Transforming growth factor β isoforms (TGF-β) are among the most recently evolved members of a signaling superfamily with more than 30 members. TGF-β play vital roles in regulating cellular growth and differentiation, and they signal through a highly restricted subset of receptors known as TGF-β type I receptor (TβR-I) and TGF-β type II receptor (TβR-II). TGF-β's specificity for TβR-I has been proposed to arise from its pre-helix extension, a five-residue loop that binds in the cleft between TGF-β and TβR-II. The structure and back<em>bone</em> dynamics of the unbound form of the TβR-I extracellular domain were determined using NMR to investigate the extension's role in binding. This showed that the unbound form is highly similar to the bound form in terms of both the β-strand framework that defines the three-finger toxin fold and the extension and its characteristic cis-Ile54-Pro55 peptide bond. The NMR data further showed that the extension and two flanking 3(<em>10</em>) helices are rigid on the nanosecond-to-picosecond timescale. The functional significance of several residues within the extension was investigated by binding studies and reporter gene assays in cultured epithelial cells. These demonstrated that the pre-helix extension is essential for binding, with Pro55 and Pro59 each playing a major role. These findings suggest that the pre-helix extension and its flanking prolines evolved to endow the TGF-β signaling complex with its unique specificity, departing from the ancestral promiscuity of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> subfamily, where the binding interface of the type I receptor is highly flexible.

Periodontal ligament stem cells (PDLSCs) are considered as potential mesenchymal stem cell sources for future clinical applications in periodontal regeneration therapy. Simvastation, widely used for lowering serum cholesterol, is known to have a <em>bone</em> stimulatory effect. However, it is not clear whether simvastation affects the differentiation of PDLSCs. This study examined the effects of simvastatin on human PDLSCs in vitro and in vivo. Using the limiting dilution technique, human PDLSCs were isolated and expanded. PDLSCs were cultured with simvastatin (0.01-<em>10</em> μM), and the proliferation was measured. The osteogenic differentiation was characterized by alkaline phosphatase (ALP) activity and Alizarin Red-S staining for calcium deposition. The gene expression levels of osteogenic markers were evaluated by RT-PCR. In addition, PDLSCs were transplanted into nude mice with ceramic bovine <em>bone</em> powders as carriers to observe the capacity of mineralized tissue formation in vivo. Simvastatin at concentrations <1 μM did not suppress the proliferation of PDLSCs. After the administration of 0.1 μM simvastatin, the expression of ALP, <em>bone</em> sialo<em>protein</em>, and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 genes were significantly upregulated, and the ALP activity and mineralized nodule formation were significantly higher in the simvastatin-treated cells than the control cells. In addition, the in vivo transplantation results showed that simvastatin treatment promoted the degree of mineralized tissue formation. Collectively, simvastatin has positive effects on osteogenic differentiation of human PDLSCs in vitro and in vivo. This suggests that simvastatin might be a useful osteogenic induction agent for periodontal <em>bone</em> regeneration.

Good bioactivity and osteogenesis of three-dimensional porous alginate scaffolds (PAS) are critical for <em>bone</em> tissue engineering. In this work, alginate and <em>bone</em>-forming peptide-1 (BFP-1), derived from <em>bone</em> <em>morphogenetic</em> <em>protein</em>-7 (BMP-7), have been combined together (without carbodiimide chemistry treatment) to develop peptide-incorporated PAS (p-PAS) for promoting <em>bone</em> repairing ability. The mechanical properties and SEM images show no difference between pure PAS and p-PAS. The release kinetics of the labeled peptide with 6-carboxy tetramethyl rhodamine from the PAS matrix suggests that the peptide is released in a relatively sustained manner. In the cell experiment, p-PAS show higher cell adhesion, spreading, proliferation and alkaline phosphatase (ALP) activity than the pristine PAS group, indicating that the BFP-1 released from p-PAS could significantly promote the aggregation and differentiation of osteoblasts, especially at <em>10</em>μg/mL of trapped peptide concentration (p-PAS-<em>10</em>). Furthermore, p-PAS-<em>10</em> was implanted into Beagle calvarial defects and <em>bone</em> regeneration was analyzed after 4 weeks. New <em>bone</em> formation was assessed by calcein and Masson's trichrome staining. The data reveal that p-PAS group exhibits significantly enhanced oseto-regenerative capability in vivo. The peptide-modified PAS with promoted bioactivity and osteogenic differentiation in vitro as well as <em>bone</em> formation ability in vivo could be promising tissue engineering materials for repairing and regeneration of <em>bone</em> defects.

