The purpose of this study was to identify the hard tissue formed early in experimental pulp exposures capped with mineral trioxide aggregate (MTA) or <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-7 using dentin sialo<em>protein</em> (DSP) as a marker. The pulps of 35 maxillary first, second, and third molar teeth from <em>10</em> male rats were experimentally exposed. The pulps were capped with MTA alone as a pulp-capping agent and final restoration or with BMP-7 followed by restoration with MTA. Five teeth with class I occlusal preparations, no exposure, and no restoration served as positive controls. Five teeth that received pulp exposures and no restoration served as negative controls. Five untreated third molars served as additional controls. The animals were killed at 2 weeks. The specimens were prepared and evaluated histologically and with immunohistochemistry using polyclonal antibodies raised against rat DSP. Pulps capped with MTA formed hard tissue that demonstrated significantly more immunostaining for DSP compared with BMP-7 (p = 0.0031). MTA-capped pulps also showed significantly more complete bridge formation compared with BMP-7 (p = 0.0008). Pulps capped with BMP-7 demonstrated a hard tissue that was <em>bone</em>-like in appearance and devoid of DSP staining.

Critical size defects of <em>bone</em> and delayed fracture healing due to metabolic disorders are still problems in orthopaedic surgery. Adenoviral vectors encoding <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (Ad.BMP-2) have been used to stimulate <em>bone</em> formation in small animals. The present study evaluated the use of direct adenoviral gene transfer for inducing <em>bone</em> formation in a large animal. Standardized iliac crest defects were created surgically on both sides of the pelvic <em>bone</em> of white mountain sheep. The efficiency of gene transfer was evaluated using recombinant adenoviruses carrying the cDNA for luciferase. High levels of transgene expression, restricted to the site of injection, were found for the 1st week. Transgene expression then fell considerably, but could still be detected for up to 5 weeks. To investigate the effect on <em>bone</em> healing, Ad.BMP-2 (<em>10</em>(11) particles in 200 mul saline) was unilaterally injected into iliac crest defects and into tibial osteotomies. The contralateral defects remained untreated to evaluate possible systemic effects. The controls were treated with saline solution. <em>Bone</em> formation within the defect, assessed by micro-computed tomography (CT) measurement at 8 weeks, and callus formation after osteotomy were significantly reduced following direct application of Ad.BMP-2. The retardation compared to untreated control animals was additionally found at the contralateral iliac crest indicating a systemic inhibitory effect. Histological analysis confirmed the CT measurement and showed an increased number of inflammatory cells within both defects. Antibodies against the adenovirus and the transgene product were detected in all treated animals. These data show a systemic retardation of <em>bone</em> formation following a single local injection of Ad.BMP-2 in sheep. This finding stands in contrast to the data obtained from small animal models. Further studies are needed to determine the contribution of the immune response to these results, and whether a lower dose of Ad.BMP-2 would be advantageous.

METHODS

Prospective, single center, nonblinded radiographic analysis of anterior and posterior adult spinal deformity fusions performed with bone morphogenetic protein (rhBMP-2).

OBJECTIVE

To determine the ability of rhBMP-2 to achieve multilevel spinal fusion in the deformity patient.

BACKGROUND

No previous study has evaluated rhBMP-2 for multilevel adult spinal deformity fusion with 2-year results. We postulated fusion could be achieved without distant autogenous graft harvest.

METHODS

Prospective analysis was performed for 98 patients (308 levels; mean age, 51.4 years) who underwent multilevel anterior or posterior spinal fusion (PSF) with minimum 2-year follow-up (average, 2.6 years). Group 1 (10 mg/level) contained 47 patients (109 levels; 2.33 levels/patient) who underwent anterior spinal fusion (ASF): BMP on an absorbable collagen sponge (ACS) with a titanium mesh cage. Group 2 (20 mg/level) included 43 patients (156 levels; 3.63 levels/patient) with PSF: BMP on an ACS with local bone graft (LBG) and bulking agent [tricalcium phosphate/hydroxyapatite (TCP-HA)]. Group 3 (40 mg/level) contained 8 patients (43 levels; 5.38 levels/patient) with PSF: rhBMP-2 and TCP-HA with no autologous bone. Confounding negative factors were present in the study population: medical comorbidities (26%), tobacco use (17%), revision surgery (34%), previous laminectomy (51%), and preoperative pseudarthrosis (27%). Postoperative films (AP, lateral, oblique) were evaluated by independent observers. Average fusion grade was based on a published scale.

