Bone Morphogenetic Protein-2 Gene Research Articles

Bacitracin promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by stimulating the bone morphogenetic protein-2/Smad axis

Bacitracin, a widely used metallopeptide antibiotic, has been reported to be locally used in treating wounds without systemic adverse reactions. Our preliminary study showed that bacitracin might enhance the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). The present study investigated whether bacitracin affects the osteogenic differentiation of HBMSCs and the molecular mechanisms involved. The proliferation of HBMSCs in the presence of bacitracin was examined using a cell counting kit-8 (CCK-8) assay. The effects of bacitracin on the cell cycle and apoptosis of HBMSCs were observed using flow cytometry assay. Staining and quantitative assays for alkaline phosphatase (ALP) staining, collagen deposition (Sirius Red), and mineralization (Alizarin Red) were used to study osteogenic differentiation of HBMSCs. The expression of osteogenic differentiation markers was detected using quantitative reverse transcription polymerase chain reaction (RT-qPCR) analyses. The activation of related signaling pathways was examined using a luciferase reporter assay and western blotting. Bacitracin treatment increased osteogenic differentiation of HBMSCs without cytotoxicity and did not adversely affect cell cycle progression or apoptosis. The luciferase reporter assay showed that bacitracin activated the transcription of bone morphogenetic protein-2 (BMP2) gene, a key gene in the BMP2/Smad signaling axis. Western blotting indicated that this axis was markedly activated by bacitracin stimulation of osteogenesis. Moreover, the activation of Smad phosphorylation and osteogenic differentiation by bacitracin was inhibited by a transforming growth factor (TGF)-β/Smad inhibitor (LDN-193189 HCl) and small interfering RNA (siRNA) gene silencing (si-BMP2). In conclusion, our results suggest that bacitracin can promote osteogenesis of HBMSCs by activating the BMP2/Smad signaling axis.

Matrix Gla protein negatively regulates calcification of human aortic valve interstitial cells isolated from calcified aortic valves

Calcified aortic valve stenosis (CAS) is a common heart valve disease in elderly people, and is mostly accompanied by ectopic valve calcification. We recently demonstrated that tumor necrosis factor-α (TNF-α) induces calcification of human aortic valve interstitial cells (HAVICs) obtained from CAS patients. In this study, we investigated the role of matrix Gla protein (MGP), a known calcification inhibitor that antagonizes bone morphogenetic protein 2 (BMP2) in TNF-α-induced calcification of HAVICs. HAVICs isolated from aortic valves were cultured, and calcification was significantly induced with 30 ng/mL TNF-α. Gene expression of the calcigenic marker, BMP2, was significantly increased in response to TNF-α, while the gene and protein expression of MGP was strongly decreased. To confirm the role of MGP, MGP-knockdown HAVICs and HAVICs overexpressing MGP were generated. In HAVICs, in which MGP expression was inhibited by small interfering RNA, calcification and BMP2 gene expression were induced following long-term culture for 32 days in the absence of TNF-α. In contrast, HAVICs overexpressing MGP had significantly decreased TNF-α-induced calcification. These results suggest that MGP acts as a negative regulator of HAVIC calcification, and as such, may be helpful in the development of new therapies for ectopic calcification of the aortic valve.

Histone-targeted gene transfer of bone morphogenetic protein-2 enhances mesenchymal stem cell chondrogenic differentiation

