Given the paucity of information on the potential roles of <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed BMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K(d) 0.28 +/- 0.02 nM; <em>10</em>0%), the relative affinities of FS for BMP-4, -6 and -7 were <em>10</em>, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.

Smad4 is the common mediator for TGFbeta signals, which play important functions in many biological processes. To study the role of Smad4 in skin development and epidermal tumorigenesis, we disrupted this gene in skin using the Cre-loxP approach. We showed that absence of Smad4 blocked hair follicle differentiation and cycling, leading to a progressive hair loss of mutant (MT) mice. MT hair follicles exhibited diminished expression of Lef1, and increased proliferative cells in the outer root sheath. Additionally, the skin of MT mice exhibited increased proliferation of basal keratinocytes and epidermal hyperplasia. Furthermore, we provide evidence that the absence of Smad4 resulted in a block of both TGFbeta and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) signaling pathways, including p21, a well-known cyclin-dependent kinase inhibitor. Consequently, all MT mice developed spontaneous malignant skin tumors from 3 months to 13 months of age. The majority of tumors are malignant squamous cell carcinomas. A most notable finding is that tumorigenesis is accompanied by inactivation of phosphatase and tensin homolog deleted on chromosome <em>10</em> (Pten), activation of AKT, fast proliferation and nuclear accumulation of cyclin D1. These observations revealed the essential functions of Smad4-mediated signals in repressing skin tumor formation through the TGFbeta/BMP pathway, which interacts with the Pten signaling pathway.

We investigated the role of AMP-activated <em>protein</em> kinase (AMPK), Akt, mammalian target of rapamycin (mTOR), autophagy and their interplay in osteogenic differentiation of human dental pulp mesenchymal stem cells. The activation of various members of AMPK, Akt and mTOR signaling pathways and autophagy was analyzed by immunoblotting, while osteogenic differentiation was assessed by alkaline phosphatase staining and real-time RT-PCR/immunoblot quantification of osteocalcin, Runt-related transcription factor 2 and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 mRNA and/or <em>protein</em> levels. Osteogenic differentiation of mesenchymal stem cells was associated with early (day 1) activation of AMPK and its target Raptor, coinciding with the inhibition of mTOR and its substrate p70S6 kinase. The early induction of autophagy was demonstrated by accumulation of autophagosome-bound LC3-II, upregulation of proautophagic beclin-1 and a decrease in the selective autophagic target p62. This was followed by the late activation of Akt/mTOR at days 3-7 of differentiation. The RNA interference-mediated silencing of AMPK, mTOR or autophagy-essential LC3β, as well as the pharmacological inhibitors of AMPK (compound C), Akt (<em>10</em>-DEBC hydrochloride), mTOR (rapamycin) and autophagy (bafilomycin A1, chloroquine and ammonium chloride), each suppressed mesenchymal stem cell differentiation to osteoblasts. AMPK knockdown prevented early mTOR inhibition and autophagy induction, as well as late activation of Akt/mTOR signaling, while Akt inhibition suppressed mTOR activation without affecting AMPK phosphorylation. Our data indicate that AMPK controls osteogenic differentiation of human mesenchymal stem cells through both early mTOR inhibition-mediated autophagy and late activation of Akt/mTOR signaling axis.

Transforming growth factor (TGF)-beta1, -beta2, and -beta3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-A resolution crystal structure of the ternary complex between human TGF-beta1 and the extracellular domains of its type I and type II receptors, TbetaRI and TbetaRII. The TGF-beta1 ternary complex structure is similar to previously reported TGF-beta3 complex except with a <em>10</em> degrees rotation in TbetaRI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-beta. TbetaRI showed significant binding to TGF-beta2 and TGF-beta3 but not TGF-beta1, and the binding to all three isoforms of TGF-beta was enhanced considerably in the presence of TbetaRII. The preference of TGF-beta2 to TbetaRI suggests a variation in its receptor recruitment in vivo. Although TGF-beta1 and TGF-beta3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-beta superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of TbetaRII on TGF-beta is unique to TGF-betas, whereas that of type II receptor for <em>bone</em> <em>morphogenetic</em> <em>protein</em> on <em>bone</em> <em>morphogenetic</em> <em>protein</em> appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.