RESULTS

Overall fusion rate was 95%. (group 1 91%, group 2 97%, group 3 100%). No confounding factor demonstrated a detrimental statistical significance to fusion.

CONCLUSIONS

In multilevel ASF, BMP (10 mg/level) generates fusion without autogenous bone. In multilevel PSF, BMP (20 mg/level) with LBG and TCP-HA produced fusion. BMP (40 mg/level) and TCP-HA without LBG achieved fusion. In multilevel spinal fusion, rhBMP-2 eliminated the necessity for iliac crest bone graft and yielded an excellent fusion rate.

BACKGROUND

Signaling mechanisms involved in early human germ cell development are largely unknown and believed to be similar to mouse germ cell development; however, there may be species specific differences. KIT ligand (KITL) and Bone morphogenetic protein 4 (BMP4) are necessary in mouse germ cell development and may play an important role in human germ cell development.

METHODS

KITL signaling studies were conducted by differentiating human embryonic stem cells (hESCs) on KITL wild-type, hetero- or homozygous knockout feeders for 10 days, and the effects of BMP signaling was determined by differentiation in the presence of BMP4 or its antagonist, Noggin. The formation of germ-like cells was ascertained by immunocytochemistry, flow cytometry and quantitative RT-PCR for germ cell markers.

RESULTS

The loss of KITL in enrichment and differentiation cultures resulted in significant down-regulation of germ cell genes and a 70.5% decrease in germ-like (DDX4+ POU5F1+) cells, indicating that KITL is involved in human germ cell development. Moreover, endogenous BMP signaling caused germ-like (DDX4+ POU5F1+) cell differentiation, and the inhibition of this pathway caused a significant decrease in germ cell gene expression and in the number of DDX4+ POU5F1+ cells. Further, we demonstrated that eliminating feeders but maintaining their secreted extracellular matrix is sufficient to sustain the increased numbers of DDX4+ POU5F1+ cells in culture. However, this resulted in decreased germ cell gene expression.

CONCLUSIONS

From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.

Several porous calcium hydroxyapatite (HA) ceramics have been used clinically as <em>bone</em> substitutes, but most of them possessed few interpore connections, resulting in pathological fracture probably due to poor <em>bone</em> formation within the substitute. We recently developed a fully interconnected porous HA ceramic (IP-CHA) by adopting the 'foam-gel' technique. The IP-CHA had a three-dimensional structure with spherical pores of uniform size (average 150 microm, porosity 75%), which were interconnected by window-like holes (average diameter 40 microm), and also demonstrated adequate compression strength (<em>10</em>-12 MPa). In animal experiments, the IP-CHA showed superior osteoconduction, with the majority of pores filled with newly formed <em>bone</em>. The interconnected porous structure facilitates <em>bone</em> tissue engineering by allowing the introduction of mesenchymal cells, osteotropic agents such as <em>bone</em> <em>morphogenetic</em> <em>protein</em> or vasculature into the pores. Clinically, we have applied the IP-CHA to treat various bony defects in orthopaedic surgery, and radiographic examinations demonstrated that grafted IP-CHA gained radiopacity more quickly than the synthetic HA in clinical use previously. We review the accumulated data on <em>bone</em> tissue engineering using the novel scaffold and on clinical application in the orthopaedic field.