<AbstractText>Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease with high heritability. Although several predisposing genes have been linked to IPAH, the genetic aetiology remains unknown for a large number of IPAH cases.</AbstractText><AbstractText>We conducted an exome-wide gene-based burden analysis on two independent case-control studies, including a total of 331 IPAH cases and <em>10</em> 508 controls. Functional assessments were conducted to analyse the effects of genetic mutations on <em>protein</em> biosynthesis and function.</AbstractText><p><div><b>RESULTS</b></div>The gene encoding human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 9 (<i>BMP9</i>) was identified as a novel genetic locus displaying exome-wide association with IPAH in the discovery cohort (OR 18.8; p=1.9×<em>10</em>^-11). This association was authenticated in the independent replication cohort (p=1.0×<em>10</em>^-5). Collectively, the rare coding mutations in <i>BMP9</i> occurred in 6.7% of cases, ranking this gene second to <i>BMPR2</i>, comprising a combined significance of 2.7×<em>10</em>^-19 (OR 21.2). Intriguingly, the patients with <i>BMP9</i> mutations had lower plasma levels of BMP9 than those without. Functional studies showed that the <i>BMP9</i> mutations led to reduced BMP9 secretion and impaired anti-apoptosis ability in pulmonary arterial endothelial cells.</p><p><div><b>CONCLUSION</b></div>We identify <i>BMP9</i> as an IPAH culprit gene.</p>

The <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) pathway is essential for the morphogenesis of multiple organs in the digestive system. Abnormal BMP signaling has also been associated with disease initiation and progression in the gastrointestinal (GI) tract and associated organs. Recent studies using animal models, tissue organoids, and human pluripotent stem cells have significantly expanded our understanding of the roles played by BMPs in the development and homeostasis of GI organs. It is clear that BMP signaling regulates GI function and disease progression that involve stem/progenitor cells and inflammation in a tissue-specific manner. In this review we discuss these new findings with a focus on the esophagus, stomach, and intestine. Expected final online publication date for the <i>Annual Review of Physiology</i>, Volume 82 is February <em>10</em>, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Demineralized <em>bone</em> matrix (DBM) reproducibly induces extraskeletal <em>bone</em> formation in rodents, but its effects in dogs and primates are negative or uncertain. In previous studies on the squirrel monkey, DBM did not induce <em>bone</em>, although the same implants were effective in nude rats. In the present study, the DBM was augmented with recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2). <em>Bone</em> was formed in <em>10</em> of 12 monkeys, as verified by histology and calcium content. However, in 4 monkeys, the induced <em>bone</em> mass appeared smaller than the original implant. DBM controls induced microscopic amounts of <em>bone</em> in 2 out of <em>10</em> monkeys. In the nude rats, all DBM controls and augmented implants induced <em>bone</em>. The difficulties in achieving <em>bone</em> induction in higher animals may be overcome, at least partially, by using a higher concentration of the inductive <em>protein</em> than is present in DBM.