The mouse prostate gland develops by branching morphogenesis from the urogenital epithelium and mesenchyme. Androgens and developmental factors, including FGF<em>10</em> and SHH, promote prostate growth (Berman, D.M., Desai, N., Wang, X., Karhadkar, S.S., Reynon, M., Abate-Shen, C., Beachy, P.A., Shen, M.M., 2004. Roles for Hedgehog signaling in androgen production and prostate ductal morphogenesis. Dev. Biol. 267, 387-398; Donjacour, A.A., Thomson, A.A., Cunha, G.R., 2003. FGF-<em>10</em> plays an essential role in the growth of the fetal prostate. Dev. Biol. 261, 39-54), while BMP4 signaling from the mesenchyme has been shown to suppresses prostate branching (Lamm, M.L., Podlasek, C.A., Barnett, D.H., Lee, J., Clemens, J.Q., Hebner, C.M., Bushman, W., 2001. Mesenchymal factor <em>bone</em> <em>morphogenetic</em> <em>protein</em> 4 restricts ductal budding and branching morphogenesis in the developing prostate. Dev. Biol. 232, 301-314). Here, we show that <em>Bone</em> <em>Morphogenetic</em> <em>Protein</em> 7 (BMP7) restricts branching of the prostate epithelium. BMP7 is expressed in the periurethral urogenital mesenchyme prior to formation of the prostate buds and, subsequently, in the prostate epithelium. We show that BMP7(lacZ/lacZ) null prostates show a two-fold increase in prostate branching, while recombinant BMP7 inhibits prostate morphogenesis in organ culture in a concentration-dependent manner. We further explore the mechanisms by which the developmental signals may be interpreted in the urogenital epithelium to regulate branching morphogenesis. We show that Notch1 activity is associated with the formation of the prostate buds, and that Notch1 signaling is derepressed in BMP7 null urogenital epithelium. Based on our studies, we propose a model that BMP7 inhibits branching morphogenesis in the prostate and limits the number of domains with high Notch1/Hes1 activity.

BACKGROUND

The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.

METHODS

Adenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 x 10(2) viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.

RESULTS

Levels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size.

CONCLUSIONS

BMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.

The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding <em>proteins</em> and between prodomains of BMP-2, -4, -7, and -<em>10</em> and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding <em>proteins</em> and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other <em>bone</em> <em>morphogenetic</em> <em>proteins</em>. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-<em>10</em> prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-<em>10</em> growth factor and suggest that the BMP-<em>10</em> complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.

Myostatin is a secreted TGF-beta family member that controls skeletal muscle growth. Humans, cattle, and dogs carrying natural loss-of-function mutations in the myostatin gene and myostatin knockout mice exhibit significant increases in skeletal muscle mass. Treatment of adult mice with antimyostatin antibodies also resulted in significant muscle mass increases. However, myostatin-knockout mice that were treated with a soluble form of the activin type II receptor (ActRII) B increased their muscle mass by an additional 15-25%, indicating that there is at least one additional ligand, in addition to myostatin, that functions to limit muscle growth. Here, both soluble ActRII and -IIB fragment-crystallizable <em>proteins</em> were used to affinity purify their native ligands from human and mouse sera. Using mass spectrometry-based proteomics and in vitro binding assays we have identified and confirmed that a number of TGF-beta family members, including myostatin, activins-A, -B, and -AB, <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) -9, -<em>10</em>, and -11, bind to both ActRIIs. Many of these factors, such as BMPs-11, -9, and -<em>10</em> were discovered in systemic circulation for the first time, indicating that these ligands may also act in an endocrine fashion. Using a promoter-specific gene reporter assay, we demonstrated that soluble ActRIIB fragment-crystallizable <em>proteins</em> can inhibit the canonical signaling induced by these ligands. In addition, like myostatin, these factors were able to block the differentiation of myoblast cells into myotubes. However, in addition to myostatin, only BMP-11, and activins-A, -B, and -AB could be blocked from inhibiting the myoblast-to-myotube differentiation with both soluble ActRIIs, thus implicating them as potential novel regulators of muscle growth.

Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) is a member of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> family involved in de novo <em>bone</em> induction. Successful use of rhBMP-2 requires implantation with a biomaterial which can act as a scaffold for cell invasion for osteoinduction and retains rhBMP-2 at a site of implantation. This study was carried out to characterize rhBMP-2 pharmacokinetics from a variety of biomaterial carriers in a rat ectopic model. Retention of rhBMP-2 within carriers after 3 h was variable among the carriers (range, 75-<em>10</em>%), with collagenous sponges retaining the highest fraction of implanted dose. A gradual loss of rhBMP-2 was subsequently observed, the kinetics of which was strongly dependent on the implanted carrier. Collagenous carriers were observed to lose rhBMP-2 gradually from the implant site, whereas some of the mineral-based carriers retained a fraction of implanted rhBMP-2 within the implants. These differences in <em>protein</em> pharmacokinetics among carriers, in addition to their physicochemical nature, are expected to affect the biological activity of implanted rhBMP-2.

Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C<em>10</em>1-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At embryonic day 11.5, primitive lung buds were dissected and cultured at either 20 or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with 20% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-1). Expression of two crucial epithelial branching factors, fibroblast growth factor-<em>10</em> and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant <em>protein</em> C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible factor-1 alpha or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.

Osteoprotegerin (OPG), a newly discovered member of the tumor necrosis factor receptor family, is a potent inhibitor of osteoclastogenesis. The overexpression of OPG in transgenic mice leads to osteopetrosis, whereas targeted ablation of OPG in knock-out mice leads to severe osteoporosis. However, the production and regulation of OPG in normal human <em>bone</em> has not been studied. Thus, we assessed OPG mRNA expression and <em>protein</em> secretion in human osteoblastic lineage cells. 1,25-Dihydroxyvitamin D3 (<em>10</em>(-7) M) increased OPG mRNA levels by 90 and 50% in a fetal osteoblastic cell line (hFOB) and normal trabecular osteoblastic cells (hOB) cells, respectively, but did not affect OPG mRNA levels in a marrow stromal preosteoblastic (hMS) cell line. Interleukin (IL)-1beta (5 x <em>10</em>(-9) M), tumor necrosis factor (TNF)-alpha (9 x <em>10</em>(-9) M), and <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2 (<em>10</em>0 ng/ml) also increased OPG mRNA levels in hFOB cells by 4-, 6-, and 4-fold, respectively. Treatment with 1,25-dihydroxyvitamin D3, IL-1beta, TNF-alpha, and BMP-2 increased OPG <em>protein</em> production by hFOB cells by 60, 390, 300, and 80%, respectively (P < 0.001). Because it is expressed in various types of human osteoblastic cells, and is stimulated by vitamin D, BMP-2 and cytokines, OPG may be an important paracrine modulator of <em>bone</em> remodeling.

Human pluripotent stem cells (hPSCs) represent a renewable source of pancreatic beta cells for both basic research and therapeutic applications. Given this outstanding potential, significant efforts have been made to identify the signaling pathways that regulate pancreatic development in hPSC differentiation cultures. In this study, we demonstrate that the combination of epidermal growth factor (EGF) and nicotinamide signaling induces the generation of NKX6-1(+) progenitors from all hPSC lines tested. Furthermore, we show that the size of the NKX6-1(+) population is regulated by the duration of treatment with retinoic acid, fibroblast growth factor <em>10</em> (FGF<em>10</em>), and inhibitors of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) and hedgehog signaling pathways. When transplanted into NOD scid gamma (NSG) recipients, these progenitors differentiate to give rise to exocrine and endocrine cells, including monohormonal insulin(+) cells. Together, these findings provide an efficient and reproducible strategy for generating highly enriched populations of hPSC-derived beta cell progenitors for studies aimed at further characterizing their developmental potential in vivo and deciphering the pathways that regulate their maturation in vitro.