We sought to develop a retroviral vector system that would produce secretion of high levels of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-4 by optimizing the expression construct and developing an improved retroviral vector. Replacement of the propeptide domain of BMP4 with that of BMP2 increased the secretion level of mature BMP4 <em>protein</em> in transduced cells. The intact BMP2 pro-peptide sequence was essential, as deletion of a small part of the propeptide sequence of BMP2 from the BMP2/4 hybrid construct diminished BMP4 expression and secretion. Addition of a hemaglutinin tag to the carboxy terminus of BMP4 abolished the bioactivity of secreted BMP4. Transduction of rat marrow stromal cells (and fibroblasts) with an MFG-based retroviral vector pseudotyped with VSV-G envelope containing this BMP2/4 hybrid expression construct led to secretion of very high levels of mature BMP4 in conditioned medium (up to 1 microg/<em>10</em>(6) cells/24 hours). The secreted BMP4 was biologically active, as it induced alkaline phosphatase expression in C2C12 cells. The transduced rat marrow stromal cells expressing mature BMP4 induced de novo ectopic <em>bone</em> formation in syngenic immune-competent rats. We have developed an MFG-based retroviral vector system that causes secretion of high levels of functionally active human BMP4 <em>protein</em>.

OBJECTIVE

To identify key genes differentially expressed in the human retinal pigment epithelium (hRPE) following low-level West Nile virus (WNV) infection.

METHODS

Primary hRPE and retinal pigment epithelium cell line (ARPE-19) cells were infected with WNV (multiplicity of infection 1). RNA extracted from mock-infected and WNV-infected cells was assessed for differential expression of genes using Affymetrix microarray. Quantitative real-time PCR analysis of 23 genes was used to validate the microarray results.

RESULTS

Functional annotation clustering of the microarray data showed that gene clusters involved in immune and antiviral responses ranked highly, involving genes such as chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X-C motif) ligand <em>10</em> (CXCL<em>10</em>), and toll like receptor 3 (TLR3). In conjunction with the quantitative real-time PCR analysis, other novel genes regulated by WNV infection included indoleamine 2,3-dioxygenase (IDO1), genes involved in the transforming growth factor-β pathway (<em>bone</em> <em>morphogenetic</em> <em>protein</em> and activin membrane-bound inhibitor homolog [BAMBI] and activating transcription factor 3 [ATF3]), and genes involved in apoptosis (tumor necrosis factor receptor superfamily, member <em>10</em>d [TNFRSF<em>10</em>D]). WNV-infected RPE did not produce any interferon-γ, suggesting that IDO1 is induced by other soluble factors, by the virus alone, or both.

CONCLUSIONS

Low-level WNV infection of hRPE cells induced expression of genes that are typically associated with the host cell response to virus infection. We also identified other genes, including IDO1 and BAMBI, that may influence the RPE and therefore outer blood-retinal barrier integrity during ocular infection and inflammation, or are associated with degeneration, as seen for example in aging.

Epithelial-mesenchymal interactions and extracellular matrix remodeling are key processes of embryonic lung development. Lung smooth muscle cells, which are derived from the mesenchyme, form a sheath around bronchi and blood vessels. During lung organogenesis, smooth muscle differentiation coincides with epithelial branching morphogenesis and closely follows developing airways spatially and temporally. The precise function of parabronchial smooth muscle (PBSM) cells in healthy adult lung remains unclear. However, PBSM may regulate epithelial branching morphogenesis during lung development by the induction of mechanical stress or through regulation of paracrine signaling pathways. Alveolar myofibroblasts are interstitial contractile cells that share features and may share an origin with smooth muscle cells. Alveolar myofibroblasts are essential for secondary septation, a process critical for the development of the gas-exchange region of the lung. Dysregulation of PBSM or alveolar myofibroblast development is thought to underlie the pathogenesis of many lung diseases, including bronchopulmonary dysplasia, asthma, and interstitial fibrosis. We review the current understanding of the regulation of PBSM and alveolar myofibroblast development, and discuss the role of PBSM in lung development. We specifically focus on the role of these cells in the context of fibroblast growth factor-<em>10</em>, sonic hedgehog, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4, retinoic acid, and Wnt signaling pathways in the regulation of lung branching morphogenesis.