Previous studies have suggested that <em>bone</em> <em>morphogenetic</em> <em>protein</em>-6 (BMP-6) has a pronounced upregulation in rat brains subjected to traumatic brain injury. <em>Bone</em> <em>morphogenetic</em> <em>protein</em>-<em>10</em> (BMP-<em>10</em>) is a newly identified cardiac-specific peptide growth factor that belongs to the TGF-β superfamily. To elucidate the dynamic expression changes and cellular localization of BMP-<em>10</em> during traumatic brain injury (TBI), we performed an acute traumatic brain injury model in adult rats. Western blot analysis, immunohistochemistry and RTPCR revealed that BMP-<em>10</em> expression in impaired cerebral cortex was more strongly induced not only at <em>protein</em> level but also at mRNA level compared to that in normal group. Double immunofluorescence labeling suggested that BMP-<em>10</em> was localized mainly in the cytoplasm of neurons, microglias, and astrocytes within 3 mm from the lesion site at day 3 post-injury. And there was a specific upregulation of BMP-<em>10</em> in astrocytes following brain injury. Besides, co-localization of BMP-<em>10</em> and proliferating cell nuclear antigen (PCNA) was detected in Glial fibrillary acidic <em>protein</em> (GFAP) (+) cells. We also examined the expression profiles of PCNA and GFAP whose change was correlated with the expression profiles of BMP-<em>10</em> in the incised injury model used here. Another experiment in which astrocytes were treated with BMP-<em>10</em> was also performed to confirm the relationship between the upregulation of BMP-<em>10</em> and proliferation of astrocytes following TBI. Taken together, this is the first description of BMP-<em>10</em> expression during the central nervous system (CNS) lesion and repair. Thus, the present data suggested that BMP-<em>10</em> may be implicated in CNS pathophysiology after TBI. But, further studies are needed to understand the cell signal pathway which can direct the exact role of BMP-<em>10</em> following traumatic brain injury.

According to 5- and <em>10</em>-year clinical follow-up studies large-scale <em>bone</em> allografts have a high failure rate, largely due to poor allograft incorporation with adjacent <em>bone</em> and subsequent poor remodeling. The goal of this study was to develop a methodology to deliver growth factors from large-scale <em>bone</em> allografts in a temporally controlled manner. Intact long <em>bone</em> allografts were coated with a micron-scale thick layer of degradable polymer that maintained inherent pore structures and acted as a delivery vehicle for <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 and vascular endothelial growth factor. VEGF was loaded onto the surface of the polymer to produce rapid release, to encourage initial vascularization at the defect site, while BMP-2 was encapsulated within the polymer layer to promote a more sustained release, to encourage <em>bone</em> formation over time. Release kinetics from factor-loaded polymer-coated allografts show an early burst release of VEGF over the first 7 days followed by a more sustained release of BMP-2 over the second and third week. In vitro cell studies using human mesenchymal stem cells confirm the bioactivity of the released BMP-2. In-vivo results show robust <em>bone</em> formation over the first 8 weeks of healing in femoral segmental defects in rats implanted with BMP-2 loaded polymer-coated allografts. A microscale thin coating of degradable polymer on a large-scale <em>bone</em> allograft provides temporal control over the delivery of growth factor loaded onto one allograft, while maintaining its microscale pore structure. Enhancing the incorporation and subsequent remodeling of allografts would reduce the incidence of allograft failure over time, and potentially speed healing at the earliest stages after implantation.

Mutations in the Endoglin (Eng) gene, an auxiliary receptor in the transforming growth factor beta (TGFβ)-superfamily signaling pathway, are responsible for the human vascular disorder hereditary hemorrhagic telangiectasia (HHT) type 1, characterized in part by blood vessel enlargement. A growing body of work has uncovered an autonomous role for Eng in endothelial cells. We will highlight the influence of Eng on distinct cellular behaviors, such as migration and shape control, which are ultimately important for the assignment of proper blood vessel diameters.

How endothelial cells establish hierarchically ordered blood vessel trees is one of the outstanding questions in vascular biology. Mutations in components of the TGFβ-superfamily of signaling molecules disrupt this patterning and cause arteriovenous malformations (AVMs). Eng is a TGFβ coreceptor enhancing signaling through the type I receptor Alk1. Recent studies identified <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) 9 and <em>10</em> as the primary ligands for Alk1/Eng. Importantly, Eng potentiated Alk1 pathway activation downstream of hemodynamic forces. New results furthermore revealed how Eng affects endothelial cell migration and cell shape control in response to these forces, thereby providing new avenues for our understanding of AVM cause.

