, HSC-3 with mesenchymal phenotype, than in HSC-2 cells without it. Notably, CCN6 interacted with bone morphogenetic protein 2 (BMP2) and suppressed the cell migration of HSC-3 cells stimulated by BMP2. Moreover, knockdown of CCN6 in HSC-2 cells led to the promotion of EMT and enhanced the effect of transforming growth factor-ß (TGF-ß) on the promotion of EMT. Furthermore, CCN6 combined with BMP2 suppressed EMT. These results suggest that CCN6 strongly suppresses EMT in cooperation with BMP2 and TGF-ß. Interestingly, CCN6 combined with BMP2 increased the gene expression of receptor activator of nuclear factor-κB ligand (RANKL) in HSC-2 and HSC-3 cells. Additionally, CCN6 interacted with RANKL, and CCN6 combined with RANKL suppressed RANKL-induced osteoclast formation. In metastatic lesions, increasing BMP2 due to the bone destruction led to interference with binding of CCN6 to RANKL, which results in the promotion of bone metastasis of OSCC cells due to continuous osteoclastogenesis. These findings suggest that CCN6 plays dual roles in the suppression of EMT and in the promotion of bone destruction of OSCC in primary and metastatic lesions, respectively, through cooperation with BMP2 and interference with RANKL.

Bone Morphogenetic Protein-9 Promotes Osteogenic Differentiation and Mineralization in Human Stem-Cell-Derived Spheroids.

Background and Objectives: Alkaline phosphatase activity, mineralized matrix, and osteogenic-related gene expression have been shown to increase in response to bone morphogenetic protein-9 (BMP-9). In this study, spheroids derived from human gingival stem cells were used to determine the effects of BMP-9 on cell survival, osteogenesis, and mineralization. Materials and Methods: Human gingival stem cells were used to produce spheroids and then grown to concentrations of 0, 0.1, 1, 10, and 100 ng/mL with BMP-9. On days 1, 3, 5, and 7, morphological examination was carried out. A live/dead assay and Cell Counting Kit-8 was used to assess the vitality of cells. On days 7 and 14, alkaline phosphatase activity assays were carried out using a commercially available kit to examine the osteogenic differentiation of cell spheroids. Alizarin Red Staining was performed on the 7th and 14th days to evaluate mineralization, and RUNX2 and COL1A1 expression levels were evaluated on the 7th and 14th days using real-time polymerase chain reactions. Results: The BMP-9 added at the measured quantities did not appear to alter the shape of the well-formed spheroids produced by stem cells on day 1. In addition, treatment with BMP-9 at doses of 0, 0.1, 1, 10, or 100 ng/mL did not significantly alter cell diameter. Throughout the whole experimental process, viability was maintained. On day 14, the alkaline phosphatase activity in the groups dosed with 0.1, 1, 10, or 100 ng/mL was statistically higher than that in the unloaded control group (p < 0.05). According to qPCR data, the mRNA expression level of RUNX2 with 1 ng/mL dosing was higher on day 7 compared to that of the unloaded control group (p < 0.05). Conclusions: These findings suggest that BMP-9 can be employed to stimulate early osteogenic differentiation in stem cell spheroids.

H3K27me3-modulated Hofbauer cell BMP2 signalling enhancement compensates for shallow trophoblast invasion in preeclampsia.

BACKGROUND: Preeclampsia (PE) is a common hypertensive pregnancy disorder associated with shallow trophoblast invasion. Although bone morphogenetic protein 2 (BMP2) has been shown to promote trophoblast invasion in vitro, its cellular origin and molecular regulation in placenta, as well as its potential role in PE, has yet to be established. Additionally, whether BMP2 and/or its downstream molecules could serve as potential diagnostic or therapeutic targets for PE has not been explored. METHODS: Placentas and sera from PE and healthy pregnant women were subjected to multi-omics analyses, immunoblots, qPCR, and ELISA assays. Immortalized trophoblast cells, primary cultures of human trophoblasts, and first-trimester villous explants were used for in vitro experiments. Adenovirus expressing sFlt-1 (Ad Flt1)-induced PE rat model was used for in vivo studies. FINDINGS: We find globally decreased H3K27me3 modifications and increased BMP2 signalling in preeclamptic placentas, which is negatively correlated with clinical manifestations. BMP2 is derived from Hofbauer cells and epigenetically regulated by H3K27me3 modification. BMP2 promotes trophoblast invasion and vascular mimicry by upregulating BMP6 via BMPR1A-SMAD2/3-SMAD4 signalling. BMP2 supplementation alleviates high blood pressure and fetal growth restriction phenotypes in Ad Flt1-induced rat PE model. INTERPRETATION: Our findings demonstrate that epigenetically regulated Hofbauer cell-derived BMP2 signalling enhancement in late gestation could serve as a compensatory response for shallow trophoblast invasion in PE, suggesting opportunities for diagnostic marker and therapeutic target applications in PE clinical management. FUNDING: National Key Research and Development Program of China (2022YFC2702400), National Natural Science Foundation of China (82101784, 82171648, 31988101), and Natural Science Foundation of Shandong Province (ZR2020QH051, ZR2020MH039).