Early embryonic lung branching morphogenesis is regulated by many growth factor-mediated pathways. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 4 (BMP4) is one of the morphogens that stimulate epithelial branching in mouse embryonic lung explant culture. To further understand the molecular mechanisms of BMP4-regulated lung development, we studied the biological role of Smad-ubiquitin regulatory factor 1 (Smurf1), an ubiquitin ligase specific for BMP receptor-regulated Smads, during mouse lung development. The temporo-spatial expression pattern of Smurf1 in mouse embryonic lung was first determined by quantitative real-time PCR and immunohistochemistry. Overexpression of Smurf1 in airway epithelial cells by intratracheal introduction of recombinant adenoviral vector dramatically inhibited embryonic day (E) 11.5 lung explant growth in vitro. This inhibition of lung epithelial branching was restored by coexpression of Smad1 or by addition of soluble BMP4 ligand into the culture medium. Studies at the cellular level show that overexpression of Smurf1 reduced epithelial cell proliferation and differentiation, as documented by reduced PCNA-positive cell index and by reduced mRNA levels for surfactant <em>protein</em> C and Clara cell <em>protein</em> <em>10</em> expression. Further studies found that overexpression of Smurf1 reduced BMP-specific Smad1 and Smad5, but not Smad8, <em>protein</em> levels. Thus overexpression of Smurf1 specifically promotes Smad1 and Smad5 ubiquitination and degradation in embryonic lung epithelium, thereby modulating the effects of BMP4 on embryonic lung growth.

Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (<em>10</em> genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface <em>proteins</em> (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID-1, ID-2, ID-3, and ID-4. These encode dominant negative helix-loop-helix (dnHLH) <em>proteins</em> that lack the nuclear binding domain of the basic HLH <em>proteins</em> and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor beta (CBFbeta), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID <em>proteins</em>, block differentiation along alternate pathways.

We have identified a new member of the transforming growth factor-beta (TGF-beta) superfamily, growth/differentiation factor-<em>10</em> (GDF-<em>10</em>), which is highly related to <em>bone</em> <em>morphogenetic</em> <em>protein</em>-3 (BMP-3). The nucleotide sequence of GDF-<em>10</em> encodes a predicted <em>protein</em> of 476 amino acids with a molecular weight of approximately 52,000. The GDF-<em>10</em> polypeptide contains a potential signal sequence for secretion, a putative RXXR proteolytic processing site, and a carboxy-terminal domain with considerable homology to other known members of the TGF-beta superfamily. In the mature carboxy-terminal domain GDF-<em>10</em> is more homologous to BMP-3 (83% amino acid sequence identity) than to any other previously identified TGF-beta family member. GDF-<em>10</em> also shows significant homology to BMP-3 (approximately 30% amino acid sequence identity) in the pro- region of the molecule. Based on these sequence comparisons, GDF-<em>10</em> and BMP-3 define a new subgroup within the larger TGF-beta superfamily. By Northern analysis, GDF-<em>10</em> mRNA was detected primarily in murine uterus, adipose tissue, and brain and to a lesser extent in liver and spleen. In addition, GDF-<em>10</em> mRNA was present in both neonatal and adult <em>bone</em> samples, with higher levels being detected in calvaria than in long <em>bone</em>. These results suggest that GDF<em>10</em> may play multiple roles in regulating cell differentiation events, including those involved in skeletal morphogenesis. Gdf<em>10</em> was mapped to the proximal region of mouse chromosome 14 close to a region known to contain a spontaneous recessive mutation that is associated with a craniofacial defect.

