Synthesis and Evaluation of BMMSC-seeded BMP-6/nHAG/GMS Scaffolds for Bone Regeneration.

Bioactive scaffolding materials and efficient osteoinductive factors are key factors for bone tissue engineering. The present study aimed to mimic the natural bone repair process using an osteoinductive bone morphogenetic protein (BMP)-6-loaded nano-hydroxyapatite (nHA)/gelatin (Gel)/gelatin microsphere (GMS) scaffold pre-seeded with bone marrow mesenchymal stem cells (BMMSCs). BMP-6-loaded GMSs were prepared by cross-linking and BMP-6/nHAG/GMS scaffolds were fabricated by a combination of blending and freeze-drying techniques. Scanning electron microscopy, confocal laser scanning microscopy, and CCK-8 assays were carried out to determine the biocompatibility of the composite scaffolds in vitro. Alkaline phosphatase (ALP) activity was measured to evaluate the osteoinductivity of the composite scaffolds. For in vivo examination, critical-sized calvarial bone defects in Sprague-Dawley rats were randomly implanted with BMMSC/nHAG/GMS and BMMSC/BMP-6/nHAG/GMS scaffolds, and compared with a control group with untreated empty defects. The BMP-6-loaded scaffolds showed cytocompatibility by favoring BMMSC attachment, proliferation, and osteogenic differentiation. In radiological and histological analyses, the BMMSC-seeded scaffolds, especially the BMMSC-seeded BMP-6/nHAG/GMS scaffolds, significantly accelerated new bone formation. It is concluded that the BMP-6/nHAG/GMS scaffold possesses excellent biocompatibility and good osteogenic induction activity in vitro and in vivo, and could be an ideal bioactive substitute for bone tissue engineering.

Promoting Induced Pluripotent Stem Cell-driven Biomineralization and Periodontal Regeneration in Rats with Maxillary-Molar Defects using Injectable BMP-6 Hydrogel.

Periodontal disease may cause considerable destruction of alveolar bone, periodontal ligaments (PDLs) and cementum and even lead to progressive oral dysfunction. Periodontal tissue regeneration is the ultimate goal of periodontal disease treatment to reconstruct both structures and functions. However, the regenerative efficiency is low, possibly due to the lack of a proper periodontal microenvironment. In this study, we applied an injectable and thermosensitive chitosan/gelatin/glycerol phosphate hydrogel to provide a 3D environment for transplanted stem cells and to enhance stem cell delivery and engraftment. The iPSCs-BMP-6-hydrogel complex promoted osteogenesis and the differentiation of new connective tissue and PDL formation. In animal models of maxillary-molar defects, the iPSCs-BMP-6-hydrogel-treated group showed significant mineralization with increased bone volume, trabecular number and trabecular thickness. Synergistic effects of iPSCs and BMP-6 increased both bone and cementum formation. IPSCs-BMP-6-hydrogel-treated animals showed new bone synthesis (increased ALP- and TRAP-positive cells), new PDL regeneration (shown through Masson's trichrome staining and a qualification assay), and reduced levels of inflammatory cytokines. These findings suggest that hydrogel-encapsulated iPSCs combined with BMP-6 provide a new strategy to enhance periodontal regeneration. This combination not only promoted stem cell-derived graft engraftment but also minimized the progress of inflammation, which resulted in highly possible periodontal regeneration.

Combined delivery of PDGF-BB and BMP-6 for enhanced osteoblastic differentiation.

Natural microenvironment during bone tissue regeneration involves integration of multiple biological growth factors which regulate mitogenic activities and differentiation to induce bone repair. Among them platelet derived growth factor (PDGF-BB) and bone morphogenic protein-6 (BMP-6) are known to play a prominent role. The aim of this study was to investigate the benefits of combined delivery of PDGF-BB and BMP-6 on proliferation and osteoblastic differentiation of MC3T3-E1 preosteoblastic cells. PDGF-BB and BMP-6 were loaded in gelatin and poly (3-hydroxybutyric acid-co-3-hydroxyvaleric acid) particles, respectively. The carrier particles were then loaded into 3D chitosan matrix fabricated by freeze drying. The fast release of PDGF-BB during 7 days was accompanied by slower and prolonged release of BMP-6. The premising release of mitogenic factor PDGF-BB resulted in an increased MC3T3-E1 cell population seeded on chitosan scaffolds. Osteogenic markers of RunX2, Col 1, OPN were higher on chitosan scaffolds loaded with growth factors either individually or in combination. However, OCN expression and bone mineral formation were prominent on chitosan scaffolds incorporating PDGF-BB and BMP-6 as a combination.