We will discuss the interplay of Eng and hemodynamic forces, such as shear stress, in relation to Alk1 receptor activation. We will furthermore detail how this signaling pathway influences endothelial cell behaviors important for the establishment of hierarchically ordered blood vessel trees. Finally, we will provide an outlook how these insights might help in developing new therapies for the treatment of HHT.

Scaffold materials for <em>bone</em> tissue engineering often are supplemented with <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs). In the current study we aimed to investigate COLLOSS, a bovine extracellular matrix product containing native BMPs. Hollow cylindrical implants were made, with a length of <em>10</em> mm, a 3 mm inner diameter, and a 5 mm outer diameter, from titanium fibre mesh. The central space of the tube was filled with 20 mg COLLOSS. Subsequently, these implants, as well as non-loaded controls, were implanted subcutaneously into the back of Wistar rats, with n=6 for all study groups. After implantation periods of 2, 8, and 12 weeks, tissue-covered implants were retrieved, and sections were made, perpendicular to the long axis of the tube. Histology showed, that all implants were surrounded by a thin fibrous tissue capsule. After 2 weeks of implantation, the COLLOSS material was reduced in size inside the loaded implants, but no <em>bone</em>-like tissue formation was evident. After 8 weeks, in two out of six loaded specimens, new-formed <em>bone</em>- and <em>bone</em> marrow-like tissues could be observed. After 12 weeks, this had increased to five out of six COLLOSS-loaded samples. The amount of <em>bone</em>-like tissue did not differ between 8 and 12 weeks, and on average occupied 15% of the central space of the tube. In the non-loaded control samples, only connective tissue ingrowth was observed. In conclusion, we can say that COLLOSS material loaded in a titanium fibre mesh tube, showed <em>bone</em>-inducing properties. The final efficacy of these osteo-inductive properties has to be confirmed in future large animal studies.

BACKGROUND

Bone morphogenetic protein-2 (BMP-2) has high potential for bone formation, but its in vivo effects are unpredictable due to the short life time. This study was designed to evaluate the effects of recombinant human (rh) BMP-2 suspended in fibrin on bone formation during distraction osteogenesis (DO) in rabbits.

METHODS

The in vitro release kinetics of rhBMP-2 suspended in fibrin was tested using an enzyme-linked immunosorbent assay. Unilateral tibial lengthening for 10 mm was achieved in 48 rabbits. At the completion of osteodistraction, vehicle, fibrin, rhBMP-2 or rhBMP-2 suspended in fibrin (rhBMP-2 + fibrin) was injected into the center of the lengthened gap, with 12 animals in each group. Eight weeks later, the distracted callus was examined by histology, micro-CT and biomechanical testing. Radiographs of the distracted tibiae were taken at both 4 and 8 weeks after drug treatment.

RESULTS

It was found that fibrin prolonged the life span of rhBMP-2 in vitro with sustained release during 17 days. The rhBMP-2 + fibrin treated animals showed the best results in bone mineral density, bone volume fraction, cortical bone thickness by micro-CT evaluation and mechanical properties by the three-point bending test when compared to the other groups (p < 0.05). In histological images, rhBMP-2 + fibrin treatment showed increased callus formation and better gap bridging compared to the other groups.

CONCLUSIONS

The results of this study suggest that fibrin holds promise to be a good carrier of rhBMP-2, and rhBMP-2 suspended in fibrin showed a stronger promoting effect on bone formation during DO in rabbits.

Pulmonary arterial hypertension (PAH) is usually a devastating condition with a poor prognosis. Nearly <em>10</em> years ago, the underlying molecular basis of heritable PAH was elucidated with the identification of mutations in the gene encoding the <em>bone</em> <em>morphogenetic</em> <em>protein</em> type II receptor (BMPR-II). This discovery is now beginning to suggest novel approaches to therapy in heritable PAH. Moreover, recent studies provide evidence that dysfunction of the BMPR-II pathway is a feature of non-familial forms of PAH, broadening the scope for intervention in this pathway.