Epimedii herba is one of the most frequently used herbs in formulas prescribed for the treatment of osteoporosis in China. The main active flavonoid glucoside extracted from Epimedium pubescens is Icariin, which has been reported to enhance <em>bone</em> healing and reduce osteoporosis occurrence. However, the detailed molecular mechanisms remain unclear. In this present study, we examine the molecular mechanisms of icariin by using primary osteoblast cell cultures obtained from adult mice. The osteoblast cells were harvested from 8-month old female Imprinting Control Region (ICR) mice. The effects of icariin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The production of nitric oxide (NO) and caspase-3 were analyzed, along with the gene expressions of <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (BMP-2), SMAD4, Cbfa1/Runx2, OPG, and RANKL. The viability of the osteoblasts reached its maximum at <em>10</em>(-8)M icariin. At this concentration, icariin increased the proliferation and matrix mineralization of osteoblasts and promoted NO synthesis. With icariin treatment, the BMP-2, SMAD4, Cbfa1/Runx2, and OPG gene expressions were up-regulated; the RANKL gene expression was however down-regulated. Concurrent treatment involving the BMP antagonist (Noggin) or the NOS inhibitor (L-NAME) diminished the icariin-induced cell proliferation, ALP activity, NO production, as well as the BMP-2, SMAD4, Cbfa1/Runx2, OPG, RANKL gene expressions. In this study, we demonstrate that in vitro icariin is a <em>bone</em> anabolic agent that may exert its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfa1/Runx2, OPG, and RANKL gene expressions. This effect may contribute to its action on the induction of osteoblasts proliferation and differentiation, resulting in <em>bone</em> formation.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) show promise in therapies for improving <em>bone</em> formation after injury; however, the high supraphysiological concentrations required for desired osteoinductive effects, off-target concerns, costs, and patient variability have limited the use of BMP-based therapeutics. To better understand the role of biomaterial design in BMP delivery, a matrix metalloprotease (MMP)-sensitive hyaluronic acid (HA)-based hydrogel was used for BMP-2 delivery to evaluate the influence of hydrogel degradation rate on <em>bone</em> repair in vivo. Specifically, maleimide-modified HA (MaHA) macromers were crosslinked with difunctional MMP-sensitive peptides to permit protease-mediated hydrogel degradation and growth factor release. The compressive, rheological, and degradation properties of MaHA hydrogels were characterized as a function of crosslink density, which was varied through either MaHA concentration (1-5wt.%) or maleimide functionalization (<em>10</em>-40%f). Generally, the compressive moduli increased, the time to gelation decreased, and the degradation rate decreased with increasing crosslink density. Furthermore, BMP-2 release increased with either a decrease in the initial crosslink density or an increase in collagenase concentration (non-specific MMP degradation). Lastly, two hydrogel formulations with distinct BMP-2 release profiles were evaluated in a critical-sized calvarial defect model in rats. After six weeks, minimal evidence of <em>bone</em> repair was observed within defects left empty or filled with hydrogels alone. For hydrogels that contained BMP-2, similar volumes of new <em>bone</em> tissue were formed; however, the faster degrading hydrogel exhibited improved cellular invasion, <em>bone</em> volume to total volume ratio, and overall defect filling. These results illustrate the importance of coordinating hydrogel degradation with the rate of new tissue formation.

The regulation of callus formation during fracture repair involves the coordinate expression of growth factors and their receptors. This article describes the temporal and spatial expression of noggin gene, an antagonist to <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP), during the fracture repair process. Noggin expression was examined by means of Northern blotting and in situ hybridization and compared with the expression pattern of BMP-4 in a model of fracture repair in adult mice. Expression levels of noggin messenger RNA (mRNA) were enhanced in the early phase of fracture callus formation. The localization of the noggin mRNA was similar to that of BMP-4 mRNA. Distinct noggin mRNA signals were located predominantly in cells lining the periosteum and the cortical endosteum near the fracture site at 2 days after fracture. At 5, <em>10</em>, and 21 days after fracture, noggin mRNA was detected in the chondrocytes and osteoblasts in the newly formed callus. The pattern of localization was indistinguishable from that of BMP-4. These results suggest that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing.

Critical size segmental <em>bone</em> defects are still a major challenge in reconstructive orthopedic surgery. Transplantation of human mesenchymal stem cells (hMSC) has been proposed as an alternative to autogenous <em>bone</em> graft, as MSC can be expanded in vitro and induced to differentiate into <em>bone</em>-regenerating osteoblasts by several <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP). The aim of this study was to investigate whether the association of hMSC and BMP-7, with providing the necessary scaffold to fill the <em>bone</em> loss, improved <em>bone</em> regeneration in a rat model of critical size segmental <em>bone</em> defect, compared to treatment with either hMSC or BMP-7 and the matrix. In addition, we tested whether pre-treatment of hMSC with cyclic ADP-ribose (cADPR), an intracellular Ca2+ mobilizer previously shown to accelerate the in vitro expansion of hMSC (Scarfì S et al, Stem Cells, 2008), affected the osteoinductive capacity of the cells in vivo. X-ray analysis, performed 2, <em>10</em> and 16 weeks after transplantation, revealed a significantly higher score in the rats treated with hMSC and BMP-7 compared to controls, receiving either hMSC or BMP-7. Microtomography and histological analysis, performed 16weeks after transplantation, confirmed the improved <em>bone</em> regeneration in the animals treated with the association of hMSC and BMP-7 compared to controls. Pre-treatment with cADPR to stimulate hMSC proliferation in vitro did not affect the <em>bone</em> regenerating capacity of the cells in vivo. These results indicate that the association of in vitro expanded hMSC with BMP-7 provide a better osteoinductive graft compared to either hMSC or BMP-7 alone. Moreover, cADPR may be used to stimulate hMSC proliferation in vitro in order to reduce the time required to obtain a transplantable number of cells, with no adverse effect on the <em>bone</em> regenerating capacity of hMSC.