Bone morphogenetic protein-2, -6, and -7 differently regulate osteogenic differentiation of human periodontal ligament stem cells.

The utility of adult stem cells for bone regeneration may be an attractive alternative in the treatment of extensive injury, congenital malformations, or diseases causing large bone defects. To create an environment that is supportive of bone formation, signals from molecules such as the bone morphogenetic proteins (BMPs) are required to engineer fully viable and functional bone. We therefore determined whether BMP-2, -6, and -7 differentially regulate the (1) proliferation, (2) mineralization, and (3) mRNA expression of bone/mineralized tissue associated genes of human periodontal ligament stem cells (hPDLSCs), which were obtained from periodontal ligament tissue of human impacted third molars. hPDLSCs from six participants were isolated and characterized using histochemical and immunohistochemical methods. A real-time cell analyzer was used to evaluate the effects of BMP-2, -6, and -7 on the proliferation of hPDLSCs. hPDLSCs were treated with Dulbecco's modified Eagle's medium containing different concentrations of BMP-2, -6, and -7 (10, 25, 50, 100 ng/mL) and monitored for 264 hours. After dose-response experiments, 50 and 100 ng/mL concentrations of BMPs were used to measure bone/mineralized tissue-associated gene expression. Type I collagen, bone sialoprotein, osteocalcin, osteopontin, and osteoblastic transcription factor Runx2 mRNA expression of hPDLSCs treated with BMP-2, -6, and -7, were evaluated using quantitative RT-PCR. Biomineralization of hPDLSCs was assessed using von Kossa staining. This study demonstrated that BMPs at various concentrations differently regulate the proliferation, mineralization, and mRNA expression of bone/mineralized tissue associated genes in hPDLSCs. BMPs regulate hPDLSC proliferation in a time and dose-dependent manner when compared to an untreated control group. BMPs induced bone/mineralized tissue-associated gene mRNA expression and biomineralization of hPDLSCs. The most pronounced induction occurred in the BMP-6 group in the biomineralization of the hPDLSCs. Our data suggest that BMP-2, -6, and -7 are potent regulators of hPDLSC gene expression and biomineralization. Employing BMPs with hPDLSCs isolated from periodontal ligament tissues provides a promising strategy for bone tissue engineering.

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.

[Advances in osteogenic mechanism and osteogenic effects of bone morphogenetic protein 6].

OBJECTIVE: To review the osteogenic mechanism and osteogenic effects of bone morphogenetic protein 6 (BMP-6) so as to provide the basis for further research of BMP-6. METHODS: The related articles about the osteogenic mechanism and the osteogenic effects of BMP-6 in experimental animals were extensively summarized. RESULTS: BMP-6 from bone matrix can transduct the osteogenic signal to bone marrow mesenchymal stem cells (BMSCs) by means of Smad protein signal transduction pathway. And the BMSCs which received the signals will differentiate into osteoblasts and chondroblasts. Therefore, BMP-6 plays an important role in the development and maturation of bone and cartilage. In addition, BMP-6 has a close relation with bone diseases, such as fracture, osteoporosis, and bone tumor. CONCLUSION: The deep research of BMP-6 is expected to provide a new therapeutic approach for treating bone diseases of nonunion, osteoarthritis, and osteoporosis.

An in vivo role of bone morphogenetic protein-6 in aldosterone production by rat adrenal gland.

Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex. We previously reported the presence of a functional BMP system including BMP-6 in human adrenocortical cells. BMP-6 contributes to Ang II-induced aldosterone production by activating Smad signaling, in which endogenous BMP-6 action is negatively controlled by Ang II in vitro. In the present study, we examined the in vivo role of BMP-6 in regulation of aldosterone by neutralizing endogenous BMP-6 in rats treated with immunization against BMP-6. Three-week-old male rats were actively immunized with rat mature BMP-6 antigen conjugated with keyhole limpet hemocyanin (KLH). The immunization treatment had no effect on bilateral adrenal weight or its ratio to body weight. Urinary aldosterone excretion was time-dependently increased during the 8-week observation period in the control group. Of note, the level of urinary aldosterone excretion in BMP-6-KLH-immunized rats was significantly reduced compared to that in the control group, suggesting that endogenous BMP-6 contributes to the induction of aldosterone production in vivo. Moreover, the level of urinary aldosterone/creatinine after 8-week treatment was significantly lowered by treatment with BMP-6-KLH. In contrast, with chronic Ang II treatment, urinary aldosterone and creatinine-corrected values at 8 weeks were not significantly different between the two groups, suggesting that the effects of BMP-6-KLH were impaired under the condition of chronic treatment with Ang II. The mRNA levels of Cyp11b2, but not those of Star, P450scc and 3ßhsd2, were significantly decreased in adrenal tissues isolated from BMP-6-KLH-immunized rats after 8-week treatment. Furthermore, the ratio of plasma aldosterone level to corticosterone was significantly decreased by immunization with BMP-6-KLH. Collectively, the results indicate that endogenous BMP-6 is functionally linked to aldosterone synthesis by the zona glomerulosa in the adrenal cortex in vivo.

Improvement of ovarian response and oocyte quality of aged female by administration of bone morphogenetic protein-6 in a mouse model.

BACKGROUND: Advancing female age remains a difficult problem in infertility treatment. Ovarian angiogenesis plays an important role in follicular development and the activation of ovarian angiogenesis has been emerged as a new strategy for the improvement of age-related decline of oocyte quality. BMP-6 affect gonadotropin signals in granulosa cells and it promotes normal fertility by enabling appropriate response to LH and normal oocyte quality. BMP-6 has a potential role in regulation of angiogenesis and regulates the expression of inhibitor of DNA-binding proteins (Ids). Ids involved in the control and timing of follicle selection and granulosa cells differentiation. Especially, Id-1 is well-characterized target of BMP-6 signaling. Therefore, this study investigated whether co-administration of BMP-6 during superovulation process improves ovarian response, oocyte quality and expression of Id-1 and vascular endothelial growth factor (VEGF) in the ovary of aged female using a mouse model. METHODS: Aged C57BL/6 female mice (26-31 weeks old) were superovulated by injection with 0.1 mL of 5 IU equine chorionic gonadotropin (eCG) containing recombinant mouse BMP-6 at various doses (0, 0.01, 0.1, 1, and 10 ng), followed by injection with 5 IU human chorionic gonadotropin (hCG) 48 h later. Then, the mice were immediately paired with an individual male. The aged control group was superovulated without BMP-6. Young mice of 6-9 weeks old were superovulated without BMP-6 as a positive control for superovulation and in vitro culture of embryos. Eighteen hours after hCG injection, zygotes were retrieved and cultured for 4 days. Both ovaries of each mouse were provided in the examination of ovarian expression of Id-1 and VEGF by reverse transcriptase-polymerase chain reaction, western blot, and immunohistochemistry. RESULTS: Administration of 0.1 ng BMP-6 significantly increased the number and blastocyst formation rate of oocytes ovulated and ovarian expression of Id-1 and VEGF compared to aged control mice. These increased levels were comparable to those of young control mice. CONCLUSIONS: This result suggests that BMP-6 during ovulation induction plays an important role in improvement of oocyte quality and ovarian response of aged female, possibly by regulating of ovarian Id-1 and VEGF expression.

Low melting point amphiphilic microspheres for delivery of bone morphogenetic protein-6 and transforming growth factor-ß3 in a hydrogel matrix.