Skeletal tissue regeneration following traumatic injury involves a complex cascade of growth factor signals that direct the differentiation of mesenchymal stem cells (MSCs) within the fracture. The necessity for controlled and localized expression of these factors has highlighted the role gene therapy may play as a promising treatment option for bone repair. However, the design of nanocarrier systems that negotiate efficient intracellular trafficking and nuclear delivery represents a significant challenge. Recent investigations have highlighted the roles histone tail sequences play in directing nuclear delivery and activating DNA transcription. We previously established the ability to recapitulate these natural histone tail activities within non-viral nanocarriers, improving gene transfer and expression by enabling effective navigation to the nucleus via retrograde vesicular trafficking. Herein, we demonstrate that histone-targeting leads to ∼4-fold enhancements in osteogenic bone morphogenetic protein-2 (BMP-2) expression by MSCs over 6 days, as compared with standard polymeric transfection reagents. This improved expression augmented chondrogenesis, an essential first step in fracture healing. Importantly, significant enhancements of cartilage-specific protein expression were triggered by histone-targeted gene transfer, as compared with the response to treatment with equivalent amounts of recombinant BMP-2 protein. In fact, an ∼100-fold increase in recombinant BMP-2 was required to achieve similar levels of chondrogenic gene and protein expression. The enhancements in differentiation achieved using histone-targeting were in part enabled by an increase in transcription factor expression, which functioned to drive MSC chondrogenesis. These novel findings demonstrate the utility of histone-targeted gene transfer strategies to enable substantial reductions in BMP-2 dosing for bone regenerative applications. Statement of significanceThis contribution addresses significant limitations in non-viral gene transfer for bone regenerative applications by exploiting a novel histone-targeting approach for cell-triggered delivery that induces osteogenic BMP-2 expression coincident with the initiation of bone repair. During repair, proliferating MSCs respond to a complex series of growth factor signals that direct their differentiation along cellular lineages essential to mature bone formation. Although these MSCs are ideal targets for enhanced transfection during cellular mitosis, few non-viral delivery approaches exist to enable maximization of this effect. Accordingly, this contribution seeks to utilize our histone-targeted nanocarrier design strategy to stimulate BMP-2 gene transfer in dividing MSCs. This gene-based approach leads to significantly augmented MSC chondrogenesis, an essential first step in bone tissue repair.

BMP2 and VEGF165 transfection to bone marrow stromal stem cells regulate osteogenic potential in vitro.

An exogenous supply of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factors 165 (VEGF165) will synergize to promote bone regeneration in vivo. The aim of this study was to confirm the role of VEGF165 on the osteogenesis potential of bone mesenchymal stem cells (BMSCs) transduced by adenovirus vector containing BMP2 gene in vitro.Rabbit BMSCs were isolated and transfected with various adenovirus vectors: Ad-BMP2-VEGF165 (BMP2+VEGF165 group), Ad-BMP2 (BMP2 group), Ad-VEGF165 (VEGF165 group), and Ad-green fluorescent protein (GFP group). The multiplicity of infection was detected by GFP expression. Expression of BMP2 and VEGF165 was detected by Western blot and ELISA, and the osteogenic biological activity of BMP2 and VEGF165 by osteogenic assay. Meanwhile, the osteogenic biological activity of BMP2 and VEGF165 was evaluated by detection of Col I (collagen type I), OC (osteocalcin), and ALP (alkaline phosphatase) activity using OC staining, ALP activity assay, and real-time PCR assay.Expression of target genes and proteins reached peak values at 5 days and then gradually declined. The OC staining, ALP activity, and real-time PCR assay of ColI, OC, and ALP were all increased in cells transfected with Ad-BMP2-VEGF165, Ad-BMP2, Ad-VEGF165, and Ad-GFP. However, the osteogenic biological activity in cells transfected with Ad-BMP2 was higher compared to cells transfected with other vectors after transfection at 14 and 21 days. We also found that BMP2 +VEGF165 group showed more osteogenic activity effect than the VEGF165 or control group. Furthermore, osteogenic assays in VEGF165 showed that a slightly lower osteogenic effect when compared to controls at 21 days.VEGF165 might be a potent inhibitor of BMSCs differentiation into osteoblasts. The strategies to use BMP2 and VEGF165 in bone regeneration and the molecular mechanism of their interaction require further investigation.

Enhanced Bone Regeneration by Diabetic Cell-Based Adenoviral BMP-2 Gene Therapy in Diabetic Animals.