Subcutaneous implantation of demineralized <em>bone</em> matrix initiates a sequence of developmental events, which culminate in endochondral <em>bone</em> formation. During early stages of development of matrix-induced implants, ED1, Ia-positive monocytes-macrophages were observed, suggesting that in the initial phases of the endochondral <em>bone</em> formation cascade, the <em>bone</em>-inductive <em>protein</em> osteogenin and related <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) might serve as potent chemoattractants to recruit circulating monocytes. In this investigation, we demonstrate that at concentrations of <em>10</em>-<em>10</em>0 fg/ml (0.3-3 fM), native bovine osteogenin and recombinant human BMP-2B (rhBMP-2B) induce the directed migration of human blood monocytes in vitro. This chemotactic response was associated with expression of BMP binding sites (receptors) on monocytes. About 750 receptors per cell were detected with an apparent dissociation constant of 200 pM. Both osteogenin and rhBMP-2B at higher concentrations (0.1-30 ng/ml) stimulated mRNA expression for an additional regulatory molecule, type beta 1 transforming growth factor (TGF-beta 1) in human monocytes. TGF-beta 1, in turn, is known to induce a cascade of events leading to matrix generation. Monocytes stimulated by TGF-beta are known to secrete a number of chemotactic and mitogenic cytokines that recruit endothelial and mesenchymal cells and promote their synthesis of collagen and associated matrix constituents. TGF-beta 1 in concert with these other cytokines and matrix components regulates chemotaxis, mesenchymal proliferation, differentiation, angiogenesis, and controlled synthesis of extracellular matrix. Our results demonstrate that osteogenin and related BMPs through their profound effects on monocyte recruitment and cytokine synthesis may promote additional successive steps in the endochondral <em>bone</em> formation cascade.

OBJECTIVE

To study the effects of recombinant human osteogenic protein-1 (rHuOP-1; bone morphogenetic protein-7) on proteoglycan and collagen synthesis by human articular chondrocytes.

METHODS

Articular chondrocytes from fetal, adolescent, and adult human donors were cultured in alginate beads for 4 days in a mixture of Ham's F-12, Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), then for an additional 3-10 days in the presence and absence of rHuOP-1, with and without FBS. Chondrocyte synthetic activity was measured as the amount of incorporation of 35S-sulfate into proteoglycans and 3H-proline into hydroxyproline. Sieve chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed to identify specific proteoglycans and collagens.

RESULTS

Recombinant human OP-1 markedly stimulated the synthesis of proteoglycans (mostly aggrecan) and collagens (predominantly type II) by all chondrocyte preparations. This did not require the presence of FBS and was associated with continued expression of the chondrocyte phenotype.

CONCLUSIONS

Recombinant human OP-1 is a more potent stimulator of the synthesis of cartilage-specific molecules by human articular chondrocytes than are other factors tested for comparison, including TGF beta 1 and activin A.