OBJECTIVE

Effective carrier technologies and dosing appear critical for the successful use of bone morphogenetic proteins (BMPs). This study evaluated radiographically the potential of a purpose-designed titanium porous-oxide implant surface combined with recombinant human BMP-2 (rhBMP-2) to stimulate alveolar ridge augmentation.

METHODS

Twelve young-adult Labrador dogs were used. Three 10-mm titanium implants per jaw quadrant were placed 5 mm into the alveolar ridge following extraction of the premolar teeth and reduction of alveolar ridge. Six animals received implants coated with rhBMP-2 at 0.75 or 1.5 mg/ml randomized to contralateral jaw quadrants. Another six animals received implants coated with rhBMP-2 at 3 mg/ml or uncoated control using the same split-mouth design. The mucoperiosteal flaps were advanced, adapted, and sutured to submerge the implants. Radiographic registrations were made immediately postsurgery (baseline), and at weeks 4 and 8 (end of study).

RESULTS

rhBMP-2-coated implants exhibited robust radiographic bone formation extending to and above the implant platform from week 4 (P<0.01). Some rhBMP-2-coated implants showed voids within the newly formed bone that gradually resolved and/or implant displacement, being severe in two animals receiving implants coated with rhBMP-2 at 3 mg/ml. Controls showed limited, if any, new bone formation at weeks 4 and 8 postsurgery. There were no significant differences among the rhBMP-2 groups in bone gain.

CONCLUSIONS

The titanium porous-oxide surface serves as an effective carrier for rhBMP-2, showing a clinically significant potential to stimulate local bone formation. With the carrier technology used, therapeutic dosage appears to be in the range of 0.75-1.5 mg/ml.

The <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) play a pivotal role in endochondral <em>bone</em> formation. Using differential display polymerase chain reaction, we have identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb), that is induced as a murine prechondroblastic cell line, MLB13MYC clone 17, acquires osteoblastic features in response to BMP-2 treatment. The 3-kilobase mRNA encodes a 34-kDa <em>protein</em> containing seven WD-40 repeats. Northern and Western analyses demonstrated that BIG-3 mRNA and <em>protein</em> were induced after 24 h of BMP-2 treatment. BIG-3 mRNA was expressed in conditionally immortalized murine <em>bone</em> marrow stromal cells, osteoblasts, osteocytes, and growth plate chondrocytes, as well as in primary calvarial osteoblasts. Immunohistochemistry demonstrated that BIG-3 was expressed in the osteoblasts of calvariae isolated from mouse embryos. To identify a role for BIG-3 in osteoblast differentiation, MC3T3-E1 cells were stably transfected with the full-length coding region of BIG-3 (MC3T3E1-BIG-3) cloned downstream of a cytomegalovirus promoter in pcDNA3.1. Pooled MC3T3E1-BIG-3 clones expressed alkaline phosphatase activity earlier and achieved a peak level of activity <em>10</em>-fold higher than cells transfected with the empty vector (MC3T3E1-EV) at 14 days. Cyclic AMP production in response to parathyroid hormone was increased <em>10</em>- and 14-fold at 7 and 14 days, respectively, in MC3T3E1-BIG-3 clones, relative to MC3T3E1-EV clones. This increase in cAMP production was associated with an increase in PTH binding. Expression of BIG-3 increased mRNA levels encoding Cbfa1, type I collagen, and osteocalcin and accelerated formation of mineralized nodules. In conclusion, we have identified a novel WD-40 <em>protein</em>, induced by BMP-2 treatment, that dramatically accelerates the program of osteoblastic differentiation in stably transfected MC3T3E1 cells.