The application of bone morphogenetic protein 2 (BMP-2) has been extensively investigated to improve diabetes-impaired bone healing; however, the delivery of BMP-2 by gene therapy for bone regeneration has rarely been investigated in diabetic animals. In this study, we aimed to evaluate which cells induce more new bone formation in diabetic animals when cell-based BMP2 gene therapy is applied. For this purpose, we harvested bone marrow stromal cells (BMSCs) twice in the same animal before (non-diabetic BMSCs; nBMSCs) and after diabetes induction (diabetic BMSCs; dBMSCs) using modified bone marrow ablation methods. And then, cells were transduced by adenoviral vectors carrying the BMP2 gene (AdBMP2). In in vitro, AdBMP2-transfected dBMSCs (B2/dBMSCs) produced higher BMP-2 mRNA levels over 48 h, whereas AdBMP2-transfected nBMSCs (B2/nBMSCs) exhibited a transient increase in BMP-2 mRNA followed by a decrease to the baseline level within 48 h. Both B2/dBMSCs and B2/nBMSCs induced secretion of BMP-2 for 3 weeks. However, B2/dBMSC BMP-2 secretion peaked from day 3 to 10, whereas B2/nBMSC BMP-2 secretion peaked from day 1 to 7. The analysis of osteogenic activity revealed that mineralization nodule formation and the expression levels of osteogenic genes were significantly higher in B2/dBMSCs than B2/nBMSCs and were accompanied by upregulation of canonical Wnt/β-catenin and Smad signaling. AdBMP2-transfected autologous cells were implanted into critical-sized calvarial defects in diabetic animals and induced significantly more bone regeneration than non-AdBMP2-transfected cells. In addition, B2/dBMSCs led to significantly more new bone formation than B2/nBMSCs. Thus, BMP2 gene therapy using diabetic cells effectively supported diabetic bone healing and it was related to the enhanced responses to AdBMP2 of dBMSCs.

Comparative expression analyses of bone morphogenetic protein 4 (BMP4) expressions in muscles of tilapia and common carp indicate that BMP4 plays a role in the intermuscular bone distribution in a dose-dependent manner

Intermuscular bones in fish negatively influence both meet processing and attractiveness to consumers. Tilapia (Oreochromis niloticus) and common carp (Cyprinus carpio) are both major farmed fish species globally, but whereas the former does not possess intermuscular bones, the latter does. Therefore, these two species might present a good model to study the genetic control of distribution of intermuscular bones in fish. Bone morphogenetic protein 4 (BMP4) gene is associated with tissue ossification and bone regeneration in mammals, but in fish its role in ossification remains understudied. To study the relationship between BMP4 and bone distribution in fish, we determined the expression of BMP4 in muscle tissues of common carp and tilapia on transcriptional and translational levels. As the gene has been merely predicted in silico from the genome of common carp, we have cloned and characterized it. The gene (GenBank: HQ446455) contains one intron and two exons, which encode a 400-amino acid protein with high homology to other known BMP4 protein sequences. Phylogenetic analysis showed that common carp clustered within the Cypriniformes clade (zebrafish was the closest ortholog) and tilapia within the Percomorpha clade. Using microCT scanning, we confirmed that intermuscular bones could be observed only in common carp (none in tilapia), but only in dorsal and caudal muscles (none in the ventral muscle). Expression levels of BMP4 in the muscles of common carp were in agreement with this observation both on transcriptional (qPCR) and translational (immunohistochemistry) level: higher in dorsal and caudal muscles, and lower in the ventral muscle. In tilapia, expression of BMP4 gene was also detectable in all three muscles, but expression levels in all three muscles were comparable to the one observed in the ventral muscle of carp, i.e., very low. Therefore, among the six studied muscles, the expression of BMP4 was high only in the two that possess intermuscular bones: dorsal and caudal muscles of common carp. The results of this study suggest that BMP4 is likely to play a key role in the determination of intermuscular bone distribution in fish in a dose-dependent manner.