Arterial calcification is a phenotype of vascular repair in atherosclerosis, diabetes, hyperphosphatemic renal failure, and aging. Arterial calcification is modulated by transition of arterial smooth muscle cells (SMCs) from contractile to chondro-osseous differentiation programmed in response to increases in P(i), <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2, and certain other stimuli. Transglutaminase (TG)2 release modulates tissue repair, partly by transamidation-catalyzed covalent crosslinking of extracellular matrix substrates. TG2 regulates cultured SMC differentiation, resistance artery remodeling to vasoconstriction, and atherosclerotic lesion size. Here, TG2 expression was required for the majority of TG activity in mouse and human aortic SMCs. TG2(-/-) SMCs lost the capacity for P(i) donor-induced formation of multicellular <em>bone</em>-like nodules and for increased expression of the type III sodium-dependent P(i) cotransporter Pit-1 and certain osteoblast and chondrocyte genes (tissue-nonspecific alkaline phosphatase, the osteoblast master transcription factor runx2, and chondrocyte-restricted aggrecan), and for P(i) donor- and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2-induced calcification. Uniquely in TG2(-/-) SMCs, P(i) donor treatment increased expression of the physiological SMC chondro-osseous differentiation and calcification inhibitors osteoprotegerin, matrix Gla <em>protein</em>, and osteopontin. Conversely, TG2(-/-) SMCs, unlike wild-type SMCs, failed to maintain contractile differentiation on laminin. Exogenous catalytically active TG2 augmented calcification by TG2(-/-) SMC in response to P(i) donor treatment. TG2 expression also drove P(i)-stimulated calcification of mouse aortic ring organ cultures, which was suppressed by the TG2 catalytic site-specific inhibitor Boc-DON-Gln-Ile-Val-OMe (<em>10</em> micromol/L). Our results suggest that TG2 release in injured arteries is critical for programming chondro-osseous SMC differentiation and calcification in response to increased P(i) and <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2.

Although isolated reports of hard-tissue reconstruction in the cranio-maxillofacial skeleton exist, multipatient case series are lacking. This study aimed to review the experience with 13 consecutive cases of cranio-maxillofacial hard-tissue defects at four anatomically different sites, namely frontal sinus (3 cases), cranial <em>bone</em> (5 cases), mandible (3 cases), and nasal septum (2 cases). Autologous adipose tissue was harvested from the anterior abdominal wall, and adipose-derived stem cells were cultured, expanded, and then seeded onto resorbable scaffold materials for subsequent reimplantation into hard-tissue defects. The defects were reconstructed with either bioactive glass or β-tricalcium phosphate scaffolds seeded with adipose-derived stem cells (ASCs), and in some cases with the addition of recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2. Production and use of ASCs were done according to good manufacturing practice guidelines. Follow-up time ranged from 12 to 52 months. Successful integration of the construct to the surrounding skeleton was noted in <em>10</em> of the 13 cases. Two cranial defect cases in which nonrigid resorbable containment meshes were used sustained <em>bone</em> resorption to the point that they required the procedure to be redone. One septal perforation case failed outright at 1 year because of the postsurgical resumption of the patient's uncontrolled nasal picking habit.

BACKGROUND

Juvenile polyposis (JP) is an inherited condition predisposing to upper gastrointestinal (UGI) polyps and colorectal cancer. Two genes are known to predispose to JP, SMAD4 and bone morphogenetic protein receptor type 1A (BMPR1A). The object of this study was to determine the differences in phenotype of patients with SMAD4 or BMPR1A mutations (MUT+) compared with those without (MUT-).

METHODS

DNA was extracted from 54 JP probands and used for polymerase chain reaction of all exons of SMAD4 and BMPR1A. Products were then sequenced and analyzed for mutations. Medical record data were used to create a JP database, and statistical analysis was performed using Fisher's exact and unpaired t-tests.

RESULTS

Nine of 54 patients had germline SMAD4 mutations, 13 had BMPR1A mutations, and 32 had neither. There were no significant differences between SMAD4+ and BMPR1A+ cases in terms of clinical factors examined, except for a family history of UGI involvement (P <.01). There was a higher prevalence of familial cases in MUT+ patients (P =.09), >10 lower gastrointestinal polyps (P =.06), and frequency of family history of gastrointestinal cancer compared with MUT- patients (P =.01).

CONCLUSIONS

Patients with germline SMAD4 or BMPR1A mutations have a more prominent JP phenotype than those without, and SMAD4 mutations predispose to UGI polyposis.

Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to <em>bone</em> tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-9 (rhBMP-9) would induce <em>bone</em> formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day <em>10</em>. <em>Bone</em> tissue was formed in the site of rhBMP-9 delivery, as was shown by micro-computerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring <em>bone</em> tissue regeneration after further development that will increase the transfection efficiency.