Tissue transglutaminase is a calcium-dependent, <em>protein</em> cross-linking enzyme that is highly expressed in cells undergoing apoptosis. The expression of tissue transglutaminase is regulated by a variety of molecules including retinoids, interleukin-6, and transforming growth factor-beta1 (TGF-beta1). Retinoid and interleukin-6 inductions of tissue transglutaminase expression are mediated by specific cis-regulatory elements located within the first 4.0 kilobase pairs of the promoter of the gene. The present studies were designed to identify the molecular mechanisms mediating the regulation of tissue transglutaminase gene expression by TGF-beta family members. Transient transfection of Mv1Lu cells with transglutaminase promoter constructs demonstrated that 0.2 nM TGF-beta1 maximally induced the activation of the promoter through a <em>10</em>-base pair TGF-beta1 response element (TRE; GAGTTGGTGC) located 868 base pairs upstream of the transcription start site. This same element mediated an inhibitory activity of TGF-beta1 on the transglutaminase promoter in MC3T3 E1 cells. The TRE through which TGF-beta1-regulated the activity of the transglutaminase promoter was necessary and sufficient for <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2- (BMP) and BMP4-dependent inhibition of the tissue transglutaminase promoter. The TGF-beta1, BMP2, and BMP4 regulation of the transglutaminase promoter activity was similar to the responses we observed for the endogenous transglutaminase activity of Mv1Lu and MC3T3 E1 cells. For BMP2 and BMP4, this regulation was paralleled by a decrease in tissue transglutaminase mRNA in MC3T3 E1 cells. The results of these experiments suggest that TGF-beta1, BMP2, and BMP4 regulation of mouse tissue transglutaminase gene expression requires a composite TRE located in the 5'-flanking DNA.

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between <em>10</em> and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.

While human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) is a promising growth factor for <em>bone</em> regeneration, its clinical efficacy has recently shown to be below expectation. In order to improve the clinical translation of rhBMP-2, there exists strong motivation to engineer better delivery systems. Hyaluronic acid (HA) hydrogel is a suitable carrier for the delivery of rhBMP-2, but a major limitation of this scaffold is its low cell adhesive properties. In this study, we have determined whether covalent grafting of an integrin-specific ligands into HA hydrogel could improve cell attachment and further enhance the osteogenic potential of rhBMP-2. A structurally stabilized fibronectin (FN) fragment containing the major integrin-binding domain of full-length FN (FN III9*-<em>10</em>) was engineered, in order to be incorporated into HA hydrogel. Compared to non-functionalized HA hydrogel, HA-FN hydrogel remarkably improved the capacity of the material to support mesenchymal stem cell attachment and spreading. In an ectopic <em>bone</em> formation model in the rat, delivery of rhBMP-2 with HA-FN hydrogel resulted in the formation of twice as much <em>bone</em> with better organization of collagen fibers compared to delivering the growth factor in non-functionalized HA hydrogel. This engineered hydrogel carrier for rhBMP-2 can be relevant in clinical <em>bone</em> repair.

OBJECTIVE

Ex vivo gene therapy with the use of human mesenchymal stem cells (hMSCs) and bone morphogenetic protein (BMP) genes provides a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules that may promote bone formation in patients with inadequate hMSC populations because of age, osteoporosis, metastatic bone disease, iatrogenic depletion, or other metabolic derangements. This study was undertaken to evaluate the efficacy of ex vivo gene therapy with the use of hMSCs and the BMP-9 gene to promote spinal fusion in the rat.

METHODS

Sixteen athymic nude rats were treated with hMSCs transduced with recombinant, replication-defective Type 5 adenovirus containing the cytomegalovirus promoter and either the BMP-9 (Ad-BMP-9) or the beta-galactosidase (Ad-beta-gal) gene. Ad-beta-gal served as the control. Each animal received a percutaneous, paraspinal injection of 10(6) hMSCs transduced with 50 plaque-forming units/cell adenovirus in the lumbar region, with Ad-BMP-9 on the left and Ad-beta-gal on the right. At 8 weeks postinjection, computed tomographic scans of the lumbosacral spine were obtained, and the lumbosacral spine specimens were examined histologically.