Stem cell-based gene delivery mediated by cationic niosomes for bone regeneration

Bone morphogenetic protein-7(BMP-7) plays a pivotal role in the transformation of mesenchymal stem cells (MSCs) into bone. However, its impact is hampered due to its short half-life. Therefore, gene therapy may be an interesting approach to deliver BMP-7 gene to D1-MSCs. In this manuscript we prepared and characterized niosomes based on cationic lipid 2,3-di(tetradecyloxy)propan-1-amine, combined with polysorbate 80 for gene delivery purposes. Niosomes were characterized and combined initially with pCMS-EGFP reporter plasmid, and later with pUNO1-hBMP-7 plasmid to evaluate osteogenesis differentiation. Additionally, specific blockers of most relevant endocytic pathways were used to evaluate the intracellular disposition of complexes. MSCs transfected with niosomes showed increased growth rate, enhanced alkaline phosphatase activity (ALP) and extracellular matrix deposition which suggested the formation of osteoblast-like cells. We concluded that hBMP-7-transfected MSCs could be considered not only as an effective delivery tool of hBMP-7, but also as proliferating and bone forming cells for bone regeneration.

Two-stage study of lung cancer risk modification by a functional variant in the 3'-untranslated region of SMAD5 based on the bone morphogenetic protein pathway.

Increasing evidence supports a key role for the bone morphogenetic protein (BMP) signaling pathway in lung vasculogenesis and angiogenesis. Genetic variations in BMP genes have been found to be correlated with cancer risk. In particular, the mutation in the 3′-untranslated region of BMPs may significantly affect gene function, leading to cancer susceptibility. The aim of the present study was to determine whether genetic variations in the components of the BMP family are associated with lung cancer risk. A total of 314 tag single-nucleotide polymorphisms were identified in 18 genes, which are considered to either compose or regulate BMPs, and their association with lung cancer risk was evaluated in a two-stage case-control study with 4,680 cases and controls. A consistently significant association of SMAD5 rs12719482 with elevated lung cancer risk was observed in the three types of sources of populations (adjusted additive model in the combined population: Odds ratio=1.32, 95% confidence interval: 1.16–1.51). The lung cancer risk statistically significantly increased with the increasing number of variant alleles of SMAD5 rs12719482 in a dose-dependent pattern (P for trend=4.9×10−5). Consistent evidence was identified for a significant interaction between the rs12719482 and cigarette smoking, performed as either a continuous or discrete variable. These findings indicated that SMAD5 rs12719482 may be a possible candidate marker for susceptibility to lung cancer in the Chinese population.

Biomimetic Functionalized Surfaces and the Induction of Bone Formation.

Tissue engineering still needs to assign the molecular basis of pattern formation, tissue induction, and morphogenesis: What next to morphogens and stem cells? Macroporous biomimetic matrices per se, without the addition of the soluble osteogenic molecular signals of the transforming growth factor-β (TGF-β) supergene family, remarkably initiate the induction of bone formation. Carving geometries within different calcium phosphate-based macroporous bioreactors we show that geometric cues imprinted within the macroporous spaces initiate the spontaneous induction of bone. Concavities biomimetize the remodeling cycle of the primate osteonic bone and are endowed with functionalized smart geometric cues that per se initiate osteoblasts' differentiation with the expression and secretion of osteogenic molecular signals that induce bone as a secondary response. To study the role of calcium ions (Ca++) and osteoclastogenesis, coral-derived calcium carbonate (CC)/hydroxyapatite (HA) bioreactors with limited conversion to HA (7% HA/CC) were preloaded with 500 μg of the L-type voltage gated calcium channel blocker verapamil hydrochloride. Bioreactors were also loaded with 240 μg of the bisphosphonate zoledronate, an osteoclast inhibitor, and implanted in heterotopic sites of the rectus abdominis muscle of Papio ursinus. Bisphosphonate-treated specimens were characterized by a delayed profoundly inhibited induction of tissue patterning with limited induction of bone. Macroporous constructs pretreated with verapamil hydrochloride yielded limited bone formation. Similarly, 125 or 150 μg human Noggin previously adsorbed onto the macroporous bioreactors resulted in minimal bone formation by induction, indirectly showing that the initiation of bone formation is through the bone morphogenetic protein (BMP) pathway. Downregulation of BMP-2 and osteogenic protein-1 (OP-1) with upregulation of Noggin correlated with limited bone induction. Angiogenesis, capillary sprouting, and Ca++ provide chemotactic signals for myoendothelial, myoblastic, and pericytic stem cell differentiation into osteoblastic-like cells expressing the osteogenic soluble molecular signals of the TGF-β supergene family. Secreted gene products are embedded directly onto the substratum within its regulatory concavities. The protected microenvironment of the concavities biomimetizes the phylogenetically ancient repetitive multitested designs and topographies of Nature. Migrating cells onto the primed substratum by osteoclastic nanotopographical geometric inductive modifications convert geometrical cues set by osteoclastogenesis into BMP gene expression pathways that ultimately set into motion the spontaneous induction of bone formation.