Clinically, it would be more convenient to use recombinant than purified preparations of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP). Recently, recombinant human BMP (rhBMP) has attracted the attention of many investigators, but it has not been fully characterized. We examined the <em>bone</em>-inducing activity of rhBMP-2 and compared it with that of purified BMP derived from human <em>bone</em> matrix (phBMP). Two, <em>10</em>, or 50 microg of rhBMP-2 or phBMP was mixed with 3 mg of atelopeptide type I collagen (carrier), and specimens were implanted in the calf muscles of Wistar rats (n=5 in each group). Four weeks later, new <em>bone</em> had formed in all the rhBMP-2- and phBMP-implanted muscles and was visible radiographically and histologically. The quantitative analysis indicated that the activity of rhBMP-2 was less than one tenth that of phBMP. It is necessary to find out why rhBMP-2 has fewer activities than phBMP.

BACKGROUND

The posterior cruciate ligament heals to some extent after injury. However, results after conservative treatment may diminish with long-term follow-up. Bone morphogenetic protein-12 can induce formation of ligament tissues.

OBJECTIVE

Bone morphogenetic protein-12 gene transfer can improve the histologic and biomechanical properties of healing posterior cruciate ligaments.

METHODS

Controlled laboratory study.

METHODS

Bilateral posterior cruciate ligaments of 32 rabbits were injured. The cut ends in 1 limb received an injection containing 3 x 10(7) pfu recombinant bone morphogenetic protein-12 adenovirus, and the posterior cruciate ligament in the contralateral limb served as an untreated control. Eight rabbits were sacrificed at each time point of 3, 6, 12, and 26 weeks after the operation. In addition, 6 rabbits receiving a sham operation were used to obtain normal control data. The posterior cruciate ligament specimens were evaluated biomechanically and histologically.

RESULTS

The repair tissue of the treatment group at 26 weeks was similar to the normal posterior cruciate ligament in collagen arrangement, collagen formation, and mechanical properties. At weeks 6, 12, and 26, the ultimate load, stiffness, and energy absorbed at failure of the treatment group were significantly greater than those of the untreated group.

CONCLUSIONS

Adenovirus-mediated bone morphogenetic protein-12 gene transfer in a partial posterior cruciate ligament laceration rabbit model resulted in an obvious improvement of histologic properties, tensile strength, and stiffness of the repaired ligaments, indicating improved posterior cruciate ligament healing.

CONCLUSIONS

Bone morphogenetic protein-12 gene transfer is a potential future strategy to improve the repair of injured posterior cruciate ligaments.

Our previous studies have demonstrated that slow release of basic fibroblast growth factor (b-FGF) or <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) induces cartilage regeneration. In the present study, we investigated whether synchronous slow release of b-FGF and BMP-2 would induce more widespread and earlier cartilage regeneration than that induced by each growth factor alone. A 1-cm defect was made in the mid-ventral portion of each of <em>10</em> consecutive tracheal rings. In four controls, the defect was left untreated. In the gelatin group (n = 4), empty gelatin sponge was implanted. In the b-FGF + BMP-2 group (n = 5), two gelatin sponges containing <em>10</em>0 microg of b-FGF or BMP-2 solution were implanted. After various periods, we euthanatized the dogs, and examined the implant sites. In the b-FGF + BMP-2 group, regenerated fibrous cartilage connected the host cartilage stumps and completely filled the defect between them at 1, 2, 3, and 12 months. Regenerated cartilage was covered by regenerated perichondrium originating from the host perichondrium, and showed neovascularization in the extracellular matrix. We succeeded in inducing more widespread and earlier cartilage regeneration using synchronous slow release of b-FGF and BMP-2 than that induced by release of each growth factor alone.