Novel bone Morphogenetic Protein (BMP)-2/4 Consensus Peptide (BCP) for the Osteogenic Differentiation of C2C12 Cells.

BACKGROUND: Despite the promising clinical potential of bone morphogenetic protein (BMP)-related therapies for bone formation, their side effects warrant the need for alternative therapeutic peptides. BMP family members can aid in bone repair; however, peptides derived from BMP2/ 4 have not yet been investigated. METHODS: In this study, three candidates BMP2/4 consensus peptide (BCP) 1, 2, and 3 were identified and their ability to induce osteogenesis in C2C12 cells was analyzed. First, an alkaline phosphatase (ALP) staining assay was performed to evaluate the osteogenic effects of BCPs. Next, the effects of BCPs on RNA expression levels and protein abundances of osteogenic markers were explored. Furthermore, the transcriptional activity of ALP by BCP1 and in silico molecular docking model on BMP type IA receptor (BRIA) were performed. RESULTS: BCP1-3 induced higher RUNX2 expression than BMP2. Interestingly, among them, BCP1 significantly promoted osteoblast differentiation more than BMP2 in ALP staining with no cytotoxicity. BCP1 significantly induced the osteoblast markers, and the highest RUNX2 expression was observed at 100 ng/mL compared to other concentrations. In transfection experiments, BCP1 stimulated osteoblast differentiation via RUNX2 activation and the Smad signaling pathway. Finally, in silico molecular docking suggested the possible binding sites of BCP1 on BRIA. CONCLUSION: These results show that BCP1 promotes osteogenicity in C2C12 cells. This study suggests that BCP1 is the most promising candidate peptide to replace BMP2 for osteoblast differentiation.

Comparison of Osteoconductive Ability of Two Types of Cholesterol-Bearing Pullulan (CHP) Nanogel-Hydrogels Impregnated with BMP-2 and RANKL-Binding Peptide: Bone Histomorphometric Study in a Murine Calvarial Defect Model.

The receptor activator of NF-κB ligand (RANKL)-binding peptide is known to accelerate bone morphogenetic protein (BMP)-2-induced bone formation. Cholesterol-bearing pullulan (CHP)-OA nanogel-crosslinked PEG gel (CHP-OA nanogel-hydrogel) was shown to release the RANKL-binding peptide sustainably; however, an appropriate scaffold for peptide-accelerated bone formation is not determined yet. This study compares the osteoconductivity of CHP-OA hydrogel and another CHP nanogel, CHP-A nanogel-crosslinked PEG gel (CHP-A nanogel-hydrogel), in the bone formation induced by BMP-2 and the peptide. A calvarial defect model was performed in 5-week-old male mice, and scaffolds were placed in the defect. In vivo µCT was performed every week. Radiological and histological analyses after 4 weeks of scaffold placement revealed that the calcified bone area and the bone formation activity at the defect site in the CHP-OA hydrogel were significantly lower than those in the CHP-A hydrogel when the scaffolds were impregnated with both BMP-2 and the RANKL-binding peptide. The amount of induced bone was similar in both CHP-A and CHP-OA hydrogels when impregnated with BMP-2 alone. In conclusion, CHP-A hydrogel could be an appropriate scaffold compared to the CHP-OA hydrogel when the local bone formation was induced by the combination of RANKL-binding peptide and BMP-2, but not by BMP-2 alone.