Genetic variations in ovulation rate which occur in different breeds of sheep provide useful models to explore the mechanisms regulating the development of antral follicles. The Booroola gene, an autosomal mutation that affects ovulation rate, has been known for over two decades and despite intensive research it has not yet been identified. Using resources from human genome mapping and known data about gene linkage and chromosome location in the sheep, we selected the gene encoding the <em>Bone</em> <em>Morphogenetic</em> <em>Protein</em> receptor (BMPR) type 1 B (ALK-6) as a candidate site for the mutation. The BMPR1B gene in the human is located at the region linked with the Booroola mutation, syntenic to chromosome 6 in the sheep. A fragment of the sheep BMPR1B gene was cloned from an ovarian cDNA and the deduced aminoacid (AA) sequence is over 98% homologous to the known mammalian sequences. cDNA and genomic DNA from 20 Booroola genotypes were screened and two point mutation were found in the kinase domain of the receptor, one at base 746 of the coding region (A in the ++ to a G in FF animals) which results in a change from a glutamine in the wild type to a arginine in the Booroola animals. Another point mutation was identified at position 1113, (C to A) but this mutation does not change the coding aminoacid. The first mutation was confirmed in genomic DNA from <em>10</em> ewes from an independent Brazilian flock which segregates the Booroola phenotype. In all instances homozygous FecB gene carrier (n=11) had only the 746 A to G mutation, non gene carriers (n=14) had only the wild type sequence and heterozygote gene carriers (n=5) had both sequences. This mutation in the subdomain 3 of the kinase domain could result in an alteration in the expression and/or phosphorylation of SMADs, resulting in the phenotype characteristic of the Booroola animals which is the 'precocious' development of a large number of small antral follicles resulting in increased ovulation rate.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are well-established agents for inducing orthotopic and ectopic <em>bone</em> formation. However, their clinical usefulness as regenerative agents may be limited by a short in vivo half-life and low specific activity. BMP gene therapy is an alternative route for exploiting the <em>bone</em>-inductive activity of this class of molecules. To test the feasibility of this approach, we examined the osteogenic activity of AdCMV-BMP7, an adenovirus containing BMP7 cDNA under control of the CMV promoter that was constructed using Cre/lox recombination (Hardy et al. [1997] J. Virol. 71:1842-1849). Adenovirus vectors were shown to readily infect a wide variety of cell types in vitro including osteoblasts, fibroblasts, and myoblasts. COS7 cells transduced with AdCMV-BMP7 produced high levels of BMP-7 (approximately 0.5 microg/<em>10</em>(6) cells). Furthermore, transduction of C2C12 murine myoblast cells with AdCMVBMP-7 suppressed the muscle phenotype and induced in vitro osteoblast differentiation. To test its in vivo biological activity, AdCMV-BMP7 was mixed with a bovine <em>bone</em>-derived collagen carrier (<em>10</em>(8) plaque-forming units virus/site) and was implanted into mouse muscle and dermal pouches. In both cases, an ossicle containing cortical and trabecular <em>bone</em> and a clearly defined marrow cavity formed at the site of virus implantation within 4 weeks. These data demonstrate that AdCMV-BMP7 transduced cells produce biologically active BMP-7 both in vitro and in vivo and show that gene therapy by direct viral transduction using a virus/matrix implant may be a viable route for stimulating <em>bone</em> regeneration.

Currently available murine models to evaluate mesenchymal stem cell (MSC) differentiation are based on cell injection at ectopic sites such as muscle or skin. Due to the importance of environmental factors on the differentiation capacities of stem cells in vivo, we investigated whether the peculiar synovial/cartilaginous environment may influence the lineage specificity of <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2-engineered MSCs. To this aim, we used the C3H<em>10</em>T1/2-derived C9 MSCs that express BMP-2 under control of the doxycycline (Dox)-repressible promoter, Tet-Off, and showed in vitro, using the micropellet culture system that C9 MSCs kept their potential to differentiate toward chondrocytes. Implantation of C9 cells, either into the tibialis anterior muscles or into the joints of CB17-severe combined immunodeficient bg mice led to the formation of cartilage and <em>bone</em> filled with <em>bone</em> marrow as soon as day <em>10</em>. However, no differentiation was observed after injection of naïve MSCs or C9 cells that were repressed to secrete BMP-2 by Dox addition. The BMP-2-induced differentiation of adult MSCs is thus independent of soluble factors present in the local environment of the synovial/cartilaginous tissues. Importantly, we demonstrated that a short-term expression of the BMP-2 growth factor is necessary and sufficient to irreversibly induce <em>bone</em> formation, suggesting that a stable genetic modification of MSCs is not required for stem cell-based <em>bone</em>/cartilage engineering.