The ability of a pool of bovine <em>bone</em> <em>morphogenetic</em> <em>proteins</em> bound to synthetic microgranular hydroxyapatite (BMPb-HA) to stimulate <em>bone</em> repair was determined in rat critical size defects. An 8-mm diameter defect was created in the calvaria of 25 rats. In 15 rats, the defects were filled with BMPb-HA homogenized with blood (experimental group), and in <em>10</em> rats the defects were filled only with blood clots (control). The calvariae of experimental rats were collected 1, 3 and 6 months after surgery and of the control rats at the end of surgery and 6 months thereafter. The morphometric results obtained in the radiographs showed an absence of new <em>bone</em> formation at 1 and 3 months post-surgery and, histologically, the defects were filled with fibrous connective tissue and numerous foci of a foreign body-type granulomatous reaction around hydroxyapatite agglomerates. At the end of 6 months, the number and size of the granulomatous foci decreased and the area of the defects was reduced by 22% compared to the 0-hour control due to the formation of new <em>bone</em> at their borders, although the mean area was similar to the 6-month control. We conclude that the use of BMPb-HA in the treatment of critical size <em>bone</em> defects of the rat skull leads to the formation of a foreign body-type granulomatous reaction that markedly inhibits new <em>bone</em> formation, suggesting that synthetic microgranular hydroxyapatite does not represent a good carrier for BMP-induced <em>bone</em> formation.

It is well known that parathyroid hormone (PTH) possesses an anabolic effect on <em>bone</em>. However, the mechanisms are not fully elucidated. So far, it is unclear whether or not PTH could stimulate the expression of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2), a strong mediator for <em>bone</em> formation. Growing evidence suggests that BMP-2 expression is regulated by the mevalonate pathway and Rho-associated <em>protein</em> kinase (ROK) activity. This study was performed to examine if PTH affects BMP-2 expression and to clarify its involvement of the mevalonate pathway. Osteoblastic MC3T3-E1 cells were treated with human PTH-(1-34) to determine BMP-2 mRNA expression levels by real-time PCR and to measure the ROK activity by the kinase assay. Incubation with <em>10</em> (-9)-<em>10</em> (-8) M of hPTH-(1-34) for 6 h induced significant upregulation of BMP-2 mRNA levels in MC3T3-E1 cells. Short-term treatment of hPTH-(1-34) suppressed Rho kinase activity and mevalonate kinase mRNA levels. PTH-induced BMP-2 mRNA upregulation was selectively reversed by geranylgeranyl pyrophosphate (GGPP) pretreatment, but not by mevalonate pretreatment. These findings suggest that BMP-2 mRNA expression was upregulated by PTH in MC3T3-E1 cells mediated by mevalonate pathway suppression followed by ROK inhibition. We have now demonstrated for the first time that PTH stimulated BMP-2 mRNA expression via the mevalonate pathway and ROK in osteoblastic MC3T3-E1 cells.

OBJECTIVE

Facial osseous defects remain a major functional and esthetic challenge for the head and neck surgeon. Tissue engineering may provide advantageous alternatives to conventional therapies. The objective of the study was to evaluate the efficacy of gene therapy in the repair of osseous facial defects.

METHODS

Blinded, controlled, prospective animal experiment.

METHODS

Thirty adult athymic nude rats were divided into five groups of six animals. Three groups were used as control subjects. Two groups were treated with 3.75 x <em>10</em>(8) viral particles containing recombinant type 5 adenoviral vectors. One group received viruses that coded for beta-galactosidase production, another received viruses that coded for <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP-2) production. After 120 days rats were examined at necropsy with precise planimetry, histological analysis of new <em>bone</em> growth, and radio-densitometric analysis of <em>bone</em> thickness.

RESULTS

Radio-densitometric measurements showed that BMP-2-treated nude athymic rats had significantly enhanced osseous repair compared with control subjects when evaluated by both radio-densitometry and histological examination.