Low melting-point poly(1,3-trimethylene carbonate-co-ε-caprolactone)-b-poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate-co-ε-caprolactone), P(TMC-CL)(2)-PEG, was employed to fabricate microspheres for sustained growth factor delivery in a photocrosslinked N-methacrylate glycol chitosan hydrogel matrix. The P(TMC-CL)(2)-PEG had a melting range such that it was solid at 10°C, yet liquid with a low degree of crystallinity at 37°C. The in vitro degradation of P(TMC-CL)(2)-PEG microspheres was slow, regardless of the triblock copolymer molecular weight and so did not influence protein release. The size of protein loaded P(TMC-CL)(2)-PEG microspheres manufactured using a low-temperature electrospray technique was between 65 and 85µm. Initial formulation work was done with the model protein lysozyme, co-lyophilized with trehalose and encapsulated as approximately 2µm particles within P(TMC-CL)(2)-PEG microspheres. This work indicated a sustained release could be achieved with high trehalose content (90% w/w) in the particles. Under these conditions, the release rate of bone morphogenetic protein-6 was more sustained than that of the excipient bovine serum albumin (BSA) and closely followed that of lysozyme. On the other hand, transforming growth factor-ß3 and the stabilizing agent BSA generated similar release profiles. This difference in release was proposed to be linked to the protein isoelectric point, with positively charged proteins possibly being more strongly adsorbed to the P(TMC-CL)(2)-PEG. Both growth factors were released in highly bioactive form, indicating the potential of the release approach.

Effect of direct ovarian infusion of bone morphogenetic protein 6 (BMP6) on ovarian function in sheep.

Bone morphogenetic protein 6 (BMP6) has been suggested as an important local factor capable of modulating the stimulatory actions of follicle-stimulating hormone in granulosa cells in vitro. The objective of this experiment was to determine the effect of direct ovarian infusion of BMP6 (2 microg/h) on ovarian function in ewes with an autotransplanted ovary. Treated ewes (n = 6) and vehicle-treated controls (n = 6) were infused during the early follicular phase, between 12 and 24 h after luteal regression, and ovarian response was determined by collection of samples of ovarian venous blood and transdermal ultrasound. In the absence of any change in circulating gonadotropins or in the antral follicle population, BMP6 infusion resulted in acute but transient increases in ovarian inhibin A, androstenedione, and estradiol secretion (P < 0.05) during the second half of the infusion period. Thereafter, treated animals had an advance in the time of the LH surge by around 10 h (43.3 +/- 2.8 h in treated vs. 53.3 +/- 2.7 h in controls; P < 0.05) and smaller preovulatory follicles (4.1 +/- 0.2 mm in treated vs. 5.3 +/- 0.1 mm in controls; P < 0.01), which gave rise to smaller corpora lutea (9.5 +/- 0.8 mm in treated vs. 11.7 +/- 0.6 mm in controls; P < 0.05). There was, however, no effect of infusion on ovulation rate. Despite the changes in the size of the ovulatory follicles, when the hormonal data were aligned to the time of the luteinizing hormone surge, there were no differences in preovulatory estradiol, androstenedione, or inhibin A between groups. This study therefore provides strong in vivo evidence to support the hypothesis that BMP6 is an important local regulator of ovarian function and that alterations in BMP6 cellular signaling may explain some of the effects of the FecB mutation in inducing precocious maturation of ovulatory follicles.

Is there a role for bone morphogenetic proteins in osteoporotic fractures?

The central role of bone morphogenetic proteins (BMPs) in the remodelling process of the human skeleton has been identified in numerous experimental and clinical studies. BMPs appear to be key agents in the osteoblastic differentiation of mesenchymal stem cells, and more recent evidence implicates them with the cells of the osteoclastic lineage. BMP-2, BMP-4, BMP-6 and BMP-7 have been studied in the context of osteoporosis and have been associated with its pathophysiological pathways. The theoretical advantages of local or systemic treatment of osteoporotic fractures with BMPs include the potential of inducing a rapid increase in bone strength locally at the fractured area and systemically in the entire skeleton, as well as accelerating the bone-healing period. Animal models of osteoporotic fractures suggested that the induction of new bone by local or systemic use of BMP-7 should be investigated as potential bone augmentation therapy to improve bone quality in symptomatic spinal osteoporosis. As our knowledge expands, new innovations may provide clinicians with advanced biologically-based therapies for the successful treatment of osteoporotic fractures.