Projection Stereolithographic Fabrication of BMP-2 Gene-activated Matrix for Bone Tissue Engineering

Currently, sustained in vivo delivery of active bone morphogenetic protein-2 (BMP-2) protein to responsive target cells, such as bone marrow-derived mesenchymal stem cells (BMSCs), remains challenging. Ex vivo gene transfer method, while efficient, requires additional operation for cell culture and therefore, is not compatible with point-of-care treatment. In this study, two lentiviral gene constructs – (1) Lv-BMP/GFP, containing human BMP-2 and green fluorescent protein (GFP) gene (BMP group); or (2) Lv-GFP, containing GFP gene (GFP group) – were incorporated with human BMSCs into a solution of photocrosslinkable gelatin, which was then subjected to visible light-based projection stereolithographic printing to form a scaffold with desired architectures. Upon in vitro culture, compared to the GFP group, cells from BMP group showed >1,000-fold higher BMP-2 release, and the majority of them stained intensely for alkaline phosphatase activity. Real-time RT-PCR also showed dramatically increased expression of osteogenesis marker genes only in the BMP group. 3.5 months post-implantation into SCID mice, the micro-computed tomography imaging showed detectable mineralized areas only in the BMP group, which was restricted within the scaffolds. Alizarin red staining and immunohistochemistry of GFP and osteocalcin further indicated that the grafted hBMSCs, not host cells, contributed primarily to the newly formed bone.

One-Step Fabrication of Bone Morphogenetic Protein-2 Gene-Activated Porous Poly-L-Lactide Scaffold for Bone Induction

Bone morphogenetic protein 2 (BMP2) is an efficacious inducer for the osteogenesis of mesenchymal stem cells (MSCs). Conventional applications of BMP2 have involved either the direct incorporation of BMP2 protein or ex vivo BMP2 gene transfer into stem cells prior to their transplantation. These approaches are able to promote bone formation to some extent; however, they are hampered by either the lack of stability and sustainability of BMP2 protein or the time-consuming and cost-prohibitive in vitro cell culture procedure. To overcome these limitations, we have developed a gene-activated poly-L-lactide acid (PLLA) scaffold with the encapsulation of recombinant adeno-associated viral (AAV) vector encoding a full-length cDNA of human BMP2 using an ice-based microparticle porogenization method that was recently developed. Results showed continuous release of AAV particles from the micropores of scaffolds for up to 1 week, subsequently transducing embedded human MSCs and producing functional BMP2. MSCs within scaffolds underwent efficacious osteogenesis, on the basis of osteoinductive gene expression and osteogenic differentiation, which resulted in robust new bone formation in vivo at 4 weeks. These findings show the potential of the technology toward developing clinical applications of a rapid, cost-effective, and potentially point-of-care approach for the repair of bone defects.

Effect of Resveratrol on Critical-Sized Calvarial Defects of Diabetic Rats: Histometric and Gene Expression Analysis