CONCLUSIONS

Gene therapy-treated, immunosuppressed rodents had an enhanced osteoinductive repair of a dorsal osseous nasal defect.

BACKGROUND

FLRG (follistatin-related gene) is a secreted glycoprotein which is very similar to follistatin. As observed for follistatin, FLRG is involved in the regulation of various biological processes through its binding to members of the TGFbeta (transforming growth factor beta) superfamily, activin, BMPs (bone morphogenetic proteins) and myostatin. Unlike follistatin, FLRG has been found to be both secreted and localized within the nucleus of many FLRG-producing cells, suggesting the existence of specific intracellular functions of the protein.

RESULTS

In order to analyse the function of the nuclear form of FLRG, we performed a yeast two-hybrid screen, in which we identified AF10 [ALL1 (acute lymphoblastic leukaemia) fused gene from chromosome 10], a translocation partner of the MLL (mixed-lineage leukaemia) oncogene in human leukaemia, as a FLRG-interacting protein. This interaction was confirmed by far-Western-blot analysis and co-immunoprecipitation with transfected COS-7 cells. The N-terminal region of AF10, including the PHD (plant homeodomain), is sufficient to mediate this interaction, and has been shown to be involved in AF10 homo-oligomerization. By immunoprecipitation experiments, we showed that FLRG enhances the homo-oligomerization of AF10. Functional studies demonstrated that FLRG enhances the transactivation properties of the AF10 protein fused to Gal4 DNA-binding domains in transient transfection assays.

CONCLUSIONS

Our present study provides novel insights into the function of the nuclear form of the FLRG protein, which is revealed as a novel regulator of transcription. The nuclear isoform of FLRG lacks an intrinsic transactivation domain, but enhances AF10-mediated transcription, probably through promoting the homo-oligomerization of AF10, thus facilitating the recruitment of co-activators.

Recently, we reported that human PDB (placental decidua basalis) is an excellent source of MSCs (mesenchymal stem cells), meanwhile, PDB-MSCs could survive under hypoxia and serum deprivation. Herein, we investigated the proliferation, clonogentic efficiency, phenotypes, metabolic activity and cytokines secretion of PDB-MSCs in hypoxia and serum deprivation. PDB-MSCs were cultured in four groups: normoxia (20% O2) and complete medium [<em>10</em>% FBS (foetal bovine serum)+DMEM-HG (Dulbecco's modified Eagle's medium-high glucose)], hypoxia and complete medium, normoxia and serum deprivation (0% FBS), and hypoxia and serum deprivation. After 96 h of culture in the above groups, PDB-MSCs maintain the phenotypes stably. Interestingly, hypoxia notably enhanced the proliferation, colony-forming potential and lactate/glucose ratio in complete medium, but suppressed the secretion of BMP-2 (<em>bone</em> <em>morphogenetic</em> <em>protein</em>-2) and bFGF (basic fibroblast growth factor), while it did not change the quantity of VEGF (vascular endothelial growth factor) and bFGF in serum deprivation. Although PDB-MSCs grew slowly and seldom formed a colony unit in hypoxia and serum deprivation, they possessed a moderate metabolism. In conclusion, our results indicate that PDB-MSCs appear to be promising seed cells for ischaemia-related tissue engineering.

OBJECTIVE

Hepatic cirrhosis is a serious clinical problem caused by the accumulation of extracellular matrix, which can ultimately progress into hepatic failure. Transforming growth factor-beta1 (TGF-β1) plays a pivotal role in extracellular matrix production. Bone morphogenetic protein-7 (BMP-7), as a member of the TGF-β1 superfamily, has been well proved to be capable of reversing renal fibrosis in mice. In this study, we aim to investigate the potential effect of BMP-7 on hepatic fibrosis in rats.