The human <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) family consists of over 20 growth factor <em>proteins</em> that are involved in <em>bone</em> formation and developmental processes. BMPs are extracellular signalling molecules that are able to regulate various cellular functions, proliferation, differentiation, apoptosis and migration. For the last <em>10</em> years, these powerful cytokines have increasingly been studied in several cancers, and aberrant expression patterns of BMPs have been reported. Functional studies have suggested that BMPs are involved in both cancer promotion and inhibition. The role these signalling molecules play in breast cancer is only starting to emerge: thus far, studies have been even contradictory. Different BMP ligands have been shown to decrease as well as increase cancer cell growth and migration. Furthermore, they are involved in <em>bone</em> metastases, which are a common feature in breast cancer. In this sense, BMPs resemble a closely related <em>protein</em> transforming growth factor beta, which possesses a bidirectional role in cancer cell regulation. In this review, we focus on the current knowledge of BMP expression, functional roles and involvement in <em>bone</em> metastasis in breast cancer.

The purpose of this prospective randomised clinical study was to compare the efficacy of recombinant <em>bone</em> <em>morphogenetic</em> <em>protein</em> 7 (rhBMP-7) and platelet-rich plasma (PRP) as <em>bone</em>-stimulating agents in the treatment of persistent fracture non-unions. One hundred and twenty patients were randomised into two treatment groups (group rhBMP-7 vs. group PRP). Sixty patients with sixty fracture non-unions were assigned to each group (median age: 44 years, range 19-65, for the rhBMP-7 group and 41 years, range 21-62, for the PRP group, respectively). In the rhBMP-7 group, there were 15 tibial non-unions, <em>10</em> femoral, 15 humeral, 12 ulnar, and 8 radial non-unions. In the PRP group, there were 19 tibial non-unions, 8 femoral, 16 humeral, 8 ulnar, and 9 radial non-unions. The median number of operations performed prior to our intervention was 2 (range 1-5) and 2 (range 1-5) with autologous <em>bone</em> graft being used in 23 and 21 cases for the rhBMP-7 and PRP groups, respectively. Both clinical and radiological union occurred in 52 (86.7%) cases of the rhBMP-7 group compared to 41 (68.3%) cases of the PRP group, with a lower median clinical and radiographic healing time observed in the rhBMP-7 group (3.5 months vs. 4 months and 8 months vs. 9 months, respectively). This study supports the view that in the treatment of persistent long <em>bone</em> non-unions, the application of rhBMP-7 as a <em>bone</em>-stimulating agent is superior compared to that of PRP with regard to their clinical and radiological efficacy.

Posterior lumbar interbody fusion (PLIF) and transforaminal lumbar interbody fusion (TLIF) create intervertebral fusion by means of a posterior approach. Both techniques are useful in managing degenerative disk disease, severe instability, spondylolisthesis, deformity, and pseudarthrosis. Successful results have been reported with allograft, various cages (for interbody support), autograft, and recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2. Interbody fusion techniques may facilitate reduction and enhance fusion. The rationale for PLIF and TLIF is biomechanically sound. However, clinical outcomes of different anterior and posterior spinal fusion techniques tend to be similar. PLIF has a high complication rate (dural tear, 5.4% to <em>10</em>%; neurologic injury, 9% to 16%). These findings, coupled with the versatility of TLIF throughout the entire lumbar spine, may make TLIF the ideal choice for an all-posterior interbody fusion.