This study investigated the influence of resveratrol (RESV) on the repair of bone critical defects in calvaria of animals with induced diabetes mellitus (DM). One hundred rats were divided into 5 groups: induced DM+RESV administration (DM+RESV; n=20); induced DM plus placebo solution administration (DM+PLAC; n=20); induced DM plus insulin therapy (DM+INS; n=20); induced DM plus administration of RES and INS (DM+RESV+INS; n=20); and nondiabetic controls (NDM; n=20). DM was induced by intraperitoneal injection of streptozotocin 50mg/kg 3days before the surgical procedures. Two critical calvarial defects were created in each animal at the start of the study (day 0). Treatments were administered from day 0 to day 30 of the experiment, when animals were euthanized. One defect was processed for histometric analysis to measure closure of the bone defect. The tissue of the other defect was analyzed for quantification of bone morphogenetic protein-2 (BMP-2), osteopontin, osteoprotegerin, receptor activator of nuclear factor-κB ligand, runt-related transcription factor-2, osterix (Osx), β-catenin, lipoprotein receptor-related protein-5, and dikkop-1 mRNA by quantitative polymerase chain reaction. Histometric results showed that the DM+RESV, DM+RESV+INS, and NDM groups exhibited greater closure of the bone defects compared with the PLAC- or INS-treated groups (P<.05). Diabetic animals treated with RESV plus INS showed higher levels of BMP-2 and Osx; Osx also was positively increased in animals treated with INS alone (P<.05). The use of RESV, regardless of the presence of INS, positively influenced bone repair in animals with induced DM. Further, the combination of INS plus RESV was necessary for the modulation of BMP-2 gene expression.

BMP4 Gene Therapy in Mature Mice Reduces BAT Activation but Protects from Obesity by Browning Subcutaneous Adipose Tissue

We examined the effect of Bone Morphogenetic Protein 4 (BMP4) on energy expenditure in adult mature mice by targeting the liver with adeno-associated viral (AAV) BMP4 vectors to increase circulating levels. We verified the direct effect of BMP4 in inducing a brown oxidative phenotype in differentiating preadipocytes invitro. AAV-BMP4-treated mice display marked browning of subcutaneous adipocytes, with increased mitochondria and Uncoupling Protein 1 (UCP1). These mice are protected from obesity on a high-fat diet and have increased whole-body energy expenditure, improved insulin sensitivity, reduced liver fat, and reduced adipose tissue inflammation. On a control diet, they show unchanged body weight but improved insulin sensitivity. In contrast, AAV-BMP4-treated mice showed beiging of BAT with reduced UCP1, increased lipids, and reduced hormone-sensitive lipase (HSL). Thus, BMP4 exerts different effects on WAT and BAT, but the overall effect is to enhance insulin sensitivity and whole-body energy expenditure by browning subcutaneous adipose tissue.

Genome wide identification, phylogeny, and expression of bone morphogenetic protein genes in tetraploidized common carp (Cyprinus carpio)

Bone morphogenetic proteins (Bmps) are a group of signaling molecules known to play important roles during formation and maintenance of various organs, not only bone, but also muscle, blood and so on. Common carp (Cyprinus carpio) is one of the most intensively studied fish due to its economic and environmental importance. Besides, common carp has encountered an additional round of whole genome duplication (WGD) compared with many closely related diploid teleost, which make it one of the most important models for genome evolutionary studies in teleost. Comprehensive genome resources of common carp have been developed recently, which facilitate the thorough characterization of bmp gene family in the tetraploidized common carp genome. We identified a total of 44 bmps from the common carp genome, which are twice as many as that of zebrafish. Phylogenetic analysis revealed that most of bmps are highly conserved. Comparative analysis was performed across six typical vertebrate genomes. It appeared that all the bmp genes in common carp were duplicated. Obviously, the expansion of the bmp gene family in common carp was due to the latest additional round of whole genome duplication and made it more abundant than other diploid teleosts. Expression signatures were assessed in major tissues, including gill, intestine, liver, spleen, skin, heart, gonad, muscle, kidney, head kidney, brain and blood, which demonstrated the comprehensive expression profiles of bmp genes in the tetraploidized genome. Significant gene expression divergences were observed which revealed substantial functional divergences of those duplicated bmp genes post the latest WGD event. The conserved synteny blocks of bmp5s revealed the genome rearrangement of common carp post the 4R WGD. The whole set of bmp gene family in common carp provides insight into gene fate of tetraploidized common carp genome post recent WGD.

A doxycycline inducible, adenoviral bone morphogenetic protein-2 gene delivery system to bone.