METHODS

Sprague-Dawley rats were randomly divided into five groups. In the hepatic fibrosis model group (n=8), rats was treated with porcine serum at 0.5 ml each time, twice a week. In the negative control group (n=10), rats were intraperitoneally injected with equal amount and frequency saline. Rats were injected with BMP-7 (100 μg/kg weight) before porcine serum intraperitoneal injection in the preventive group (n=9). For the early (n=10) and late (n=8) treatment group, rats were received with BMP-7 (100 μg/kg weight) every other day since the second and fourth week respectively after porcine serum injection. After eight weeks, the degree of liver fibrosis in rats was evaluated and the expression of TGF-β1 in liver tissues was detected by Western blot and immunohistochemistry.

RESULTS

The grade of hepatic fibrosis was significant attenuated by BMP-7 prevention and treatment compared with the rats in negative control group (P<0.05). In addition, the expression of TGF-β1 greatly decreased in the BMP-7 preventive and treatment groups detected by both Western blot and immunohistochemistry.

CONCLUSIONS

BMP-7 can attenuate and even prevent the level of hepatic fibrosis in rats through inhibiting the expression of TGF-β1 in the liver fibrotic tissues. Therefore, it may be a potential clinical drug for the prevention and treatment of hepatic fibrosis.

OBJECTIVE

The development of biomaterials for secondary <em>bone</em> grafting to the alveolar cleft, recombinant human <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) may have great potential to promote osseous regeneration. The purpose of this study was to investigate the osteoinductive activity of low-dose rhBMP-2 (<em>10</em> microg/<em>10</em>0 microL) and the biological response of newly generated <em>bone</em> to orthodontic stimulation, compared with previous results using high-dose rhBMP-2 (40 microg/<em>10</em>0 microL).

METHODS

After extraction of the maxillary first premolars, bone defects were surgically created in eight adult beagle dogs using a 5-mm-diameter trepan bar. The rhBMP-2 with a PLGA/gelatin sponge complex (PGS) made of gelatin and polylactic acid polyglycolic acid copolymer was immediately implanted. The osteoinductive activity of rhBMP-2 and tooth movement into the newly generated bone was evaluated based on histological and morphometric examination.

RESULTS

Dramatic histological and morphometric differences were found in the time course of bone remodeling between low- and high-dose rhBMP-2. The onset of bone remodeling occurred significantly earlier in low-dose rhBMP-2. During experimental tooth movement, in contrast to high-dose rhBMP-2, with which root resorption occurred, only partial cementum resorption occurred on the pressure side with low-dose rhBMP-2.

CONCLUSIONS

These results indicated that the proper dosage of rhBMP-2 should be determined for successful clinical application.

Recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (rhBMP2) has been used clinically to treat <em>bone</em> fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and <em>bone</em> healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration <em>10</em>-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is <em>10</em>0-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union <em>bone</em> fractures.

All cellular phenomena and developmental events, including inner ear development, are modulated through harmonized signaling networks. Phosphatase and tensin homolog deleted on chromosome <em>10</em> (PTEN), a tumor suppressor, is a major signaling component involved in cross talk with key regulators of development; i.e., Wnt, Notch, and <em>bone</em> <em>morphogenetic</em> <em>proteins</em>. Although Pten function has been studied in various systems, its role in inner ear development is poorly understood. Here, we used inner ear-specific Pten conditional knockout mice and examined the characteristics of the inner ear. In a detailed analysis of the phenotype, reduced cochlear turning and widened epithelia were observed. Phalloidin staining of sensory epithelium revealed that hair cell patterns were disturbed; i.e., additional rows of hair cells were discovered. The neural abnormality revealed a reduction in and disorganization of nerve fibers, including apoptosis at the neural precursor stage. Pten deficiency induced increased phosphorylation of Akt at Ser473. The elevation of inhibitory glycogen synthase kinase 3β Ser9 phosphorylation (pGSK3β) was sustained until the neuronal differentiation stage at embryonic day 14.5, instead of pGSK3β downregulation. This is the first report on the influence of Pten/Akt/GSK3β signaling on the development of spiral ganglia. These results suggest that Pten is required for the maintenance of neuroblast number, neural precursors, and differentiation in the inner ear.