RESULTS

Both computed tomographic studies and histological analysis clearly demonstrated large volumes of ectopic bone at the Ad-BMP-9-transduced hMSC injection sites, resulting in successful spinal fusion and no evidence of nerve root compression or local or systemic toxicity. The contralateral regions that were treated with Ad-beta-gal-transduced hMSCs showed no evidence of osteogenesis.

CONCLUSIONS

The results of this study suggest that hMSC and BMP-9 ex vivo gene therapy may be useful in inducing spinal fusion as well as other related procedures and certainly warrants further clinical development.

OBJECTIVE

The limitations and morbidity associated with autogenous bone grafting have driven the search for predictable bone substitutes and bioimplants. A novel method of reconstruction was tested in this case series.

METHODS

Ten patients with major mandibular defects following resection of biopsy-proven ameloblastoma lesions or osteomyelitis of the mandibular body or ramus were included in this study. The resection defects were spanned with rigid reconstruction plates to hold the remaining mandibular segments in the correct position. The defects were filled with a bioimplant containing bone morphogenetic protein-7 (BMP-7) in a demineralized bone matrix (DBM) suspended in a reverse-phase medium to effect sustained BMP delivery.

RESULTS

The postoperative course for all 10 patients was uneventful. Radiographic evidence of mandibular bone formation was found in all cases. At the end of 1 year, functional and esthetic reconstruction of the mandible was complete.

CONCLUSIONS

Bioimplants containing BMP-7 in DBM suspended in a reverse phase medium were successful in restoring major mandibular defects in nonirradiated beds in this series of 10 patients.

Growth/differentiation factor-<em>10</em> (GDF-<em>10</em>) is a TGF-beta family member highly related to <em>bone</em> <em>morphogenetic</em> <em>protein</em>-3. In order to determine the biological function of GDF-<em>10</em>, we carried out a detailed analysis of the expression pattern of GDF-<em>10</em> and characterized GDF-<em>10</em>-null mice that we generated by gene targeting. During embryogenesis GDF-<em>10</em> is expressed prominently in developing skeletal structures both in the craniofacial region and in the vertebral column. In adult animals, GDF-<em>10</em> is expressed at high levels in the brain, where GDF-<em>10</em> is localized primarily to cells in the Purkinje cell layer of the cerebellum, and in the uterus, where the expression levels of GDF-<em>10</em> are regulated both during the menstrual cycle and during pregnancy. Despite the high levels of GDF-<em>10</em> expression in these tissues, we found no obvious abnormalities in GDF-<em>10</em>-knockout mice with respect to the development of these tissues. These findings suggest either that GDF-<em>10</em> plays no regulatory role in these tissues or that its function is redundant with that of other growth factor-like molecules.

Previously, we showed that newborn forkhead box (Fox)f1(+/-) mice with diminished pulmonary FoxF1 levels died of severe lung hemorrhage and exhibited abnormal formation of alveolar sacs and capillaries. Another group recently reported that Foxf1(+/-) mouse embryos displayed a number of organ and skeletal defects including fusion of lung lobes. However, identification of pulmonary genes whose altered expression was associated with the lobular fusion defect in Foxf1(+/-) lungs remains uncharacterized. The present study was conducted to determine the nature of the malformations leading to lung fusions in the FoxF1 embryos and to identify potential signaling pathways influenced by FoxF1 haploinsufficiency. We show that Foxf1(+/-) embryos exhibit defects in formation and branching of primary lung buds, which causes fusion of the right lung lobes and vessels. The severity of the Foxf1(+/-) lung fusions was correlated with decreased levels of FoxF1 mRNA. In situ hybridization studies demonstrated that the defective primary lung-bud development in early Foxf1(+/-) embryos was associated with fewer pulmonary mesenchymal-epithelial interfaces. Defects in branching morphogenesis in the Foxf1(+/-) embryos were associated with altered expression of the fibroblast growth factor-<em>10</em>, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4, and the Gli3 transcription factor, which are known to influence primary lung-bud development.