We report the novel use of a tuneable, non-integrating viral gene delivery system to bone that can be combined with clinically approved biomaterials in an 'off-the-shelf' manner. Specifically, a doxycycline inducible Tet-on adenoviral vector (AdTetBMP-2) in combination with mesenchymal stromal cells (MSCs), fibrin and a biphasic calcium phosphate ceramic (MBCP®) was used to repair large bone defects in nude rats. Bone morphogenetic protein-2 (BMP-2) transgene expression could be effectively tuned by modification of the doxycycline concentration. The effect of adenoviral BMP-2 gene delivery upon bone healing was investigated in vivo in 4mm critically sized, internally fixated, femoral defects. MSCs were transduced either by direct application of AdTetBMP-2 or by pre-coating MBCP granules with the virus. Radiological assessment scores post-mortem were significantly improved upon delivery of AdTetBMP-2. In AdTetBMP-2 groups, histological analysis revealed significantly more newly formed bone at the defect site compared with controls. Newly formed bone was vascularized and fully integrated with nascent tissue and implanted biomaterial. Improvement in healing outcome was achieved using both methods of vector delivery (direct application vs. pre-coating MCBP). Adenoviral delivery of BMP-2 enhanced bone regeneration achieved by the transplantation of MSCs, fibrin and MBCP in vivo. Importantly, our in vitro and in vivo data suggest that this can be achieved with relatively low (ng/ml) levels of the growth factor. Our model and novel gene delivery system may provide a powerful standardized tool for the optimization of growth factor delivery and release for the healing of large bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs.

More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

Determination of cell fate in skeletal muscle following BMP gene transfer by in vivo electroporation.

We previously developed a novel method for gene transfer, which combined a non-viral gene expression vector with transcutaneous in vivo electroporation. We applied this method to transfer the bone morphogenetic protein (BMP) gene and induce ectopic bone formation in rat skeletal muscles. At present, it remains unclear which types of cells can differentiate into osteogenic cells after BMP gene transfer by in vivo electroporation. Two types of stem cells in skeletal muscle can differentiate into osteogenic cells: muscle-derived stem cells, and bone marrow-derived stem cells in the blood. In the present study, we transferred the BMP gene into rat skeletal muscles. We then stained tissues for several muscle-derived stem cell markers (e.g., Pax7, M-cadherin), muscle regenerationrelated markers (e.g., Myod1, myogenin), and an inflammatory cell marker (CD68) to follow cell differentiation over time. Our results indicate that, in the absence of BMP, the cell population undergoes muscle regeneration, whereas in its presence, it can differentiate into osteogenic cells. Commitment towards either muscle regeneration or induction of ectopic bone formation appears to occur five to seven days after BMP gene transfer.

Icaritin, a novel plant-derived osteoinductive agent, enhances the osteogenic differentiation of human bone marrow- and human adipose tissue-derived mesenchymal stem cells.

For the treatment of diseases affecting bones using bone regenerative medicine, there is an urgent need to develop safe, inexpensive drugs that can strongly induce bone formation. In the present study, we systematically investigated the effects of icaritin, a metabolic product of icariin, on the osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells (hBMSCs) and human adipose tissue‑derived stem cells (hADSCs) invitro. After treatment with icaritin at concentrations of 10‑8-10‑5M, hBMSCs and hADSCs were examined for alkaline phosphatase activity, osteocalcin (OC) secretion, matrix mineralization and expression levels of bone‑related mRNA and proteins. Data showed that icaritin at concentrations 10‑7-10‑5M significantly increased alkaline phosphatase activity, OC secretion at different time points, and calcium deposition at day21. In addition, icaritin upregulated the mRNA expression of genes for bone morphogenetic proteins (BMP‑2,‑4and‑7), bone transcription factors (Runx2 and Dlx5) and bone matrix proteins (ALP, OC and Col‑1). Moreover, icaritin increased the protein levels of BMPs, Runx2 and OC, as detected by western blot analysis. These findings suggest that icaritin enhances the osteogenic differentiation of hBMSCS and hADSCs. Icaritin exerts its potent osteogenic effect possibly by directly stimulating the production of BMPs. Although the osteogenic activity of icaritin invitro was inferior to that of rhBMP‑2, icaritin displayed better results than icariin. Moreover, the low cost, simple extraction procedure, and an abundance of icaritin make it appealing as a bone regenerative medicine.

The Effects of Tocotrienol and Lovastatin Co-Supplementation on Bone Dynamic Histomorphometry and Bone Morphogenetic Protein-2 Expression in Rats with Estrogen Deficiency.

Both tocotrienol and statins are suppressors of the mevalonate pathway. Supplementation of tocotrienol among statin users could potentially protect them against osteoporosis. This study aimed to compare the effects of tocotrienol and lovastatin co-supplementation with individual treatments on bone dynamic histomorphometric indices and bone morphogenetic protein-2 (BMP-2) gene expression in ovariectomized rats. Forty-eight female Sprague-Dawley rats were randomized equally into six groups. The baseline was sacrificed upon receipt. All other groups were ovariectomized, except for the sham group. The ovariectomized groups were administered orally daily with (1) lovastatin 11 mg/kg/day alone; (2) tocotrienol derived from annatto bean (annatto tocotrienol) 60 mg/kg/day alone; (3) lovastatin 11 mg/kg/day, and annatto tocotrienol 60 mg/kg/day. The sham and ovariectomized control groups were treated with equal volume of vehicle. After eight weeks of treatment, the rats were sacrificed. Their bones were harvested for bone dynamic histomorphometry and BMP-2 gene expression. Rats supplemented with annatto tocotrienol and lovastatin concurrently demonstrated significantly lower single-labeled surface, but increased double-labeled surface, mineralizing surface, mineral apposition rate and bone formation rate compared to individual treatments (p < 0.05). There was a parallel increase in BMP-2 gene expression in the rats receiving combined treatment (p < 0.05). The combination of annatto tocotrienol and lovastatin exerted either additively or synergistically on selected bone parameters. In conclusion, tocotrienol can augment the bone formation and mineralization in rats receiving low-dose statins. Supplementation of tocotrienol in statin users can potentially protect them from osteoporosis.

Carboxymethylcellulose with phenolic hydroxyl microcapsules enclosinggene-modified BMSCs for controlled BMP-2 release in vitro

Objectives: The present study aimed to develop microparticles of phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) via Co-flow microfluidics technology and encapsulated gene-modified rat bone mesenchymal stem cells (BMSCs) for the detection of the growth factor release was controlled by Tet-on system. Meanwhile, we investigated the effect of the CMC-Ph microcapsules and Lentiviral transduction on osteogenesis of BMP2–BMSCs.Methods: The middle size of CMC-Ph microcapsules was prepared by optimized co-flow microfluidics through ejecting fluid CMC-Ph suspension (mixed with HRP) into co-flowing liquid paraffin which blends H2O2 at priority. The Lentivirus-encoding hBMP-2 and Tet-On system were constructed and amplified by RT-PCR, then encapsulated in the microcapsules. The cellular viability of CMC-Ph microparticles was assessed by Live/dead staining and metabolic activity was estimated by colorimetric assay kit. In addition, BMP-2 secretion and kinetic studies were determined by ELISA, alkaline phosphatase (ALP) activity was evaluated using ALP assay kit, and ALP staining as well as mineral calcium deposition was detected by alizarin red S staining.Key findings: The diameter of CMC-Ph microparticles was controlled between 100 and 150 μm by altering the flow speed of liquid paraffin and then encapsulated bone morphogenetic protein 2 (BMP-2) gene modified BMSCs transduced by a lentiviral vector. Moreover, the mitochondrial activity of the encapsulated cells was maintained at least 24 d and BMP-2 protein secretion into the supernatant sustained for 35 d without significant loss of efficiency under the induction of the doxycycline. Furthermore, mineral deposition staining and ALP activity detection showed that encapsulated lentiviral-BMP2 transduced BMSCs possess more osteogenic differentiation potential than normal cells.Conclusions: Co-flow microfluidics and phenolic hydroxyl derivative of carboxymethylcellulose (CMC-Ph) provide a promising strategy for cell-enclosed microcapsules in combination with BMP-2 gene and Tet-on system modified BMSCs and then controlled BMP-2 protein released effectively as well as promoted the osteogenic differentiation of BMSCs.